Atherosclerosis, the principal cause of heart attack, stroke and gangrene of the extremities, is responsible for 50% of all mortality in the USA, Europe and Japan. The lesions result from an excessive, inflammatory-fibroproliferative response to various forms of insult to the endothelium and smooth muscle of the artery wall. A large number of growth factors, cytokines and vasoregulatory molecules participate in this process. Our ability to control the expression of genes encoding these molecules and to target specific cell types provides opportunities to develop new diagnostic and therapeutic agents to induce the regression of the lesions and, possibly, to prevent their formation.

The pathogenesis of atherosclerosis: a perspective for the 1990s

Nitric oxide contrasts with most intercellular messengers because it diffuses rapidly and isotropically through most tissues with little reaction but cannot be transported through the vasculature due to rapid destruction by oxyhemoglobin. The rapid diffusion of nitric oxide between cells allows it to locally integrate the responses of blood vessels to turbulence, modulate synaptic plasticity in neurons, and control the oscillatory behavior of neuronal networks. Nitric oxide is not necessarily short lived and is intrinsically no more reactive than oxygen. The reactivity of nitric oxide per se has been greatly overestimated in vitro because no drain is provided to remove nitric oxide. Nitric oxide persists in solution for several minutes in micromolar concentrations before it reacts with oxygen to form much stronger oxidants like nitrogen dioxide. Nitric oxide is removed within seconds in vivo by diffusion over 100 microns through tissues to enter red blood cells and react with oxyhemoglobin. The direct toxicity of nitric oxide is modest but is greatly enhanced by reacting with superoxide to form peroxynitrite (ONOO-). Nitric oxide is the only biological molecule produced in high enough concentrations to out-compete superoxide dismutase for superoxide. Peroxynitrite reacts relatively slowly with most biological molecules, making peroxynitrite a selective oxidant. Peroxynitrite modifies tyrosine in proteins to create nitrotyrosines, leaving a footprint detectable in vivo. Nitration of structural proteins, including neurofilaments and actin, can disrupt filament assembly with major pathological consequences. Antibodies to nitrotyrosine have revealed nitration in human atherosclerosis, myocardial ischemia, septic and distressed lung, inflammatory bowel disease, and amyotrophic lateral sclerosis.

Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and ugly.

Evolution has resorted to nitric oxide (NO), a tiny, reactive radical gas, to mediate both servoregulatory and cytotoxic functions. This article reviews how different forms of nitric oxide synthase help confer specificity and diversity on the effects of this remarkable signaling molecule.

https://faseb.onlinelibrary.wiley.com/doi/pdf/10.1096/fasebj.6.12.1381691

Nitric oxide as a secretory product of mammalian cells.

Accumulating evidence suggests that oxidant stress alters many functions of the endothelium, including modulation of vasomotor tone. Inactivation of nitric oxide (NO(.)) by superoxide and other reactive oxygen species (ROS) seems to occur in conditions such as hypertension, hypercholesterolemia, diabetes, and cigarette smoking. Loss of NO(.) associated with these traditional risk factors may in part explain why they predispose to atherosclerosis. Among many enzymatic systems that are capable of producing ROS, xanthine oxidase, NADH/NADPH oxidase, and uncoupled endothelial nitric oxide synthase have been extensively studied in vascular cells. As the role of these various enzyme sources of ROS become clear, it will perhaps be possible to use more specific therapies to prevent their production and ultimately correct endothelial dysfunction.

Endothelial Dysfunction in Cardiovascular Diseases: The Role of Oxidant Stress

Nitric oxide (NO.), a potentially toxic molecule, has been implicated in a wide range of biological functions. Details of its biochemistry, however, remain poorly understood. The broader chemistry of nitrogen monoxide (NO) involves a redox array of species with distinctive properties and reactivities: NO+ (nitrosonium), NO., and NO- (nitroxyl anion). The integration of this chemistry with current perspectives of NO biology illuminates many aspects of NO biochemistry, including the enzymatic mechanism of synthesis, the mode of transport and targeting in biological systems, the means by which its toxicity is mitigated, and the function-regulating interaction with target proteins.

https://science.sciencemag.org/content/sci/258/5090/1898.full.pdf

Biochemistry of nitric oxide and its redox-activated forms

Studies of cultured bovine aortic endothelial cells using quantitative chemiluminescence techniques have shown that the amount of nitric oxide released under basal conditions, or in response to either bradykinin or the calcium ionophore A23187 is insufficient to account for the vasorelaxant activities of the endothelium-derived relaxing factor (EDRF) derived from the same source. This observation contradicts previous suggestions that nitric oxide and EDRF are the same compound, but may be explained if EDRF is a compound that contains nitric oxide within its structure but is a much more potent vasodilator than nitric oxide. Such a molecule could be one of several nitrosothiols which may yield nitric oxide after a one-electron reduction. The present experiments were carried out to test the possibility that the biological activities of the endothelium-derived relaxing factor might more closely resemble those of one of these compounds, S-nitrosocysteine, than nitric oxide. Nitric oxide release from cultured bovine aortic endothelial cells was detected by chemiluminescence and bioassay experiments compared the vasodilator potencies of nitric oxide, S-nitrosocysteine, and EDRF. The results suggest that EDRF is much more likely to be a nitrosylated compound such as a nitrosothiol than authentic nitric oxide.

Vasorelaxant properties of the endothelium-derived relaxing factor more closely resemble S-nitrosocysteine than nitric oxide

We examined the hypothesis that impaired endothelium-dependent vasodilation in atherosclerosis is associated with decreased synthesis of nitrogen oxides by the vascular endothelium. The descending thoracic aortae of rabbits fed either normal diet, a high cholesterol diet for 2-5 wk (hypercholesterolemic, HC), or a high cholesterol diet for 6 mo (atherosclerotic, AS) were perfused in a bioassay organ chamber with physiologic buffer containing indomethacin. Despite a dramatic impairment in the vasodilator activity of endothelium-dependent relaxing factor (EDRF) released from both HC and AS aortae (assessed by bioassay), the release of nitrogen oxides (measured by chemiluminescence) from these vessels was not reduced, but markedly increased compared to NL. Thus, impaired endothelium-dependent relaxation in atherosclerosis is neither due to decreased activity of the enzyme responsible for the production of nitrogen oxides from arginine nor to arginine deficiency. Because the production of nitrogen oxides increased in response to acetylcholine in both hypercholesterolemic and atherosclerotic vessels, impairments in signal transduction are not responsible for abnormal endothelium-dependent relaxations. Impaired vasodilator activity of EDRF by cholesterol feeding may result from loss of incorporation of nitric oxide into a more potent parent compound, or accelerated degradation of EDRF.

/pdf/diet-induced-atherosclerosis-increases-the-release-of-25moudnaen.pdf

Diet-induced atherosclerosis increases the release of nitrogen oxides from rabbit aorta.

Nitric oxide generation from nitroprusside by vascular tissue. Evidence that reduction of the nitroprusside anion and cyanide loss are required.

The present studies were performed to determine if abnormal endothelium-dependent vascular relaxation in atherosclerosis is due to decreased production or release of endothelium-derived relaxing factor (EDRF) by atherosclerotic rabbit vessels or if atherosclerotic vessels are less sensitive to the relaxing effects of EDRF. EDRF release was quantified using two approaches, by the response of bioassay detector vessels and also by the activation of guanylate cyclase within cultured endothelial cells. Using these assays, atherosclerotic vessels were found to release significantly less EDRF than normal vessels in response to both receptor- and nonreceptor-mediated stimuli. Relaxations of normal and atherosclerotic vessels to luminally applied EDRF (derived from normal rabbit aortas stimulated by the calcium ionophore, A23187) and nitric oxide, a putative EDRF, were also studied. Atherosclerotic vessels were more sensitive to EDRF than normal vessels, and equally sensitive to nitric oxide. Additional studies performed in organ chambers failed to demonstrate augmented constriction of atherosclerotic vessels in response to acetylcholine in the presence or absence of methylene blue or LY83583, compounds which inhibit the effect of EDRF. We conclude that decreased EDRF release is the principal underlying mechanism responsible for abnormal endothelium-dependent vascular relaxation in atherosclerosis.

Mechanisms of Abnormal Endothelium-Dependent Vascular Relaxation in Atherosclerosis: Implications for Altered Autocrine and Paracrine Functions of EDRF

The endothelium-derived relaxing factor (EDRF) has recently been reported to be nitric oxide. This study was performed to determine whether the vasorelaxant activity of EDRF could be accounted for by nitric oxide released from cultured endothelial cells. Nitric oxide released from cultured bovine aortic endothelial cells was monitored using a chemiluminescence technique with and without reflux processing of the effluent with a strong reducing environment (1% sodium iodide in glacial acetic acid). In the presence of indomethacin, basally released EDRF was sufficient to relax a bioassay detector vessel (preconstricted with prostaglandin F2 alpha) by 41 +/- 8%. Bradykinin (0.01 microM) and A23187 (10 microM) produced relaxation of the bioassay detector vessel equal to 74 +/- 7 and 69 +/- 13%, respectively. In both the absence and the presence of reflux preprocessing of the cell effluent in a reducing environment, the amount of nitric oxide detected by chemiluminescence was 7-10-fold less than that required to account for the detector vessel relaxation (determined from responses of the detector vessel to standard infusions of authentic nitric oxide). These experiments contradict the view that nitric oxide is the sole or principal EDRF. It is likely that either other nonprostanoid vasodilator substances are released in addition to nitric oxide or that EDRF is a nitric oxide containing or forming compound that is substantially more potent than authentic nitric oxide.

Ricardo Guerra

Papers

Vasorelaxant properties of the endothelium-derived relaxing factor more closely resemble S-nitrosocysteine than nitric oxide

Diet-induced atherosclerosis increases the release of nitrogen oxides from rabbit aorta.

Nitric oxide generation from nitroprusside by vascular tissue. Evidence that reduction of the nitroprusside anion and cyanide loss are required.

Mechanisms of Abnormal Endothelium-Dependent Vascular Relaxation in Atherosclerosis: Implications for Altered Autocrine and Paracrine Functions of EDRF

Release of NO and EDRF from cultured bovine aortic endothelial cells.