Section II: A primer on the endothelium Risk factors lead to endothelial dysfunction Content Points: Components of CDS, such as dyslipidemia, hypertension and diabetes, cause physiological and structural changes that lead to CVD. One of the earliest changes occurring with diabetes is an alteration of the oxidative metabolism in the endothelium leading to an increase in the level of oxidative stress. This change causes the endothelial cells to decrease production of some compounds and increase production of others. Thus, nitric oxide (NO) production is decreased, facilitating vasoconstriction. Other compounds are released that allow plaque and thrombosis formation.3 Hypertension and elevated LDL-C levels cause a similar cascade of physiological and structural changes within the endothelium. In subsequent slides, changes in the endothelium will be discussed in greater detail.

Oxidative stress Content Points: Normal endothelial function is dependent on a balance between NO activity, which causes vessel dilation, and angiotensin II (A II) activity, which causes vasoconstriction. Normal oxidative metabolism in the arterial wall is also necessary. Oxidative stress, created by overproduction of superoxide anions, leads to breakdown of NO and vasoconstriction. A II can facilitate production of superoxide anions, thus increasing oxidative stress, impeding endothelium-dependent vasodilation and causing hypertension.4,5 Increasing oxidative stress with subsequent decreases in NO levels leads to other malfunctions within the endothelium that contribute to coronary artery disease. These malfunctions include increased expression of adhesion molecules and increased leukocyte adhesion, acute inflammation, smooth muscle cell proliferation and increased production of extracellular matrix.6

Factors in endothelial dysfunction—early stage plaque Content Points: As shown in earlier slides, the CDS causes endothelial dysfunction by a variety of mechanisms. Over time, alterations in the dsyfunctional endothelium lead to the development of atherosclerotic plaques.7 This figure illustrates some of the earliest changes that occur within the endothelium. Permeability of the endothelium to lipoproteins and plasma constituents is enhanced. This alteration is mediated by nitric oxide, A II, endothelin and other compounds. Production of endothelial adhesion molecules, such as E-selectin and P- selectin, is increased. Leukocyte adhesion molecules are produced in greater quantity, thus increasing the uptake of leukocytes and their adhesion to the vessel wall. Oxidized LDL-C is one of the factors that mediates this movement of leukocytes.

Factors in endothelial dysfunction—late stage plaque Content Points: As the dysfunctional endothelium progresses from early to more mature stages of atherosclerotic lesions, abnormal activity within the blood vessel increases.7 A fatty streak forms within the vessel wall. Initially, it contains foam cells (macrophages that have engulfed lipids). Later, smooth muscle cells begin to migrate into the developing plaque. Leukocytes continue to adhere to the endothelium and enter it, moving into the vessel wall. T cells become activated by binding antigen processed and presented by macrophages. The activated T cells within the growing atherosclerotic lesion secrete cytokines that amplify the inflammatory response. Platelets adhere to the dysfunctional endothelium and release cytokines and growth factors. These compounds contribute to the migration and proliferation of smooth muscle cells and monocytes, which may become macrophages. Activated platelets also release a precursor of thromboxane A2, a potent vasoconstrictor that also facilitates platelet aggregation. Platelets accumulating on the vessel wall also contribute to thrombus formation. As the atherosclerotic plaque progresses to late stages, a fibrous cap forms over the plaque and walls of the lesion from the vessel lumen. The core is a mixture of leukocytes, lipid and debris that may be necrotic. This fibrous cap may then become unstable and rupture causing thrombus formation and, often, CV events.

Regulation of the endothelium: Classic understanding of the RAS Content Points: There are 2 renin-angiotensin systems (RASs) that regulate endothelial function.8,9 The system illustrated in the slide is the classic or circulating RAS. The RAS is thought to be responsible for acute blood pressure regulation. The first step in regulation occurs in the kidneys. When the volume or flow of blood through the kidneys changes, cells within the kidneys detect the change and release renin. Sodium or volume depletion, shock, hemorrhage, heart failure or renal damage can change flow or volume of blood. This leads to conversion of angiotensinogen to A I and, with the help of angiotensin-converting enzyme (ACE), A I is converted to A II. A II is a potent vasoconstrictor and thus raises blood pressure to compensate for the decrease in blood volume or flow. This increase in flow or volume then decreases renin production through a feedback mechanism.10

Regulation of the endothelium: Circulating vs tissue ACE Content Points: Only about 10% of the ACE in the body circulates in the plasma. As shown in the previous slide, the circulating ACE is responsible for acute blood pressure changes. Approximately 90% of the ACE is found in tissues, for example, blood vessels, the heart and the central nervous system.11-13 In this system circulating or local A I is converted to A II by local ACE. This local production of A II by vascular ACE is thought to be involved in vascular and cardiac structure and function over the long term.8,9,14,15

Regulatory function of ACE Content Points: One class of pharmacologic agents that can regulate the RAS and thereby regulate endothelial function is the ACE inhibitors. Agents that inhibit ACE interfere with both the conversion of A I to A II and with the degradation of bradykinin.16 By halting breakdown of bradykinin, ACE inhibitors increase levels of NO, resulting in vasodilation and blood pressure reduction. Additionally, blood pressure is reduced through a decrease in the level of the vasoconstrictor A II. ACE plays a critical role in maintaining a balance between vasoconstriction/cell growth and vasodilation/cell growth inhibition.17 ACE inhibition can help to restore this balance when it has been tipped too far towards vasoconstriction/cell growth.

Options for regulating the angiotensin system ACE inhibitors and A II receptor antagonists lower blood pressure by different mechanisms.18 These mechanistic differences may play a significant role in their effectiveness at improving endothelial function and reducing risk of CVD. ACE inhibitors act on the renin-angiotensin-aldosterone system where they block formation of A II and facilitate conversion of bradykinin to NO. This system has been reviewed in previous slides. A II receptor antagonists act to specifically block the A II receptor site, thus preventing responses normally generated by A II. One site where some of these drugs act is the A I receptor subtype that is involved in responses to vasoconstriction and aldosterone stimulation by A II. This blocking action is what makes these agents effective at reducing blood pressure. However, A II receptors do not affect the bradykinin-NO system. As will be shown in the slides to follow, this system is important for maintaining vascular health. Since the A II receptor antagonists are new, there is a lack of clinical data that might translate the differences in mechanisms of action between A II receptor antagonists and ACE inhibitors into clinical characteristics of these agents. Future clinical trials are necessary to distinguish patient subtypes appropriate for A II receptor antagonists. Numerous clinical trials have been performed with ACE inhibitors and some of these will be presented in upcoming slides. A third option for regulation of the angiotensin system is emerging—angiotensin-converting enzyme/neutral endopeptidase (ACE/NEP) inhibitors. NEP is a metalloendopeptidase that degrades a number of vasoactive peptides that enhance the natriuretic, diuretic and blood pressure-lowering effects of natriuretic peptides. Combining ACE and NEP inhibitors should reduce blood pressure in a broader range of conditions than is feasible with either drug class alone. Recent studies are demonstrating that this hypothesis is correct. In a rat model of hypertension and diabetes, an ACE/NEP inhibitor was more effective than either treatment alone.19 An ACE/NEP inhibitor was proved effective in treating African-Americans with hypertension who had been resisitant to ACE inhibitor therapy.20 Future studies may further clarify patient subsets that would benefit from ACE/NEP inhibitor therapy.

Some contrasts between angiotensin receptor blockade and ACE inhibition ACE inhibitors and AT1 receptor anatagonists both reduce hypertension effectively but do so via differing mechanisms.21-23 AT1 receptor anatagonists specifically block the AT1 receptor so that A II does not bind. Most of the cardiovascular effects appear to be mediated by the AT1 receptor. Other receptors, such as the AT2 receptor, exist; however, little is known about how they function in humans. AT1 receptor antagonists reduce A II mediated vasoconstriction. Since they block the AT1 receptor but do not affect AII production, A II plasma levels increase and plasma renin activity increases. Bradykinin is not affected by AT1 receptor antagonists, therefore, prostaglandin E2, prostacyclin or nitric oxide are not altered as these compounds are all influenced by bradykinin. Cough is not observed since this is thought to be caused by elevated bradykinin levels. ACE inhibitors also reduce A II mediated vasoconstriction; however, they do not raise plasma A II levels. Rather, A II levels are reduced because ACE inhibitors block breakdown of A I to A II. Plasma renin activity is increased as is the case with AT1 receptor antagonists. ACE inhibitors, unlike AT1 receptor antagonists, reduce the breakdown of bradykinin into inactive peptides. This action increases bradykinin, prostaglandin E2, prostacyclin and NO levels. ACE inhibitors, therefore, have 2 pathways by which to influence vascular tone, one that affects A II production and one that affects bradykinin levels. Uric acid levels are not affected by ACE inhibition. Elevated bradykinin levels, while reducing vasoconstriction, also appear to cause coughing in some patients. Cough is a class-specific side effect of ACE inhibition.

Drugs shown to improve endothelial function in patients Three classes of drugs have beneficial effects on endothelial function: calcium channel blockers, ACE inhibitors and HMG-CoA reductase inhibitors. Calcium channel blockers, such as verapamil and nifedipine, reverse vasoconstriction caused by endothelin-1. This compound is produced by endothelial cells. Elevated levels have been observed in a number of cardiovascular disorders including acute renal failure, acute myocardial infarction (MI), cardiogenic shock and severe hypertension.24 The effect of many ACE inhibitors on endothelial function has been equivocal; however, quinapril has shown strong and persistent binding to tissue ACE. This effect was demonstrated in the Trial on Reversing Endothelial Dysfunction (TREND) study, which will be discussed in subsequent slides.25 Treatment with HMG-CoA reductase inhibitors such as lovastatin and pravastatin has also been shown to have a favorable effect on vasomotor tone.26-28 Recent studies have demonstrated beneficial effects of troglitazone on the endothlium. One study by Avena et al found that 4 months of troglitazone therapy normalized brachial artery vasoactivity in patients with peripheral vascular disease and diabetes.29 Recent studies indicate that estrogen replacement therapy can improve endothelial function in animal models of CAD30 and in post-menopausal women.31 Several groups of investigators have found that intracoronary infusion of L-arginine, the substrate for NO, improves coronary blood flow in patients with CAD or with CAD risk factors.32 Other studies demonstrate beneficial effects of L-arginine on endothelial function in animal models and in humans with disorders including hypercholesterolemia, hypertension and diabetes.33 These treatments will be explored in greater detail in the sections to follow. Antioxidants, such as vitamin E, C and probucol, appear to reduce cardiovascular morbidity and mortality. Epidemiologic studies support this relationship, although results of clinical studies are mixed. It appears that some of the discrepancies in study results may be due to complex mechanisms of action. Antioxidants seem to do more than inhibit oxidation of LDL-C; they may also stabilize plaques, improve vascular tone and reduce thrombosis. A combination of antioxidants with lipid-lowering agents appears to be particularly effective.34