Section 3: Role of lipids in endothelial function Risk factors and endothelial dysfunction: Mediator role of oxidative stress Content Points: The mechanisms by which CV factors lead to endothelial malfunction are gradually being revealed. A common denominator among these risk factors is oxidative stress.

Mechanisms of oxidative stress Content Points: The L-arginine/nitric oxide synthase (NOS) system is affected by a number of pathophysiological conditions: for example, hypertension, hypercholesterolemia, aging, smoking and heart failure.17 L-arginine is the substrate for NOS, thus linking it to NO, a powerful vasodilator. However, L-arginine appears to have some independent beneficial effects on vascular tone that are not presently fully understood. NOS is altered by several conditions including high levels of oxidized LDL-C (Ox LDL-C). NO bioactivity is reduced under conditions of oxidative stress. Infusion of ascorbic acid, an antioxidant, has been shown to improve vascular responses in people with hypertension, smokers and diabetic individuals. These findings clearly indicate that the L-arginine/NOS system is damaged by oxidative stress and this damage probably arises from insults at numerous points in the system. Tetrahydrobiopterin is a vital cofactor in the L-arginine/NOS system. It seems to affect the ability of the enzymes to bind L-arginine. Recent physiological studies indicate that levels of tetrahydrobiopterin may be abnormal in diabetes and hypercholesterolemia. Superoxide is a free radical generated under conditions of oxidative stress that impairs the function of a variety of substances including NO. Research conducted within the past 5 years has uncovered the existence of membrane-bound oxidases within the endothelium and the vascular smooth muscle that utilize NADH and NADPH as substrates for electron transfer to molecular oxygen. This vascular oxidase seems to modulate NO activity. Angiotensin II (A II), in addition to several cytokines, regulates the activity of vascular oxidase. P22phox is a small protein subunit of the membrane oxidase cytochrome. Studies indicate that p22phox has an important role in control of vascular superoxide production and its modulation by angiotensin and hypertension.

Oxidized LDL-C disturbs the endothelium over long-term Content Points: A number of risk factors can injure the endothelium, rendering it dysfunctional, which may or may not involve denudation.18 These risk factors include elevated LDL-C, free radicals resulting from cigarette smoking, hypertension, diabetes, infectious micro-organisms, elevated homocysteine and genetic alterations. In particular, however, elevated LDL-C levels are a major predisposing factor for development of atherosclerosis. Injury increases the adhesiveness of the endothelium to leukocytes and platelets, and induces the endothelium to form vasoactive molecules, cytokines, and growth factors. It also increases endothelial permeability, allowing entry of LDL-C and inflammatory cells. In individuals with elevated LDL-C levels, diabetes or hypertension, oxidative stress seems to occur within the blood vessels and contributes to the development of atherosclerosis. In a state of oxidative stress, there is increased production of oxygen free radicals. One compound that is oxidized is LDL-C. Chronic exposure to Ox LDL-C is now thought to play an important role in the development of atherosclerosis as well as in initiation of cardiac events.

Some consequences of oxidation of LDL-C Content Points: Ox LDL-C is not recognized by the classic LDL-C receptor. Instead it is taken up by macrophage scavenger cell receptors.18 As the macrophages take up Ox LDL-C they fill up with lipids and become foam cells which can migrate into the endothelium and form the lipid core of plaques. Ox LDL-C is toxic to endothelial cells, damaging them and leading to increase expression of endothelial-derived adhesion molecules. This in turn causes the endothelium to become “sticky.” The damaged endothelial cells also attract and facilitate migration of monocytes (cells that can transform into macrophages) into the vascular intima. Ox LDL-C may also contribute to a thrombogenic state and platelet aggregation.19

Factors in endothelial dysfunction—early stage plaque Content Points: As discussed, endothelial dysfunction can result from a variety of mechanisms. Over time, alterations in the dsyfunctional endothelium lead to the development of atherosclerotic plaques.18 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 NO, 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. Ox 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.18 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.

Oxidized LDL-C: A common initiating factor for cardiac events Content Points: High levels of Ox LDL-C occur in states of oxidative stress such as dyslipidemia, hypertension and diabetes.20 Ox LDL-C instigates a chronic inflammatory reaction in atherosclerotic plaques.20,21 Mature plaques have a large, lipid-filled core. They develop a protective fibrous cap.22 Influx of activated macrophages and T lymphocytes secrete cytokines that degrade and weaken the connective-tissue framework of the plaque. Mechanical stresses may also weaken the fibrous cap, leading to a fragile plaque. Endothelial dysfunction is common in conditions that lead to formation of Ox LDL-C and may contribute to plaque formation. Clinical sequelae can result from lipid rich, vulnerable plaques, including transient ischemia, unstable ischemic syndromes, MI and death.

Effect of high-fat meal on endothelial activity in normocholesterolemic patients Content Points: To investigate the effect of fat consumption on endothelial function, normocholesterolemic volunteers were studied before and after consumption of a single high- or low-fat meal.23 Within 2 hours after consuming the high-fat meal, serum triglycerides increased significantly (P = 0.05). After the low-fat meal, there was no change in serum triglycerides. As shown in the graph, this increase in triglycerides translated into impaired endothelial function. Flow dependent vasoactivity decreased continuously over a 4-hour time period following consumption of the high-fat meal. At 4 hours, flow dependent vasoactivity had decreased by nearly 50% (P < 0.05). Flow dependent vasoactivity was partially restored 6 hours after the meal. No change in flow dependent vasoactivity was observed following the low-fat meal. These results indicate that excessive dietary consumption of saturated fat correlates with endothelial dysfunction.

Endothelial function in patients with and without dyslipidemia Content Points: The normal response of an artery to methacholine is a dose-dependent dilation.24 In patients with dyslipidemia this response is diminished. At higher doses, a significant difference (P < 0.05) is seen in the response observed in dyslipidemic individuals as compared with normal subjects. This study demonstrates the endothelial dysfunction that occurs in people with dyslipidemia.

Improvement within hours in endothelial function following ischemia with LDL-C apheresis Content Points: Another investigation has demonstrated that endothelial dilatation is impaired by elevated LDL-C levels.25 In this study, forearm blood flow was measured in response to a dose of acetylcholine in 7 hypercholesterolemic patients before and after LDL-C apheresis. This technique, LDL-C apheresis, is commonly used to lower LDL-C levels in individuals with familial hypercholesterolemia. As shown in the slide, vasodilatation was significantly improved (P < 0.01) following a single session of LDL-C apheresis. Investigators also found that reduction of LDL-C by this technique improved NO levels (as measured by NOX production). This study reinforces the importance of elevated lipid levels in causing endothelial dysfunction. Further, aggressive reduction in LDL-C reverses this dysfunction.

Vitamins C & E attenuate reduction in endothelial function caused by high-fat meal Content Points: In this study by Plotnick et al, high-fat and low-fat meals similar to those in a prior study by Vogel et al23 were given to 20 healthy volunteers. One week after the initial baseline meal, subjects were given 1 of the meals followed immediately by antioxidant vitamin C (1000 mg) and vitamin E (800 IU).26 Endothelial function was measured by determination of flow-mediated brachial artery dilation 6 hours after eating. Individuals who ate the high-fat meal exhibited impaired vasodilatation. Impairment was significantly different from baseline at 2, 3 and 4 hours after eating (P < 0.001). During this time period mean serum triglyceride levels increased significantly (P < 0.005). When consumption of the high-fat meal was followed by antioxidant vitamin administration, no significant change in vasodilatation was observed although triglyceride levels rose significantly (P < 0.005). No significant change in either vasodilatation or postprandial triglyceride levels was observed in subjects consuming the low-fat meal. A single high-fat meal reduces endothelial function, probably via elevated levels of triglycerides. Antioxidant vitamins can block endothelial dysfunction, therefore, an oxidative mechanism is likely involved.