Influence of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitors on endothelial nitric oxide synthase and the formation of oxidants in the vasculature

Abstract

HMGCoA reductase inhibitors (statins) can have effects outside the target tissue, liver, including serious side-effects such as rhabdomyolysis as well as beneficial pleiotrophic effects. One such effect is upregulation of endothelial nitric oxide synthase (e-NOS) which generally leads to vasorelaxation. However, changing the balance between localized NO and O₂⁻ fluxes can also lead to oxidant stress and cellular injury through formation of reactive secondary oxidants such as peroxynitrite. We compared different statins for e-NOS subcellular localization, formation of pro-oxidants, and endothelial-dependent vascular function. Vascular relaxation in aortas of statin-dosed rats was inhibited with simvastatin (sevenfold higher EC50 for acetyl-choline induced relaxation) and atorvastatin (twofold increase) but not pravastatin. Ex vivo oxidation of the fluorescent redox probe dihydrorhodamine-123 (DHR-123) was increased in aortas from simvastatin treated rats, indicating increased reactive nitrogen and oxygen species. Human aortic endothelial cells incubated with simvastatin exhibited up to threefold higher intracellular oxidation of DHR-123 along with a twofold increase in total e-NOS protein. The elevated e-NOS was found in the Golgi/mitochondrial fraction and not in the plasma membrane, and using immunofluorescence greater e-NOS was observed proximal to Golgi and cytoskeletal structures and away from plasma membrane in simvastatin-treated cells. The data suggest that the action of lipophilic statins in endothelium can shift e-NOS localization towards intracellular domains, thereby increasing the encounter with metabolically generated O₂⁻ to produce peroxynitrite and related oxidants. Thus, under some conditions the direct action of lipophilic HMGCoA reductase inhibitors may unbalance NO and O₂⁻ fluxes and promote oxidant stress, compromising potentially beneficial vascular effects of e-NOS upregulation and increasing the potential for damage to muscle and other tissues.

Introduction

The statins exert their principle effects to lower circulating LDL cholesterol levels through inhibition of HMGCoA reductase activity in the liver. Inhibition of the reductase elsewhere can be a benefit or a liability in the efficacy/safety profile of statins [1]. The serious though infrequent occurrence of statin-associated myotoxicity and rhabdomyolysis is believed to be related to blockade of HMGCoA reductase in skeletal muscle myocytes. While the exact mechanism is unresolved, differences in myotoxic potential are apparent among the individual statins [2]. Although reducing LDL-cholesterol improves endothelial function, cholesterol independent effects including stimulated endothelial nitric oxide synthase (e-NOS) protein expression and improved vascular relaxation have been described for several statins [3], [4], [5], [6]. NO bioavailability may be augmented by statins through only partly understood mechanisms attributed to blockade of isoprenoid synthesis, distinct from statin suppression of biosynthesis of the pathway endproduct, cholesterol. This blockade can alter prenylation and localization of small GTPases involved in e-NOS activity and membrane/caveolar interactions as well as other cellular processes [3], [4], [5], [6]. However, prenylation of a large number of intracellular proteins changes following statin treatment [7], and cytoskeletal organization and membrane trafficking may be affected in complex ways.

Vascular endothelial cells as well as myocytes are subjected to locally generated reactive nitrogen and oxygen species, produced endogenously and by adjacent vessel wall and tissue cells. If they are generated in excess these oxidants injure cells, while at moderate levels they modulate intracellular signaling pathways. In the context of pleiotrophic actions of statins and myotoxicity, a broader view of e-NOS-derived NO in the possible formation of reactive nitrogen and oxygen species and redox balance in the tissues could be considered. It has been shown that NO-derived oxidants and other reactive oxidant species (ROS) originating from sources external to the myocyte, such as from endothelium, can disrupt the intracellular oxidant status in skeletal muscle fibers [8], suggesting that muscle oxidant stress could be contributed to by endothelial NO-derived oxidants. Further, NO-dependent relaxation of skeletal muscle microvessels has been shown to be diminished by superoxide in muscles from the Zucker rat model [9].

Reactive oxygen and nitrogen species derived from metabolic processes in the vasculature and tissues is balanced by anti-oxidant defenses in cells and plasma. Disruption of this balance can result in diminished endothelial function, associated with loss of vascular relaxation, impaired tissue perfusion, and a pro-inflammatory and pro-atherogenic state [10], [11], [12]. Unquenched NO can oxidize and inactivate intracellular iron-containing enzymes of energy metabolism, such as aconitase and cytochrome oxidase [13]. NO is one of the few biological molecules which can effectively compete with superoxide dismutase (SOD) for the superoxide anion (O₂⁻), and the very fast reaction of NO with O₂⁻ producing peroxynitrite is diffusion rate-limited, such that the relative fluxes and localization of the two radical species controls peroxynitrite formation [14]. The subsequent reactions of peroxynitrite with biological molecules are comparatively slow and modulated by pH and bicarbonate/carbonate, making peroxynitrite a selective oxidant which attacks protein aromatic and sulfhydryl residues, membrane and LDL lipid, and nucleic acids [14], [15]. Peroxynitrite has also been associated with increased cyclooxygenase (COX) activity [16].

Multiple sources of superoxide anion (O₂⁻) are present in endothelium, include mitochondrial respiration, peroxisomal oxidases, xanthine oxidase, COXs, and NAD(P)H oxidases.Regulation of e-NOS involves translocation between plasmalemma in caveolar structures in association with caveolin, and the cytoplasmic compartment in association with Golgi and other intracellular membranes and cytoskeletal structures [4], [17], [18], [19]. Reactive oxygen species have been shown to alter e-NOS localization and caveolin interaction [20]. However, the influence of the mevalonate pathway and statin drugs on the localization of e-NOS and the intracellular production of ROS and NO-related oxidants is incompletely understood. Therefore, we studied the effects of statins on models of endothelial function, and used the fluorescent redox probe dihydrorhodamine-123 (DHR-123) and other assays in vivo and in vitro to evaluate the relationship of HMGCoA reductase inhibitors to e-NOS localization, oxidant generation, and redox balance in the vessel wall.