Mitsuhiro Yokoyama

Factors that are related to atherosclerosis and cardiovascular morbidity and mortality, including hypercholesterolemia, hy- pertension, cigarette smoking, and diabetes mellitus, are found to be associated with endothelial dysfunction. Some studies have shown that the risk for developing endothelial dysfunction increases with the number of risk factors present in an individual. Endothelial dysfunction is not confined to the coronary arteries but rather represents a systemic disorder that also affects peripheral vascular beds, including both conduit arteries and small resistance vessels in the extremi- ties. Therefore, a uniform underlying mechanism(s) is sug- gested to account for the impaired endothelial function. See page 1614 Recent studies have demonstrated that increased produc- tion of reactive oxygen species in human blood vessels is associated with endothelial dysfunction. The major source of superoxide anion in the vasculature is NAD(P)H oxidase family of enzymes. NAD(P)H oxidases are present in the vascular cells including endothelial cells, smooth muscle cells, fibroblasts, and macrophages. 2 Vascular NAD(P)H oxidase is a multisubunit enzyme complex that differs struc- turally and biochemically from the phagocytic NAD(P)H oxidase. This superoxide-generating enzyme includes the membrane-bound flavocytochrome b558 formed by gp 91 phox and p22 phox and the cytosolic proteins p47 phox, p67 phox, and Rac. Recently, novel gp 91 phox(Nox2) homo- logues, termed Nox 1, Nox 3, Nox 4, and Nox 5, were identified in nonphagocytic cells, including Nox1 and Nox 4 in the vasculature.3 The simultaneous presence of multiple Nox proteins are demonstrated in one cell type. In vascular smooth muscle cells, gp 91 phox is expressed at very low levels, and Nox 1 and Nox 4 are coexpressed at much higher levels. Nox 4 as well as Nox 2 is expressed in vascular endothelial cells. Each Nox is involved in superoxide pro- duction. In cells that express more than one gp 91phox homologue, it is possible that each Nox protein serves a specific biological function and that different intracellular localization of each Nox protein determines the distinct role of each Nox protein in cell functions. The localization of Nox 1 in caveolae could be involved in cell growth-promoting actions by angiotensin II type 1 receptors. The localization of Nox 4 in focal adhesions and in the nucleus could be associated with integrin-linked signaling (eg, stretch) and aspects of gene expression (eg, growth, differentiation, in- flammatory reactions, senescence, and apoptosis), respective- ly.4 Nox 1 is inducible and upregulated by growth factors and hormones, whereas Nox 4 is downregulated by these ago- nists. Demonstration of the nox response to vascular injury indicates that the expression of nox 1 and nox 4 in the vascular wall is temporary and spatially regulated in vivo. 5

Atherosclerosis is associated with an impairment of endothelium-dependent relaxations, which represents the reduced bioavailability of nitric oxide (NO) produced from endothelial NO synthase (eNOS). Among various mechanisms implicated in the impaired EDR in atherosclerosis, superoxide generated from dysfunctional eNOS has attracted attention. Under conditions in which vascular tissue levels of tetrahydrobiopterin (BH4), a cofactor for NOS, are deficient or lacking, eNOS becomes dysfunctional and produces superoxide rather than NO. Experimental studies in vitro have revealed that NO from eNOS constitutes an anti-atherogenic molecule. A deficiency of eNOS was demonstrated to accelerate atherosclerotic lesion formation in eNOS knockout mice. In contrast, eNOS overexpression with hypercholesterolemia may promote atherogenesis via increased superoxide generation from dysfunctional eNOS. Thus, eNOS may have 2 faces in the pathophysiology of atherosclerosis depending on tissue BH4 metabolisms. An improved understanding of tissue BH4 metabolisms in atherosclerotic vessels is needed, which would help in developing new strategies for the inhibition and treatment of atherosclerosis.

Objective— Lysophosphatidylcholine (LPC), a major phospholipid component of oxidized low density lipoprotein, has been demonstrated to induce multiple functional alterations of vasculature that are potentially involved in atherosclerosis. Recently, an orphan G-protein-coupled receptor, G2A, has been identified as a high-affinity receptor for LPC. Although it has been demonstrated that G2A is expressed predominantly in lymphoid tissues and lymphocytes, there are no reports to determine whether G2A is expressed in atherosclerotic lesions and cardiovascular cells. Methods and Results— Immunohistochemistry with an anti-G2A antibody revealed that G2A was expressed predominantly by macrophages within atherosclerotic lesions at the aortic root of apolipoprotein E-deficient mice and the thoracic aortas of Watanabe heritable hyperlipidemic rabbits. In atherosclerotic plaques of human coronary arterial specimens, G2A was expressed by macrophages within the lipid-rich plaques, whereas no immunoreactivity of G2A was observed in fibrous plaques where macrophages did not exist. Reverse transcription-polymerase chain reaction analysis demonstrated that G2A mRNA was highly expressed in human and murine monocytes/macrophages. The expression of G2A protein was detected in human and murine monocytes/macrophages by immunoblotting. Conclusions— These findings demonstrate that monocytes/macrophages abundantly express G2A and suggest that G2A may play a role in the formation and progression of atherosclerotic lesions.

Background—Augmented vasoconstriction to serotonin (5-hydroxytryptamine [5-HT]) in atherosclerotic vessels plays a crucial role in the development of myocardial ischemia. We investigated mechanisms for serotonin-evoked hypercontraction in atherosclerotic rabbit coronary arteries.Methods and Results—Contractile responses to serotonergic agents of endothelium-denuded coronary arteries from control and Watanabe heritable hyperlipidemic rabbits (WHHL) were examined. WHHL coronary arteries exhibited hypercontraction to 5-HT₁–receptor agonists; the constrictor threshold concentrations andED₅₀to serotonin, 5-carboxamidotryptamine, and sumatriptan in WHHL were significantly lower, and theE_maxin WHHL to these agents were increased 55% to 59% above those of the control. Serotonin-evoked contractions in both groups were inhibited by GR127935 (5-HT_1B/1Dantagonist; 0.1 to 1 nmol/L) and pertussis toxin but not by ketanserin (5-HT₂antagonist; 0.01 to 1 μmol/L), suggesting that the hypercontraction is most likely mediated by 5-HT_1B/1Dreceptors through a pertussis toxin–sensitive pathway. Furthermore, simultaneous measurements of [Ca²⁺]_iand isometric tension of fura-2–loaded arteries revealed that the hypercontraction was concomitant with the augmented elevation of [Ca²⁺]_iin the smooth muscle. The 5-HT_1BmRNA levels in WHHL coronary arteries increased to 2.5-fold over those in control arteries, whereas neither 5-HT_1Dnor 5-HT_2AmRNA was detected in either group.Conclusions—Atherosclerotic rabbit coronary arteries exhibited the enhancement in contraction and Ca²⁺mobilization in response to serotonin. The 5-HT_1Breceptor, which is upregulated by atherosclerosis, most likely mediates the augmenting effects of serotonin.

Abstract:It is apparent that vascular tissues can produce reactive oxygen species, including the superoxide anion, and that their increased production can contribute to altered control of vasomotor tone and atherosclerosis. The NADH/NADPH oxidase system, which includes a 22 kD subunit (p22 phox), is the major source of superoxide production in vascular tissues. The superoxide radical oxidizes LDL and oxidized LDL is shown to be a key component in atherogenesis. Superoxide anion inactivates the NO radical, an anti‐atherogenic molecule. Lysophosphatidylcholine, which accumulates during oxidative modification of LDL, has multiple effects on vascular cells, including cell proliferation, migration, apoptosis, and gene expression. Lysophosphatidylcholine stimulates superoxide production in endothelial cells through the NADH/NADPH oxidase‐dependent mechanism. To investigate the expression of p22 phox in normal and atherosclerotic coronary arteries, samples were obtained from autopsy and examined using immunohistochemistry. In normal vessels, weak positive staining of p22 phox was detectable only in the adventitial layer. In contrast, strong immunoreactivity for p22 phox was present in atherosclerotic lesions around lipid core and shoulder regions. P22 phox was localized in the macrophages, fibroblasts, endothelial cells, and some smooth muscle cells which was identified by immunofluorescence double staining. The genetic analysis of the p22 phox gene by restriction fragment length polymorphism (RFLP) for control subject and patients with coronary artery disease revealed that the prevalence of the TC+TT genotype of the C242T polymorphism of the p22 phox gene in control subjects was significantly more frequent than in coronary artery disease patients, indicating that the mutation of the p22 phox gene might reduce the susceptibility for coronary artery disease, which is independent of other coronary risk factors. These observations suggest that oxidative stress, mainly via the NADH/NADPH oxidase system in the vasculature, may play an important role in the pathogenesis of atherosclerosis.