Oxidative Stressgenerating Systems In Hypercholesterolemia

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Under normal physiological conditions, nitric oxide (NO) predominates over O2~ in vascular endothelial cells. The excess NO regulates endothelial cell function, modulates CAM expression, inhibits leukocyte adhesion, and limits platelet activation (adhesion and aggregation). However, a large amount of evidence has amassed in the literature that implicates oxidative stress, a consequence of increased reactive oxygen species (ROS) generation and decreased NO bioavail-ability, in the chronic inflammatory responses observed in all segments of the microcirculation when blood cholesterol levels are elevated. Many of the enzymes involved in ROS production during hypercholesterolemia-induced oxidative stress have been identified, including NAD(P)H oxidase, xanthine oxidase (XO), myeloperoxidase, lipoxygenase, and endothelial nitric oxide synthase (eNOS). These pathways produce substantial quantities of ROS such as O2~ and HOCl, overwhelming the capacity of endogenous antioxidants such as superoxide dismutase (SOD), catalase, and NO, thereby resulting in an oxidative stress. Evidence for the participation of the above mechanisms is derived from several animal models and human studies, using both direct measurements and pharmacological inhibitors. Indeed, the excess levels of oxi-dants may propagate the response to hypercholesterolemia by promoting the oxidative modification of LDL, which exacerbates many of the inflammatory consequences.

An increased production of O2~ has been demonstrated in arteries of hypercholesterolemic animals (5). This can be attributed to hypercholesterolemia per se since it can be corrected by dietary means (6). The response is blunted when the vessels are denuded, implicating vascular endothelium as a major source of O2. In order to elucidate the enzyme(s) responsible for the O2~ production, several inhibitors have been tested. Treatment with either XO inhibitors (allopurinol or oxypuri-nol) (7) or heparin (which competes with XO for binding sites on the vascular endothelium), each diminish O2~ production, implicating a role for XO. Studies using blockers of flavanoid-containing oxidases suggest that NAD(P)H oxidase may also be important in the O2~ generation observed in hypercholesterolemic vessels of rabbits (8). Guzik et al. (5) demonstrated that hypercholesterolemia was independently associated with O2~ generation from an NAD(P)H-containing enzyme in human saphenous veins, and that XO did not participate in this response. Furthermore, several studies using genetically engineered mice deficient in either p47phox or gp91phox (also known as NOX-2), subunits of NAD(P)H oxidase, have supported these findings.

During acute hypercholesterolemia, a p47phox-containing NAD(P)H oxidase enzyme has been implicated in the generation of O2~ from both leukocytes and the vascular endothelium of postcapillary venules (9). Crossing atherosclerosis-prone ApoE-deficient mice with p47phox-deficient mice led to reduced lesion development in the descending aorta but not at the origin of the aorta, suggesting that site-specific differences may exist for ROS production during hypercholesterolemia (10). No protection was seen in gp91phox-=- mice (11), although this may simply reflect the fact that many cells are capable of producing O2~ via NAD(P)H oxidases containing other NOX isotypes. In humans, for instance, gp91phox and NOX-4 are expressed in atherosclerotic lesions and are associated with macrophages and smooth muscle cells, respectively (12).

Nitric oxide presents a more complicated picture, such that under normal conditions, endothelial-derived NO exerts a homeostatic influence, generally viewed as anti-inflammatory. During inflammation, the bioavailability of NO is reduced, although this does not necessarily reflect decreased production. The imbalance between NO and O2~ generation favors a proinflammatory environment (Fig. 1). The reduced bioavailability of NO may be explained by several mechanisms. Under normal physiological conditions, endothelium-derived NO counteracts the actions of O2; however, during hypercholesterolemia, eNOS may become uncoupled. This could occur as a result of decreased availability of the substrate l-arginine, or diminished cofactors such as tetrahydrobiopterin (BH4). During hypercholesterolemia, l-arginine levels remain within the normal range; however, l-arginine supplementation partially reverses the accompanying endothelial dysfunction (13). This suggests that the ability of endothelial cells to transport l-arginine to the cell interior may be impaired during hypercholesterolemia or that the endogenous inhibitor asymmetric dimethylarginine (ADMA) is competitively inhibiting the interaction between l-arginine and eNOS (14). The latter possibility is supported by the fact that ADMA levels are raised during hypercholes-terolemia, perhaps as a result of diminished degradation by dimethylarginine dimethylaminohydrolase (DDAH), which is reduced in hypercholesterolemic rabbits.

Studies with BH4-knockout mice have revealed a role for this cofactor in the pathogenesis of atherosclerosis, such that

Figure 1 Inflammatory alterations in arterioles and venules elicited by hypercholesterolemia. Basal nitric oxide (NO) release maintains arteriolar smooth muscle tone and prevents cell-cell interactions in venules. During hypercholesterolemia, NO bioavailability is reduced and reactive oxygen species (ROS) generation is elevated, promoting smooth muscle contraction in arterioles and inducing a proinflammatory and prothrombogenic phenotype in the venular segment of the microcirculation. l-arginine (l-arg); endothelial nitric oxide synthase (eNOS); superoxide (O2~); catalase (cat); glutathione peroxidase (GSH); hydrogen peroxide (H2O2); superoxide dismutase (SOD); dimethylarginine dimethylaminohy-drolase (DDAH); asymmetric dimethylarginine (ADMA); intercellular adhesion molecule-1 (ICAM-1); hypoxanthine (HX); xanthine oxidase (XO); interleukin-12 (IL-12); interferon-y (IFN-y).

Figure 1 Inflammatory alterations in arterioles and venules elicited by hypercholesterolemia. Basal nitric oxide (NO) release maintains arteriolar smooth muscle tone and prevents cell-cell interactions in venules. During hypercholesterolemia, NO bioavailability is reduced and reactive oxygen species (ROS) generation is elevated, promoting smooth muscle contraction in arterioles and inducing a proinflammatory and prothrombogenic phenotype in the venular segment of the microcirculation. l-arginine (l-arg); endothelial nitric oxide synthase (eNOS); superoxide (O2~); catalase (cat); glutathione peroxidase (GSH); hydrogen peroxide (H2O2); superoxide dismutase (SOD); dimethylarginine dimethylaminohy-drolase (DDAH); asymmetric dimethylarginine (ADMA); intercellular adhesion molecule-1 (ICAM-1); hypoxanthine (HX); xanthine oxidase (XO); interleukin-12 (IL-12); interferon-y (IFN-y).

these mice have accelerated lesion development. Supplementation of ApoE~=- mice with sepiapterin, a precursor of BH4

(15), or infusion of BH4 into hypercholesterolemic patients

(16), blunts the endothelial cell dysfunction. It was also found that ONOO~ and to a lesser degree O2~ accelerated the decay of BH4, suggesting that the interaction between O2~ and NO

serves to exacerbate the oxidative stress not only by its oxidizing action but also by promoting the uncoupling of eNOS (15). In a murine model of atherosclerosis, a protective role was found for eNOS, when eNOS/ApoE double-knockout mice demonstrated exaggerated atherosclerosis development (17). In contrast to eNOS, iNOS has been shown to exert a proin-flammatory influence in many models of injury. Support for this comes from studies using mice that are genetically deficient in both iNOS and ApoE. These exhibit a reduction in fatty streak size when compared to ApoE~/- mice (18).

Despite the evidence implicating a role for oxidative stress in the pathogenesis of hypercholesterolemia-induced vascular responses, there are conflicting reports concerning the effects of hypercholesterolemia on endogenous anti-oxidant levels. Some studies have found antioxidants such as catalase, SOD, and glutathione peroxidase are decreased, while others have failed to find such a reduction. This may reflect differences between the cellular sources investigated, the time-point of assessment, or interspecies variability (19). Regardless of whether these enzymes are altered during hypercholesterolemia, it certainly appears that their antioxi-dant capacity is overwhelmed, thereby contributing to the oxidative stress found in hypercholesterolemia.

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