Actions of “antioxidants” in the protection against atherosclerosis

Abstract

This review addresses the role of oxidative processes in atherosclerosis and its resulting cardiovascular disease by focusing on the outcome of antioxidant interventions. Although there is unambiguous evidence for the presence of heightened oxidative stress and resulting damage in atherosclerosis, it remains to be established whether this represents a cause or a consequence of the disease. This critical question is complicated further by the increasing realization that oxidative processes, including those related to signaling, are part of normal cell function. Overall, the results from animal interventions suggest that antioxidants provide benefit neither generally nor consistently. Where benefit is observed, it appears to be achieved at least in part via modulation of biological processes such as increase in nitric oxide bioavailability and induction of protective enzymes such as heme oxygenase-1, rather than via inhibition of oxidative processes and lipid oxidation in the arterial wall. Exceptions to this may be situations of multiple/excessive stress, the relevance of which for humans is not clear. This interpretation is consistent with the overall disappointing outcome of antioxidant interventions in humans and can be rationalized by the spatial compartmentalization of cellular oxidative signaling and/or damage, complex roles of oxidant-producing enzymes, and the multifactorial nature of atherosclerosis.

Introduction

Cardiovascular disease (CVD) remains the major cause of mortality in the world, typically claiming a third of all deaths. The primary cause of CVD is atherosclerosis, which literally refers to the hardening of arteries. In atherosclerosis, medium to large arteries are affected and hardening is caused by the buildup of waste products, such as lipids including cholesterol, cellular debris, and calcium in the affected artery wall. Together these substances form plaque, which, when large enough, can restrict blood flow or, upon rupture, can give rise to a blood clot with the risk of a heart attack.

Atherosclerosis is a disease that progresses slowly during a lifetime and typically begins before adulthood. This slow disease progression and complex etiology have challenged attempts to identify the initial atherogenic event such that it remains elusive. The healthy artery consists of three distinct layers [1]. The innermost layer facing the arterial lumen consists of a monolayer of endothelial cells, followed by the inner elastic lamina. On the peripheral side of the inner elastic lamina reside layers of vascular smooth muscle cells (VSMC) that make up the media, bordered by the outer elastic lamina. The outer layer of the artery, the adventitia, consists of connective tissue.

The vascular endothelium is the barrier between the blood and the arterial vessel wall. One of the first clinically detectable changes in the vasculature during atherogenesis is a dysfunctional endothelium (reviewed in [2]). This is characterized by a decreased response to endothelium-dependent relaxation and has been reported for human arteries with atherosclerotic lesions [3] as well as for arterial segments isolated from animal models of atherosclerosis. A dysfunctional endothelium promotes many processes involved in atherogenesis [2]. These include decreased bioavailability of nitric oxide (^•NO) and VSMC migration and proliferation that can contribute to the formation of a neointima between the endothelium and the inner elastic lamina [1]. A key process commonly thought to be involved in endothelial dysfunction is the “uncoupling” of endothelial nitric oxide synthase (eNOS), a term used to describe the change in function of the synthase from an ^•NO- to a superoxide anion radical (O₂^•−)-producing enzyme [4], [5], [6]. Oxidative processes can contribute to the uncoupling of eNOS [7], although the relevance of this to endothelial dysfunction in vivo remains largely unknown.

Atherosclerotic lesions occur preferentially at regions exposed to disturbed blood flow, such as branch points and curvatures of arteries. The disturbed blood flow allows the endothelial cells that line the blood vessel to adopt a proinflammatory phenotype, and this generates arterial regions of higher permeability toward macromolecules [1], [8]. These facilitate the entry of lipoproteins, including low-density lipoprotein (LDL), into the arterial wall. This results in the accumulation of lipid, primarily cholesterol as well as polar, oxidized fatty acids [9], [10], and contributes to arterial inflammation [11], [12]. The initial lipid deposits have long been thought to consist of LDL retained in the intima that becomes internalized by macrophages [1] and thereby leads to foam cell formation and intimal cell proliferation. However, recent studies suggest that in the normal murine aortic intima, dendritic cells preferentially reside in areas predisposed to lesion formation [13] and that dendritic cells are important in the development of foam cells and early atherogenesis [14].

To characterize the initiating cause of atherosclerosis, three different but somewhat overlapping hypotheses have been proposed. Thus, the initiating event in atherosclerosis is considered to be a response to injury/inflammation, LDL retention, or the oxidative modification of LDL. These hypotheses overlap in that they all invoke LDL retention and modification in the intima as a key event (reviewed in [2]).

In the response-to-injury hypothesis, endothelial dysfunction is a key event. Initially it was thought that injury to the endothelium simply leads to increased permeability and hence passive deposition of LDL lipid in the subendothelial space. Subsequently, an active inflammatory response was invoked to contribute to endothelial cell injury and to drive lipid accumulation and alter arterial homeostasis [11], [12]. However, as atherosclerotic lesions can occur underneath a physically intact endothelium, the hypothesis has been refined to include the uncontrolled entry of atherogenic (i.e., altered) lipoproteins by dysfunctional endothelial cells.

Circulating lipoproteins move through the arterial wall in accordance with their molecular size. In rabbits, injection of human LDL results in lipoprotein aggregates within the arterial wall [15], a key observation based on which the response-to-retention hypothesis was formulated [16], [17]. Accordingly, proteoglycans in the arterial wall matrix retain extracellular lipoproteins containing apolipoprotein B-100 for subsequent cellular uptake [18]. In support of this theory, genetic manipulations aimed at interrupting the interaction between apolipoprotein B-100 and proteoglycans decrease atherosclerosis in animals [19], [20].

The basis of the oxidative modification hypothesis is the discovery by Brown et al. that chemically modified LDL is taken up by macrophage scavenger receptors at a 20-fold higher rate than native LDL [21]. Steinberg and co-workers proposed that “oxidation renders a chemical modification to LDL that increases its atherogenicity” [22]. Accordingly, LDL in the intima is subject to cell-induced oxidation that modifies the lipoprotein such that it is prevented from leaving the arterial wall and instead induces further lipid accumulation, endothelium dysfunction, and inflammation. Indeed, the presence of oxidized lipids in atherosclerotic lesions has been known for at least 40 years [23], and it has since been confirmed and extended to proteins in numerous studies. It is also clear that LDL oxidized in vitro by various means can lead to foam cell formation [24], [25], [26]. As reviewed previously, such “oxidized LDL” has a myriad of potential atherogenic properties (see e.g., [2], [22], [27], [28], [29], [30]). Of concern, however, “oxidized LDL” and “oxidatively modified LDL” are terms that do not refer to chemically defined entities [31], such that the meaning of studies employing in vitro-generated oxidized LDL remains unclear. Based on functional rather than chemical properties, a broad distinction has been made by some between “minimally modified LDL” (i.e., LDL still recognized by the LDL receptor) and “extensively/fully oxidized LDL” (i.e., oxLDL, recognized by scavenger receptors) [32]. However, the meaningfulness of this functional distinction can be argued, as lesion LDL is taken up avidly by macrophages independent of scavenger receptors [33]. Thus, despite more than 20 years of intense research, clear evidence for how, where, and when LDL is oxidized in the intima during atherogenesis, and to what extent this contributes directly to the initiation of atherosclerosis, remains largely unknown (Table 1).

Steinberg recently extended the original oxidative modification hypothesis by proposing that oxidative stress independent of, or accompanied by, LDL oxidation may contribute to atherogenesis [30]. Indeed, current research now more broadly examines how increased oxidative stress and oxidative events may participate in arterial pathology and how antioxidants may be involved in or regulate these processes. This review focuses on oxidative events, antioxidants, and their molecular and cellular effects in the context of atherosclerosis.