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From ST segments to endothelial pathophysiology: hypercholesterolemia and endothelial superoxide production
David G. Harrison
Division of Clinical Pharmacology, Department of Medicine, Vanderbilt University, Nashville, Tennessee, USA.
Address correspondence to: David G. Harrison, Betty and Jack Bailey Professor of Medicine, Pharmacology and Physiology, Director of Clinical Pharmacology, Room 536 Robinson Research Building, Vanderbilt University, Nashville, Tennessee 37232-6602, USA. Phone: 615.875.3049; Fax: 615.875.3297; E-mail: david.g.harrison@vanderbilt.edu.
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Published February 3, 2014 - More info
J Clin Invest. 2014;124(2):473–475. https://doi.org/10.1172/JCI70336.
© 2014 The American Society for Clinical Investigation
While a second-year internal medicine resident on the Duke coronary care unit in the 1970s, I admitted a young lady who was having repeated episodes of chest pain and profound ST-segment elevation on her electrocardiogram. During several of these episodes, she developed ventricular fibrillation that required cardioversion. An emergency cardiac catheterization revealed that her coronary arteries were overtly normal, but her left anterior descending coronary artery spontaneously developed spasm to the point of closure. Thankfully, these episodes eventually resolved. She obviously had variant angina, and I was impressed with the dramatic nature of her illness and the idea that diseases could alter vasomotion in such a striking fashion.
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Identifying a role of ROS
Our understanding of disease-dependent changes in EDRF production was advanced by the nearly concurrent observations that the EDRF was NO (7) and that the EDRF could be inactivated by superoxide (8). Because NO and superoxide react with one another at a near-diffusion-limited rate (9), we hypothesized that the hypercholesterolemia-associated defect in endothelium-dependent vasodilation was due to oxidative inactivation of NO by superoxide. At the time (late 1980s), apolipoprotein E–null mice, which develop severe hypercholesterolemia, were not available; therefore, we used cholesterol-fed rabbits as a model of hypercholesterolemia and early atherosclerosis. Fortunately, around this time I met Jim Bates, a faculty member in the Department of Anesthesia at the University of Iowa, who contributed substantially to these experiments (Figure 1). We used ozone-linked chemiluminescence to measure NO release from isolated perfused vascular segments and observed that vessels from cholesterol-fed rabbits produced NO, but it was largely released in the oxidized form of nitrite (NO2–) (10). Furthermore, cholesterol-fed rabbits treated with the superoxide scavenger polyethylene-glycolated superoxide dismutase exhibited improved endothelium-dependent vasodilation (11). Together, our results strongly suggested superoxide involvement in hypercholesterolemia-associated vascular dysfunction and in other molecular events in atherosclerosis.
Figure 1Collaboration. David Harrison (left), David Ku (center), and James Bates (right) at a Keystone Conference, circa 1990.
We next established methods to measure superoxide production by intact vessels. This was challenging, because many commonly used assays to study isolated enzymes or chemical reactions were either insensitive or subject to interference. After exploring several techniques, we settled on chemiluminescence-based methods to study intact vessels, and Dr. Yuichi Ohara, a postdoctoral fellow in our laboratory at the time, was instrumental in adapting this technique. We were surprised to discover that vessels from cholesterol-fed rabbits produced three times more superoxide than did normal vessels (12). Strikingly, removal of the vessel endothelium completely normalized superoxide production. Thus, hypercholesterolemia stimulated superoxide production in the very cells that were synthesizing NO, leading to NO oxidation prior to leaving the endothelium. A consequence of this reaction is formation of the strong oxidant peroxynitrite (OONO-), which oxidizes LDL, depletes antioxidants, and leads to the formation of isoprostanes. The isoprostanes are particularly interesting. These compounds, discovered in the early 1990s by my current colleague Jack Roberts (13), are potent vasoconstrictors and could therefore contribute to abnormal vasomotion in conditions of excessive oxidation. ROS are also implicated in many aspects of vascular disease, including lipid oxidation (14), adhesion molecule expression (15), platelet aggregation (16), thrombosis, matrix remodeling, and plaque rupture (17). Thus, superoxide production by the endothelium not only affects vasomotor tone, but also contributes to lesion formation.
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Subsequent studies and complexity
Our discoveries led to a simple concept (Figure 2A): hypercholesterolemia stimulates an increase in superoxide, which oxidizes NO, preventing endothelium-dependent vasodilation and in some cases unmasking vasoconstriction. The phrase, “reduced NO bioavailability” became commonly used in reference to conditions in which NO is produced but biologically unavailable through oxidation. Subsequent work from many groups expanded this concept, and it is now clear that as atherosclerosis progresses, plaque-associated and vascular smooth muscle cells produce ROS (18, 19). We initially found that oxypurinol reduced vascular superoxide production and endothelium-dependent vasodilation, suggesting a role for xanthine oxidase in early atherosclerosis (12). Subsequently, we learned that additional sources of ROS are important in vascular diseases, including the NADPH oxidases, uncoupled NOSs, and mitochondria. Our laboratory and others have shown that these various sources are interdependent (Figure 2B), and ROS from one source can activate ROS production by others in a feed-forward fashion (20–22). The NADPH oxidases appear to lead these interactions. For example, NADPH oxidase–produced ROS can oxidize tetrahydrobiopterin, an NOS cofactor, and this event uncouples these enzymes, resulting in the production of superoxide rather than NO (23). My colleague, Hua Cai, revealed that hydrogen peroxide inhibits the expression of dihydrofolate reductase, an enzyme involved in maintaining cellular levels of tetrahydrobiopterin, leading to eNOS uncoupling (24). Furthermore, NADPH oxidase–associated ROS disrupt mitochondrial electron transfer, leading to ROS production (25). The feed-forward nature of ROS production is recapitulated in several pathophysiological states (20).
Figure 2Evolving concepts of vascular ROS production. (A) Healthy vascular ECs respond to the presence of vasodilators by producing NO, which diffuses into smooth muscles and promotes vasodilation. Under hypercholesterolemia conditions, vascular ECs produce superoxide in addition to NO in response to vasodilators. Superoxide and NO interact within vascular ECs to produce OONO–, which is a strong oxidant that contributes to vascular dysfunction in individuals with hypercholesterolemia. (B) Interplay between various sources of ROS in vascular cells. NADPH oxidase–produced ROS can oxidize the eNOS cofactor tetrahydrobiopterin (purple), which uncouples these enzymes, resulting in ROS production. NADPH oxidase–associated ROS also disrupt mitochondrial electron transfer, leading to ROS production. Furthermore, ROS from other sources promote the formation of ROS by xanthine oxidoreductase. This feed-forward nature of ROS production promotes several pathophysiological states.
It should be noted that all methods for measuring superoxide and related ROS are imperfect. Dr. Sergey Dikalov joined our group and helped us adapt additional methods for ROS detection and quantification, including electron spin resonance, HPLC to monitor oxidation of dihydroethidium, and cytochrome c reduction, as well as hydrogen peroxide–detecting methods. Use of these methods confirmed our initial chemiluminescence-based measurements and proved extremely useful.
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