Vascular free radical release. Ex vivo and in vivo evidence for a flow-dependent endothelial mechanism.

Abstract

Mechanisms underlying production of vascular free radicals are unclear. We hypothesized that changes in blood flow might serve as a physiological stimulus for endothelial free radical release. Intact isolated aortas from 45 rabbits were perfused with the spin trap a-phenyl-N-tertbutylnitrone (PBN, 20 mmol/L) and formed radical adducts detected by electron paramagnetic resonance spectroscopy (EPR). Sequential perfusion at 2, 7.5, and 12 mL/min changed cumulative vascular PBN radical adduct yields, respectively, from 3.2+0.9 to 4.1±0.7 (P<.05) and 7.0±1.5 (P<.005) pmol/mg with endothelium and from 3.6±1.6 to 3.8±1.4 and 2.2±0.8 pmol/mg without endothelium (P=NS). In endothelialized aortas, superoxide dismutase (SOD) completely blocked flow-induced free radical production, whereas inactivated SOD, indomethacin, and the nitric oxide synthetase antagonist nitro-L-arginine methyl ester (L-NAME) had no effect; relaxations to acetylcholine remained unchanged with higher flows. To assess the role of flow on in vivo radical production, femoral arterial plasma levels of the ascorbyl radical, a stable ascorbate oxidation product, were measured by direct EPR in 56 other rabbits. Ascorbyl levels were assessed at baseline (30.2±0.7 nmol/L) and at peak-induced iliac flow changes. Flow increases from 25% to 100% due to saline injections through an extracorporeal aortic loop induced significant dose-dependent increases in ascorbyl levels (n=5). In addition, after papaverine bolus injections, flow increased by 114±8% versus baseline, and ascorbyl levels increased by 5.4±0.7 nmol/L (n=31, P<.001); similar results occurred with adenosine, isoproterenol, or hyperemia after 30-second occlusions (P<.05, n=4 or 5 in each group). Active SOD completely blocked papaverine-induced ascorbyl radical increase, despite preserved flow response (Aascorbyl=0.02±1.6 nmol/L, P=NS); inactivated SOD, catalase, indomethacin, and L-NAME had no effect. Blood flow decreases of 65% to 100% due to phenylephrine or 60-second balloon occlusions were accompanied by an average decrease of 4.4 nmol/L (P<.05) in ascorbyl levels. No change in ascorbyl signal was observed when rabbit blood alone was submitted to in vitro flow increases through a tubing circuit. Thus, increases in blood flow trigger vascular free radical generation; such a response seems to involve endothelium-derived superoxide radicals unrelated to cyclooxygenase or nitric oxide synthetase activities. This mechanism may contribute to explain vascular free radical generation in physiological or pathological circumstances. (Circ Res. 1994;74:700-709.) Key Words * ascorbyl radical * superoxide dismutase * nitric oxide * endothelium * oxidative stress T he identification of the gaseous free radical nitric oxide (QN=O) as a major signal transducer molecule and endothelium-derived vascular relaxing factor1 suggests that oxyreduction reactions are important effector steps for autocrine or paracrine regulation of vessel tone, permeability, and structure in physiological or pathological conditio...

The mechanism of flow-induced dilation in human adipose arterioles involves hydrogen peroxide during CAD

“…Cardiovascular disease states are known to reduce NO bioavailability by elevating the production of ROS, namely, superoxide (30). Many studies demonstrating reduced FID in the presence of CAD involve examination of large conduit arteries (8) that are much more prone to develop atherosclerosis than smaller arterioles.…”

“…Similar to HE, microvessels were incubated with and without a cell-permeable PEG-CAT (500 U/ml) to identify that portion of fluorescence specifically attributable to H 2O2. DCFH oxidizes rapidly to the highly fluorescent 2Ј,7Ј-dichlorofluorescein (DCF) in the presence of ROS (30). DCF fluorescence was excited by light at 488 nm and visualized utilizing fluorescence microscopy described above.…”

“…For example, in animals, cardiovascular disease and its risk factors are associated with impaired flow-induced release of NO due to production of ROS (27,30). In contrast, FID in coronary arterioles from patients with CAD is maintained primarily by the ROS, hydrogen peroxide (H 2 O 2 ), and epoxyeicosatrienoic acid (33, 38).…”

Temporal Gradient in Shear But Not Steady Shear Stress Induces PDGF-A and MCP-1 Expression in Endothelial Cells

“…One speculative possibility regards the involvement of superoxide (O 2 Ϫ ). Fluid shear can induce sustained production of reactive oxygen species, 44 which induces the expression of ICAM-1 and c-fos. 45,46 The breakdown of O 2 Ϫ occurs mainly by reaction with NO to form peroxynitrite (ONOO Ϫ ), 47 which has cytoprotective effects similar to those of NO at low concentration.…”

Effects of the Kupffer cell inactivator gadolinium chloride on rat liver oxygen uptake and content of mitochondrial cytochromes

“…These ¢ndings may be due to the previous observation that GdCl Q leads to an intrahepatic shift in carbon distribution, decreasing the uptake of carbon by liver macrophages and enhancing that of endothelial cells [8]. Although cultured endothelial cells exhibit a respiratory burst in response to several pathological stimuli in vitro (reviewed in [22]), it is uncertain whether they do so in vivo under physiological stimuli or in the intact liver perfused with colloidal carbon. Depression of basal O P consumption by perfused livers at 24 h after GdCl Q treatment (Fig.…”