Timothy E. Peterson

Hypercholesterolemia increases endothelial superoxide anion production.

Indirect evidence suggests accelerated degradation of endothelium-derived nitric oxide (ENDO) by superoxide anion (O2-) in hypercholesterolemic vessels (HV). To directly measure O2- production by normal vessels (NV) and HV, we used an assay for O2- based on the chemiluminescence (CL) of lucigenin (L). HV (1 mo cholesterol-fed rabbits) produced threefold more O2- than NV (1.47 +/- 0.20 nM/mg tissue/min, n = 7 vs. 0.52 +/- 0.05 nmol/mg tissue/min, n = 8, P < 0.001). Endothelial removal increased O2- production in NV (0.73 +/- 0.08, n = 6, P < 0.05), while decreasing it in HV (0.76 +/- 0.15, n = 5, P < 0.05). There was no difference between denuded HV and denuded NV. Oxypurinol, a noncompetitive inhibitor of xanthine oxidase, normalized O2- production in HV, but had no effect in NV. In separate isometric tension studies treatment with oxypurinol improved acetylcholine induced relaxations in HV, while having no effect on responses in normal vessels. Oxypurinol did not alter relaxations to nitroprusside. Thus, the endothelium is a source of O2- in hypercholesterolemia probably via xanthine oxidase activation. Increased endothelial O2- production in HV may inactivate endothelium-derived nitric oxide and provide a source for other oxygen radicals, contributing to the early atherosclerotic process.

Dietary Correction of Hypercholesterolemia in the Rabbit Normalizes Endothelial Superoxide Anion Production

BACKGROUND We have shown that hypercholesterolemia increases vascular superoxide anion (O2-) production, which could be responsible for augmented inactivation of endothelium-derived vascular relaxing factor. We sought to determine whether this increased vascular O2- production is due to infiltration of macrophages into the intima and whether dietary treatment of hypercholesterolemia normalizes O2- production. METHODS AND RESULTS A specific and sensitive assay for O2- based on chemiluminescence of lucigenin was used; the amount of O2- produced by vascular ring segments was quantified based on known quantities of O2- produced by xanthine-xanthine oxidase standards. O2- production of aortic segments from normal rabbits (n = 9), cholesterol-fed rabbits (1% cholesterol diet for 1 month, n = 7), and rabbits fed a 1% cholesterol diet for 1 month followed by a normal diet for 1 month (regression rabbits, n = 5) was measured. At the end of these diets, serum cholesterol levels were 1.5 +/- 0.2, 26.0 +/- 3.9, and 1.8 +/- 0.5 mmol/L (58 +/- 6, 1000 +/- 150, and 71 +/- 19 mg/dL) in the normal, cholesterol-fed, and regression animals, respectively. Vessels from normal rabbits with endothelium produced 0.32 +/- 0.06 nmol O2-/mg dry wt per minute, whereas those without endothelium produced approximately twice as much O2- (0.66 +/- 0.12 nmol O2- mg dry wt per minute. Vessels with endothelium from cholesterol-fed rabbits produced 4.5-fold more O2- than vessels from normal animals. This increased production of O2- was normalized by endothelial removal. This increased production of O2- was not due to infiltration of macrophages in the intima, because there was no correlation between vascular O2- production and macrophage infiltration assessed by immunohistochemistry with use of a specific antibody against rabbit macrophage. O2- production by vessels from regression rabbits was similar to that observed in normal animals, and as in the normal rabbits, endothelial removal increased O2- production. Aortic rings from these animals also were studied in organ chambers. Dietary lowering of cholesterol dramatically improved vasodilator responses to acetylcholine and A23187 (P < .05 versus cholesterol-fed rabbits). CONCLUSIONS Dietary lowering of cholesterol not only improves endothelium-dependent vascular relaxation but also normalizes endothelial O2- production. Decreases of O2- production by dietary lowering of cholesterol not only may improve vasomotor control but also may improve other aspects of vascular integrity in atherosclerosis.

Lysophosphatidylcholine increases vascular superoxide anion production via protein kinase C activation.

We tested the hypothesis that lysophosphatidylcholine (lyso-PC) could activate protein kinase C in intact vascular segments and sought to examine some of the physiological consequences of this activation. In segments of rabbit aorta, the patterns of protein phosphorylation determined by two-dimensional electrophoresis stimulated by lyso-PC and 12-O-tetradecanoylphorbol 13-acetate (TPA) were similar. Activation of protein kinase C can stimulate superoxide anion (O2-) production in other tissues, and we found that lyso-PC-treated rabbit aortas produced twofold more O2- than control vessels. Calphostin C, a potent and specific inhibitor of protein kinase C, attenuated O2- production in lyso-PC-treated vessels but had no effect in control vessels. The effect of lyso-PC on O2- production was mimicked by TPA. In separate bioassay studies, release of the endothelium-derived vascular relaxing factor (EDRF) quantified by the response of detector vessels was markedly impaired after exposure of donor rabbit aortic segments to lyso-PC. After incubation with calphostin C, EDRF release in response to acetylcholine from lyso-PC-treated donor vessels was restored significantly. Thus, lyso-PC can activate protein kinase C in intact vessels, leading to an increase in O2- production. Activation of protein kinase C by lyso-PC may also play a role in altering the release of EDRF in response to acetylcholine. Increased O2- production in response to lyso-PC may have important consequences in the atherogenic process.

Intermittent hypoxia regulates vasoactive molecules and alters insulin-signaling in vascular endothelial cells

Vascular dysfunction and insulin resistance (IR) are associated with obstructive sleep apnea (OSA), which is characterized by frequent episodes of nocturnal intermittent hypoxia (IH). While it is recognized that the balance between vasoconstrictive (endothelin-1) and vasodilatory molecules (nitric oxide, NO) determine vascular profile, molecular mechanisms contributing to vascular dysfunction and IR in OSA are not completely understood. Caveolin-1 is a membrane protein which regulates endothelial nitric oxide synthase (eNOS) activity which is responsible for NO generation and cellular insulin-signaling. Hence, we examined the effects of IH on caveolin-1, eNOS, and endothelin-1 in human coronary artery endothelial cells in the context of IR. Chronic 3-day IH exposure up-regulated caveolin-1 and endothelin-1 expression while reducing NO. Also, IH altered insulin-mediated activation of AKT but not ERK resulting in increased endothelin-1 transcription. Similarly, caveolin-1 overexpression attenuated basal and insulin-stimulated NO synthesis along with impaired insulin-dependent activation of AKT and eNOS, with no effect on insulin-stimulated ERK1/2 phosphorylation and endothelin-1 transcription. Our data suggest that IH contributes to a vasoconstrictive profile and to pathway-selective vascular IR, whereby insulin potentiates ET-1 expression. Moreover, IH may partly mediate its effects on NO and insulin-signaling via upregulating caveolin-1 expression.