Activation of reactive oxygen species leading the atherosclerosis

Activation of ROS leading the atherosclerosis

Allie Always

Hiromi Ohara 1, Shigeaki Ito 1 1 Japan Tobacco Inc. 6-2, Umegaoka, Aoba-ku, Yokohama, Kanagawa, 227-8512, Japan

BY-SA

2.5

The pathogenesis of atherosclerosis is initiated by the production of reactive oxygen species (ROS: MIE), which elicit oxidative stress in the vasculature (KE1). Oxidative stress responses elicit further endothelial dysfunction (KE2). This impairs the endothelium readily causing the penetration of low-density lipoprotein (LDL) into the intima, which is directly oxidized by ROS, forming oxidized LDL (KE5). The impaired endothelium has an increased expression of adhesion molecules, which recruit blood monocytes, which adhere and then infiltrate into the intima region (KE3). The microenvironment of the impaired endothelium induces the differentiation of monocytes into macrophages (KE4). Macrophages in the intima uptake oxidized LDL intracellularly, forming foam cells (KE6) and the accumulation of lipid-rich foam cells and their debris after necrosis form plaques (i.e., lipid core, KE7)). These conditions promote the migration of fibroblasts and trans-differentiation of smooth muscle cells into myofibroblasts, which form a fibrous cap on the apical side of the plaque (KE7). Then, the increased expression of collagenase causes thinning of the fibrous cap by the degradation of collagen in the fibrous cap (KE8). These plaques are unstable and this eventually leads to rapture. The aggregation of platelets occurs around the raptured endothelium leading to the formation of three-dimensional clots (i.e. thrombosis, AO).

Life Stage Applicability Age is a significant independent risk factor for CVD because it is associated with an increased likelihood of developing any number of other additional cardiac risk factors, including obesity and diabetes [1]. The prevalence of most types of CVDs is considerably higher among older adults compared with the general population [2]. An increase in the production of ROS occurs with advancing age [3, 4], and is linked to persistent inflammation and progression to chronic disease status. Therefore, conditions that induce ROS activation and result in thrombosis may be more applicable to adults. Sex Applicability Estrogen was shown to have a cardioprotective role and to be directly associated with a lower overall incidence of CVD in premenopausal women compared with age-matched men [5-7]. The decline of sex hormones has an important role in the development of CVD with advanced age, in men and women [6]. Therefore, ROS activation resulting in thrombosis may be moderate in premenopausal women.

Evidence for the essentiality of Key Events (KEs) has been confirmed mostly by the attenuation of KEs by inhibitory substances, targeted gene silencing. Attenuation of KEs by inhibitory substances and modification of target gene expression (i.e., knockdown, knockout, and overexpression). Rationale for essentiality includes: Reactive oxygen species (ROS) lead to oxidative stress [High] Reactive oxygen species (ROS) are a group of highly reactive molecules derived from O2 metabolism [8]. Members of the ROS family include superoxide (O2-), alkoxyl radical (RO-), peroxyl radical (ROO-), hydroxyl radical (OH-), peroxynitrate (ONOO-), hydrogen peroxide (H2O2), ozone (O3), and hypochlorous acid (HOCl). Although physiological concentrations of ROS are important signaling molecules that maintain vascular homeostasis, excessive ROS production can lead to oxidative stress and the progression of vascular disease. ROS maintain vascular cell homeostasis by regulating the phenotype and fate of multiple cell types, including endothelial cells (ECs), vascular smooth muscle cells (SMCs), outer membrane cells, bone marrow cells, and resident stem/progenitor cells [9, 10]. The administration of the antioxidant N-acetylcysteine, a known inhibitor of oxidative stress, reduced the severity of atherosclerosis [11]. Oxidative stress leads to endothelial dysfunction [High] Endothelial dysfunction is considered an early indicator of atherosclerosis characterized by the overexpression of adhesion molecules including intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) [12]. Extracellular as well as intracellular ROS functions as signaling molecules that, produced either intracellularly, extracellularly or through ligand-receptor interactions, function as signaling molecules that activate ICAM-1 and regulate immune cells migration through the vascular endothelium to sites of inflammation and injury [13]. Endothelial dysfunction leads to monocyte infiltration [Moderate] Vascular endothelial functions are critical; thus, genetic modification to disrupt their function is lethal. Maintaining nitric oxide (NO) bioavailability is one of the key functions of the vascular endothelium and is achieved by the expression of endothelial NO synthase (eNOS). The overexpression of eNOS in a diet-induced atherosclerosis model resulted in a significant reduction in atherosclerotic lesions [14]. Monocyte infiltration is associated with the induction of ICAM-1 on the endothelial cell surface, which captures circulating blood monocytes. A deficiency of ICAM-1 in apolipoprotein E deficient mice significantly reduced atherosclerotic lesions [15]. Monocyte infiltration leads to macrophage differentiation [Moderate] The monocyte subpopulations, CC chemokine receptor 2 (CCR2)highLy6C+ inflammatory monocytes and CCRlowLy6C− resident monocytes, are generally thought to preferentially differentiate into M1 inflammatory macrophages and M2 anti-inflammatory macrophages, respectively, in early inflammation [16]. Ly6C− monocytes dominate the early phase of myocardial infarction and exhibit phagocytic, proteolytic, and inflammatory functions, as well as digesting damaged tissues. However, Ly6C− monocytes, recruited at a later phase of inflammation, have attenuated inflammatory properties and differentiate toward M2 macrophages and contribute to angiogenesis, genesis of myofibroblasts, and collagen deposition [17]. Oxidative stress leads to LDL oxidation [Moderate] Under oxidative stress, the oxidation of LDL occurs by lipid peroxidation, primarily involving phospholipid molecules. Under pathological conditions, apolipoprotein B-containing lipoproteins in the plasma penetrate through the damaged endothelium into the vascular subendothelial intima where they are oxidized by ROS [18, 19]. Macrophage differentiation leads to foam cell formation [Moderate] Monocytes migrate into the intima guided by chemokines [20] and differentiate into macrophages. These macrophages then take up modified lipoproteins and form foam cells as they accumulate excess lipids [21]. LDL oxidation leads to foam cell formation [Moderate] Oxidized LDL (ox-LDL) is the archetypal source of cholesterol and inducer of foam cell formation [22]. The formation of fatty streaks is a major characteristic of atherosclerosis caused by the conversion of macrophages into foam cells. Foam cell formation is characterized by an accumulation of lipids, predominantly cholesterol esters [22, 23]. Foam cell formation leads to plaque formation [Moderate] Subsequent cell apoptosis and necroptosis, complicated by failed efferocytosis (dead cell removal by phagocytes), lead to the formation of a lipid-rich necrotic core (NC) and production of thrombogenic tissue factors [24]. NC components and inflammatory cells promote the degradation of plaque-stabilizing extracellular fibrous matrix-like collagen and proteoglycans and thinning of the fibrous cap [25]. Hypoxia-inducible factors produced by cells contained in the NC promote pathologic neoangiogenesis, which favors intraplaque hemorrhage and further expansion of the NC. Unresolved inflammation triggers plaque calcification, which reduces further the mechanical stability of the plaque [25]. Plaque formation leads to plaque instability [Moderate] After foam cell formation, the release of substances including matrix metalloproteinases (MMPs) increases monocyte mobilization and promotes the degradation of extracellular matrix proteins including collagen and fibronectin [26]. This process leads to plaque instability and eventually plaque rupture [27, 28]. Plaque instability leads to thrombosis [Moderate] Mature atherosclerotic plaques are composed of a lipid core that is separated from the vessel lumen by a cap composed of fibrillar collagen [29]. Disruption of this cap exposes the plaque’s underlying thrombogenic core to the bloodstream, resulting in thromboembolism. This process of ‘plaque rupture’ is the main cause of acute coronary syndromes [30-33] and ischemic cerebral events [34-36].

Biological plausibility and empirical support for KERs Although ROS are generated under normal daily activity, they can be eliminated by homeostasis. Dysfunctional homeostasis and unhealthy daily habits can promote the persistent generation of ROS, resulting in chronic and high-level oxidative stress, which eventually leads to thrombosis. In general, the biological plausibility of causal linkage from reactive oxygen species as well as oxidative stress through various diseases including thrombosis is well established. Support for the biological plausibility of KERs is summarized in the table below.   <table cellspacing="0" class="Table" style="border-collapse:collapse; border:none; width:100%"> <tbody> <tr> <td colspan="3" style="background-color:white; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; height:40px; width:100%"> Support for biological plausibility of KERs </td> </tr> <tr> <td rowspan="2" style="background-color:white; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:25px; width:15%"> MIE => KE 1 </td> <td rowspan="2" style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:55%"> ROS generally activates the anti-oxidant system to maintain homeostasis of cellular functions. An imbalance between ROS and the anti-oxidant system leads to cellular oxidative stress, whereby cellular components are oxidized to be malfunctional. </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:28%"> Biological plausibility of the MIE => KE1 is high. </td> </tr> <tr> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:46px; width:28%"> There is a well-established mechanistic understanding between MIE-->KE1. </td> </tr> <tr> <td rowspan="2" style="background-color:white; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:203px; width:15%"> KE 1 => KE 2 </td> <td rowspan="2" style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:203px; width:55%"> Oxidative stress promotes an intracellular oxidative environment, which causes an uncoupling of endothelial nitric oxide synthase (eNOS). NO bioavailability is crucial to maintain vascular tone. Oxidative stress also alters the barrier integrity of endothelial cells and irregular expression of adhesion molecules such as ICAM-1. </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:203px; width:28%"> Biological plausibility of KE1 => KE2 is high. </td> </tr> <tr> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:57px; width:28%"> Oxidative stress is one cause of endothelial dysfunction. Inflammatory responses are also involved in endothelial dysfunction. However, a direct relationship between KE1 and KE2 is consistent with current biological knowledge. </td> </tr> <tr> <td rowspan="2" style="background-color:white; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:43px; width:15%"> KE 1 => KE 5 </td> <td rowspan="2" style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:43px; width:55%"> Oxidative stress leads to the generation of excess ROS, which causes the lipid peroxidation of LDL as well as apoB modification. The final product is oxidized LDL. </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:43px; width:28%"> Biological plausibility of KE1 => KE5 is high. </td> </tr> <tr> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:28%"> Strong relationship between KE1 => KE5 is well-established and consistent with current biological knowledge. </td> </tr> <tr> <td rowspan="2" style="background-color:white; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:25px; width:15%"> KE 2 => KE 3 </td> <td rowspan="2" style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:55%"> Endothelial dysfunction enables the easy access of circulating immune cells to the vascular intima, accompanied by the upregulation of adhesion molecules and disruption of the barrier integrity. Circulating monocytes attached to dysfunctional endothelial cells via adhesion molecules then penetrate into the intima. </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:28%"> Biological plausibility of KE2 => KE3 is high. </td> </tr> <tr> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:28%"> The functional relationship between KE 2 and KE 3 is consistent with current biological knowledge. </td> </tr> <tr> <td style="background-color:white; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:25px; width:15%"> KE 3 => KE 4 </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:55%"> Infiltrated monocytes are differentiated into macrophages by autocrine and paracrine mechanisms. Dysfunctional endothelial cells allow various immune cells to penetrate into the intima region. Oxidative stress also contributes to the activation of these immune cells, leading to the secretion of inflammatory cytokines, which promote the differentiation of monocytes into macrophages </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:28%"> Biological plausibility of KE3 => KE4 is high. </td> </tr> <tr> <td style="background-color:white; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:25px; width:15%"> KE 4 and KE 5=> KE 6 </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:55%"> Activated macrophages express receptors for LDL uptake. Representative receptors are the scavenger receptor and LOX-1. Macrophages uptake lipoproteins, especially oxidized-LDL, which accumulates inside the cells, termed foam cells. </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:28%"> Biological plausibility of KE4 => KE5 is high. </td> </tr> <tr> <td style="background-color:white; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:25px; width:15%"> KE6 =>KE7 </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:55%"> Accumulated foam cells then die via necrosis. Cell debris and lipids released from the necrotic foam cells form plaques. During plaque formation, smooth muscle cells migrate underneath endothelial cells and express extracellular matrix to form a fibrous cap. </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:28%"> Biological plausibility of KE5 => AO is high. </td> </tr> <tr> <td style="background-color:white; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:25px; width:15%"> KE7 =>KE8 </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:55%"> Cell death of the migrated smooth muscle cells causes a thinning of the fibrous cap. Extracellular matrix in the fibrous cap is also degraded by proteinases including matrix metalloproteinases. An unstable fibrous cap causes vulnerable plaques. </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:28%"> Biological plausibility of KE6 => AO is high. </td> </tr> <tr> <td style="background-color:white; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:25px; width:15%"> KE8 =>KE9 </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:55%"> Vulnerable plaques finally rupture and components in the plaques are eroded. Platelets accumulate and form a thrombus. </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:28%"> Biological plausibility of KE7 => AO is high. </td> </tr> <tr> <td colspan="3" style="background-color:white; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:46px; width:100%"> Empirical Support for KERs </td> </tr> <tr> <td style="background-color:white; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:25px; width:15%"> MIE => KE 1 </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:55%"> Oxidative stress is a condition whereby excess intracellular ROS is not scavenged. The source of ROS varies, for example, chemical substances induce ROS via biological processes including metabolism and mitochondrial activity. ROS inhibitors such as N-acetylcysteine attenuate oxidative stress. </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:28%"> Empirical support of the KER between MIE and KE1 is high. </td> </tr> <tr> <td style="background-color:white; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:25px; width:15%"> KE 1 => KE 2 </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:55%"> Treatment with the causative substances of ROS induction as well as oxidative stress, including hydrogen peroxide, causes endothelial dysfunction. For example, the increased expression of adhesion molecules and monocyte-endothelial adhesion are observed. These phenomena are caused by oxidative stress-induced inflammatory responses, and the scavenging of exogenous ROS with inhibitors such as N-acetylcysteine ameliorates oxidative stress-inducible endothelial dysfunction. </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:28%"> Empirical support of the KER between KE1 and KE2 is high. </td> </tr> <tr> <td style="background-color:white; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:25px; width:15%"> KE 1 => KE 5 </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:55%"> Circulating native LDL infiltrates into the intima region of the vasculature, where ROS oxidizes LDL to form oxidized-LDL (Ox-LDL). ROS inhibitors (e.g., NAC, resveratrol, ascorbate) attenuate the oxidative modification of LDL. </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:28%"> Empirical support of the KER between KE1 and KE5 is high. </td> </tr> <tr> <td style="background-color:white; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:25px; width:15%"> KE 2 => KE 3 </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:55%"> Monocyte infiltration is initiated by monocyte-endothelial adhesion via adhesion molecules expressed on the apical surface of endothelial cells. Adherent monocytes then infiltrate into the intima region of the vasculature. Increased expression of adhesion molecules and a leaky endothelial barrier are representative features of endothelial dysfunction. ICAM-1 deficiency in ApoE KO mice resulted in fewer atherosclerotic lesions, possibly because of the reduced recruitment of monocytes into the intima. However, few studies have provided direct evidence for a relationship between endothelial dysfunction and monocyte infiltration. </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:28%"> Empirical support of the KER between KE2 and KE3 is low. </td> </tr> <tr> <td style="background-color:white; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:25px; width:15%"> KE 3 => KE 4 </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:55%"> Monocytes differentiate into macrophages. Many studies reported that various stimuli such as proinflammatory cytokines promote this differentiation. Atherogenic vasculature produces proinflammatory cytokines; thus, theoretically, the infiltrated monocytes should be differentiated into macrophages in the intima. An increase in macrophage-like cells is observed in atherosclerotic lesions; however, direct evidence demonstrating infiltrated monocyte differentiation is limited. </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:28%"> Empirical support of the KER between KE3 and KE4 is moderate. </td> </tr> <tr> <td style="background-color:white; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:25px; width:15%"> KE 4 and KE 5=> KE 6 </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:55%"> Cumulative evidence suggests macrophages change their appearance related to the intracellular accumulation of lipids, leading to the formation of foam cells. The uptake of lipids, especially ox-LDL, is crucial for atherosclerotic lesions and is promoted by its receptors LOX-1 and scavenger receptor (CD36). A deficiency in these receptors results in reduced areas of lipid staining in arteries. </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:28%"> Empirical support of the KER between KE4-KE5 and KE6 is moderate. </td> </tr> <tr> <td style="background-color:white; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:25px; width:15%"> KE6 =>KE7 </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:55%"> Inhibition of foam cell formation via the attenuation of scavenger receptors resulted in atherosclerotic plaque formation in ApoE KO mice. </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:28%"> Empirical support of the KER between KE6 and KE7 is moderate. </td> </tr> <tr> <td style="background-color:white; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:25px; width:15%"> KE7 =>KE8 </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:55%"> Plaque instability is caused by several metalloproteinases, and their inhibition was reported to prevent vulnerable atherosclerotic plaques [1]. </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:28%"> Empirical support of the KER between KE7 and KE8 is moderate. </td> </tr> <tr> <td style="background-color:white; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:25px; width:15%"> KE8 =>AO </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:55%"> Various blood components aggregate at the site of endothelial rupture. Tissue factors induced by inflammation and eroded by endothelial rupture have a critical role in this region, and their inhibition effectively reduced thrombosis [37]. </td> <td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:25px; width:28%"> Empirical support of the KER between KE8 and AO is moderate. </td> </tr> </tbody> </table> Concordance of dose-response relationships Studies presenting a clear dose-response relationship in the late stages of this AOP are limited because the later KEs are caused by the cumulative or constitutive effects of the earlier stages. Much evidence of dose-response relationships in the earlier KEs was reported; however, data of the later stages are sparse. In brief, oxidative stress (MIE) is caused by an imbalance between oxidants and their scavenging by anti-oxidant systems such as glutathione. The disruption of an anti-oxidant system was reported to be dose-dependent. Similarly, endothelial dysfunction was also dose-dependent; oxidative and proinflammatory chemical substances lead to eNOS instability, impaired barrier integrity, and increased adhesion molecule expression. However, the acute incidence of the early phase KEs including endothelial dysfunction is normally cleared by the homeostatic capacity of the vasculature; therefore, elicitation of the later KEs needs the consecutive or frequent occurrence of the earlier KEs rather than the strength of stimuli. Monocyte infiltration (KE3) requires the expression of adhesion molecules on the apical surface of endothelial cells, but at the appropriate time. Although macrophage differentiation (KE4) is promoted by proinflammatory cytokines, this occurs in a consecutive manner, not by a single stimulus. In this sense, consecutive inflammation in the vasculature is necessary for the differentiation of infiltrated monocytes into macrophages. LDL oxidation appears to be dose-dependent because the level of oxidants determines the fate of LDL. However, LDL is generally oxidized in the intima region of the vasculature; therefore, the causative oxidants are probably generated in the intima under oxidative conditions. KE7 through AO is a biological event that is cumulative of the earlier Kes; therefore, a dose-response relationship between apical exposure to chemicals and biological events is not obvious. Nevertheless, strong stimulation of the vascular system requires a long time to clear the early key events; thus, consecutive unhealthy conditions in the vasculature are likely to occur in a dose-dependent manner. Temporal concordance among the key events and adverse effects Few studies have evaluated the temporal concordance throughout this AOP. However, key event relationships between the KEs acutely elicited by stressors are well studied and established. Endothelial dysfunction (KE1) is strongly associated with oxidative stress, because excess ROS is generated in cells under oxidative stress conditions, and ROS decreased nitric oxide, inflammation, and apoptosis in the vasculature, which eventually impair endothelial functions. Temporal concordance of the relationship between MIE and KE1 has been elucidated by time-course analyses and ROS scavenging. Temporal concordance among other key events is empirically or theoretically understood; however, robust evidence for most KERs is lacking from the viewpoint of time-course analyses or inhibition of specific KEs. Uncertainties, inconsistencies, and data gaps Uncertainties underlying this AOP include individual variability in the anti-oxidant capacity. As mentioned previously, oxidative stress is a condition whereby excess ROS is generated intracellularly, leading to the oxidation of intracellular components. Intracellular ROS levels are generally determined by the balance between ROS and the anti-oxidant capacity of cells. However, the anti-oxidant capacity varies between individuals related to their age, dietary behavior, smoking habits, and exercise.

<div> <table class="table table-bordered table-fullwidth"> <thead> <tr> <th>Modulating Factor (MF)</th> <th>Influence or Outcome</th> <th>KER(s) involved</th> </tr> </thead> <tbody> <tr> <td> </td> <td> </td> <td> </td> </tr> </tbody> </table> </div>

ROS induction is well-understood in terms of the dose-response relationship and various studies elucidated the quantitative relationship between intracellular ROS levels, endothelial dysfunction, and stressors [38]. However, this AOP is based upon chronic exposure to stressors, and not a single exposure. In addition, the level of stressors should be varied over time. Therefore, the progress of the AOP is influenced by environmental conditions and individual homeostatic capacity. Because there are many confounding factors for this AOP, a quantitative understanding of KERs is needed to determine how chronic elevation of intracellular ROS levels induced by a stressor could influence the downstream KEs and AO. Currently, there is a good quantitative understanding of how ROS activation influences oxidative stress, endothelial dysfunction, monocyte infiltration, macrophage differentiation, LDL oxidation, and foam cell formation. In addition, in most of the previous studies, the summary evidence indicates dose-response relationships, time-response relationships, and causality for ROS activation leading to increased oxidative stress, lending strong support for these KERs. However, quantitative knowledge is lacking with respect to the identity of thrombosis undergoing plaque formation and plaque instability, which makes empirical support for these KERs weak. Furthermore, data on plaque formation and plaque instability at the biological level were mainly obtained from surrogate measures, which are accepted in clinical practice as indicators of thrombosis, although they may not adequately reflect quantitative values. Taken together, quantitative evidence for the KERs at the tissue and organism levels is moderate at best.

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