Nitric Oxide Mechanisms in the Pathogenesis of Global Risk

Abstract

Identification and management of cardiovascular risk factors, such as hypertension, diabetes mellitus, and dyslipidemia, is essential not only for prevention of cardiovascular disease, but also for slowing the progression of existing cardiovascular disease. A major underlying mechanism that links various cardiovascular risk factors and manifestations of cardiovascular disease is endothelial dysfunction, characterized by impaired nitric oxide bioactivity. Oxidative stress is an important cause of impaired nitric oxide bioactivity, and a major pathogenic mechanism of atherosclerosis. Several pharmacologic therapies, including angiotensin‐converting enzyme inhibitors, calcium channel blockers, statins, and the vasodilating β blocker nebivolol, have been shown to enhance nitric oxide bioactivity and improve endothelial function. This effect may help explain the cardioprotective benefits of these agents and may stimulate further use of nitric oxide modulation for the treatment of cardiovascular risk factors and manifestations of cardiovascular disease.

A wide range of laboratory and clinical research data suggest that established cardiovascular (CV) risk factors and comorbidities share common and comprehensive pathogenic mechanisms at the cellular level. ¹ These risk factors, such as aging, hypertension, dyslipidemia, smoking, obesity, diabetes mellitus, and nephropathy, all contribute to endothelial dysfunction, oxidative stress, and inflammation, which ultimately conspire to accelerate the development and progression of CV disease (CVD). ¹

Of particular interest, and the focus of this article, is endothelial dysfunction, which is an early stage of atherogenesis and may represent a major pathogenic link between the various CV risk factors and manifestations of CVD. ¹ , ² One of the first characteristic changes associated with endothelial dysfunction is the shift in endothelial cells from having a smooth and resistant surface to a permeable one. ¹ With this change, the barrier formed by the endothelium becomes increasingly irregular and porous, allowing adhesion cells to bind to it; these adhesion cells then recruit circulating white blood cells to migrate into the vessel wall, where they adhere and participate in early inflammatory changes leading to endothelial dysfunction. ¹ Advancing plaque development causes progressive damage to the endothelium as well as dramatic changes to the morphology of the vessel wall, including the excessive accumulation of cholesterol and oxidized lipids. ¹ These lipids are subjected to oxidative damage, resulting in the recruitment of macrophages and foam cell formation, apoptosis, and the formation of plaque with a necrotic lipid core and a fibrous cap; inflammatory enzymes ultimately degrade the fibrous cap, predisposing to plaque rupture and thrombosis. ¹

MECHANISMS OF ENDOTHELIAL DYSFUNCTION

The initial damage to endothelial cells may be caused by the stresses resulting from CV risk factors such as hypertension, smoking, dyslipidemia, and diabetes mellitus. ¹ Indeed, these risk factors drive the mechanisms of CVD from the earliest stages of endothelial dysfunction to the more advanced stages of unstable plaque. ³ The endothelium is an essential mediator of vascular tone, structure, and function; endothelial dysfunction is a major pathogenic mechanism underlying the development of CVD. ¹ , ² , ³

The endothelium releases and regulates a number of vasoactive substances, such as the vasodilators nitric oxide (NO) and prostacyclin, and the vasoconstrictors endothelin‐1 and angiotensin II. ³ , ⁴ The most important of these vasoactive factors appears to be NO which, in addition to its vasodilatory action, exerts a number of antiatherogenic effects, including the inhibition of platelet aggregation and adhesion, proliferation of vascular smooth muscle cells, and leukocyte adhesion and migration into the arterial wall. ² Prostacyclin has also been associated with inhibition of platelet activation. ² Endothelin‐1 and angiotensin II, by contrast, are associated with vascular smooth muscle cell growth and inflammation. ⁴ , ⁵ By maintaining these vasoactive factors in homeostasis, the endothelium not only mediates vascular tone, but also protects against atherogenic processes, including lipid accumulation and oxidation. ¹ , ³ , ⁵ Endothelial dysfunction, and particularly the impairment of NO bioactivity, represents an imbalance of these vasodilating and vasoconstrictive factors leading to vasoconstriction; increased platelet and leukocyte adhesion; vascular smooth muscle cell migration and proliferation; and increased lipid deposition and accumulation. ³ , ⁴ , ⁵

Multiple animal and clinical studies have demonstrated the association of hypertension with endothelial dysfunction and impaired NO bioactivity. ⁶ One clinical study compared the forearm blood flow (FBF) response to infusion of acetylcholine, which induces endothelium‐dependent vasodilation, in 15 patients with essential hypertension (≥145/95 mm Hg) and 15 normotensive subjects. ⁶ The hypertensive patients (while taking no antihypertensive medications) had significantly lower FBF response to acetylcholine, as measured by plethysmography, than the normotensive subjects, indicating endothelial dysfunction among the hypertensive patients (Figure 1A). The FBF response in hypertensive patients to the highest acetylcholine dose (30 (μg/min) was 7.0 mL/min/100 mL of forearm volume, compared with 16.7 mL/min/199 mL forearm volume in the normotensive subjects (p<0.0001). No significant differences between the study groups were found in the FBF response to infusion of sodium nitroprusside, which induces endothelium/NO‐independent vasodilation. ¹⁵ In another study, FBF response to acetylcholine was found to be significantly reduced (p<0.001) in 13 normotensive offspring of hypertensive parents, compared with 13 normotensive subjects with no family history of hypertension, suggesting that endothelial dysfunction may precede hypertension (Figure 1B). ⁷

Figure 1.

A) The forearm blood flow (FBF) response to infusion of acetylcholine, measured by plethysmography, was significantly lower in IS patients with essential hypertension (≥145/95 mm Hg), compared with that in 15 normotensive patients (p<0.0001). B) In another study, FBF response to acetylcholine was found to be significantly reduced (p<0.001) in 13 normotensive offspring of hypertensive parents, compared with 13 normotensive subjects with no family history of hypertension, suggesting endothelial dysfunction may precede hypertension. *p<0.001. Reproduced with permission from Circulation. 1993;87:1468–1474 ⁶ and J Cardiovasc Pharmacol. 1992;20(suppl 12):S193–S19S. ⁷

NITRIC OXIDE AND OXIDATIVE STRESS

Endothelial dysfunction, as manifested by reduced arterial vasodilation, is characterized by impaired NO bioactivity. ² A major cause of impaired NO bioactivity is an imbalance between NO and oxidative stress factors, which leads to the increased reaction of NO with superoxide (O₂ ⁻) to produce peroxynitrite (ONOO⁻), a highly toxic oxygen‐free radical, in a process referred to as NO synthase (NOS) “uncoupling.” ⁸ , ⁹ Therefore, maintaining this balance between oxidative stress and NO bioactivity is a key therapeutic goal, and management of CV risk factors is essential to achieving this goal.

The direct and accurate measurement of NO bioactivity, as opposed to indirect estimation through such indicators as endothelium‐dependent vasodilation, has only recently become available. Using nanosensor technology and electrochemical approaches, the release of NO molecules, as well as the release of O₂ ⁻ and ONOO⁻, from individual endothelial cells can be measured precisely and in real time, providing a direct and powerful method for assessing endothelial function and the vascular redox state. ⁹ Nanotechnologic techniques have also allowed for a more detailed view of fundamental cellular processes in vascular disease and of the effects of such factors as hypercholesterolemia and oxidative stress in the endothelium. ¹⁰

With cholesterol enrichment, for example, fundamental changes have been observed in the organization of the cell, particularly within the membrane. ¹⁰ These changes include an elaboration of caveolae—invaginations along the membrane surface—which become enriched with lipids. ¹⁰ , ¹¹ The main component of caveolae is the protein caveolin‐1, which binds with endothelial NOS (eNOS), the enzyme that catalyzes NO synthesis by stimulating the conversion of the NO substrate L‐arginine to L‐citrulline and NO. ¹¹ Because caveolin‐1 blocks access of eNOS to its cofactor, calcium/calmodulin, it modulates NO bioactivity in the endothelium. ¹⁰ With hypercholesterolemia and cholesterol enrichment, however, expression of caveolin‐1 is markedly elevated, leading to excessive binding of caveolin‐1 with eNOS, which impairs NOS activity and reduces release of NO. ¹⁰ , ¹¹ In addition, cholesterol enrichment and oxidative stress can also lead to the formation of unique membrane microdomains consisting entirely of unesterified cholesterol, or lipid “rafts.” ¹⁰ These microdomains can then precipitate the formation of extracellular cholesterol crystals, a prominent feature of advanced atherosclerotic lesions, which contribute to cell injury and death and make lesions more resistant to regression through therapeutic intervention. ¹⁰

Experimental in vitro and in vivo studies have illuminated the integral role of oxidative stress throughout the various phases of the atherosclerotic process. ¹² It has been shown, for example, that even minimally oxidized phospholipids can promote recruitment of monocytes and other inflammatory cells to the vessel wall, which then take up oxidized low‐density lipoprotein (LDL) through scavenger receptors to become foam cells. ¹² Apoptotic cell membranes further stimulate endothelial cells to recruit and bind monocytes. ¹² Moreover, oxidized phospholipids promote thrombosis by decreasing thrombomodulin, tissue‐factor‐pathway inhibition, and plasminogen activator inhibitors, and by increasing tissue factor. ¹² Thus, the key role of lipid oxidation in atherogenesis has become increasingly well elucidated.

The implications of this lipid/oxidative stress interaction for treatment of atherogenesis were also clarified by a meta‐analysis of clinical trials evaluating the long‐term effects of α‐tocopherol (vitamin E), beta‐carotene, or both on CV morbidity and mortality. ¹³ Observational trials had previously associated these antioxidant vitamins with reduced CV events, but such benefits had not been confirmed in clinical trials. ¹³ The meta‐analysis reviewed seven randomized, large‐scale trials of vitamin E treatment, 50–800 IU (n=81,788), and eight trials with beta‐carotene, 15–50 mg (n=138,113), with follow‐up ranging from 1.4 to 12 years. The results showed that vitamin E had no significant effect on all‐cause mortality, CV death, or stroke compared with control, while beta‐carotene had a small but significant increase in all‐cause mortality and CV death (p=0.003 for both). This outcome posed the following question: if vitamin E is a potent antioxidant, and oxidative stress plays a vital role in atherogenesis, then why does vitamin E have no effect on CV events? A clue to the answer was found in a recent study in a model of oxidative stress‐induced cholesterol domain formation. ¹⁴ This study showed that vitamin E reduced lipid oxidation in normal tissue, but had no such benefit in atherogenic cells because cholesterol enrichment and the resulting reorganization of the cell membrane appeared to prevent vitamin E from penetrating the cell membranes of the diseased tissue. ¹⁴ Therefore, vitamin E may be useful as a preventive agent, but not for the treatment of existing disease.

EFFECTS OF PHARMACOLOGIC AGENTS ON NITRIC OXIDE SYNTHESIS

The increased evidence of the role of oxidative stress, endothelial dysfunction, and impaired NO bioactivity in atherosclerosis has stimulated interest in pharmacologic approaches to modulate these disease processes. ¹⁵ Therapies of particular interest in this regard include angiotensin‐converting enzyme (ACE) inhibitors, statins, calcium channel blockers (CCBs), and β blockers. ¹⁵

ACE Inhibition

A number of trials have studied the effect of ACE inhibitors on endothelial function and NO bioactivity. ¹⁵ In the Trial on Reversing Endothelial Dysfunction (TREND), treatment with the ACE inhibitor quinapril 40 mg daily for 6 months was shown to significantly improve coronary artery diameter responses to acetylcholine, compared with placebo (p<0.002), among normotensive patients (n=105) with coronary heart disease but without heart failure, cardiomyopathy, or major lipid abnormalities (Figure 2). ¹⁶ A major postulated mechanism for this effect is the action of ACE inhibitors in preserving plasma and tissue levels of bradykinin, since ACE promotes bradykinin breakdown. ¹⁶ , ¹⁷ , ¹⁸ Bradykinin binds to specific receptors on the endothelial cell surface that promote activation of NOS, thus enhancing synthesis and release of NO; by preserving bradykinin in vascular tissue, ACE inhibitors potentiate this process. ¹⁷ , ¹⁸ Moreover, ACE inhibitors may also enhance endothelial function and NO bioactivity through their suppression of angiotensin II, which promotes vasoconstrictive and inflammatory effects that oppose the actions of NO. ¹⁵ , ¹⁶

Figure 2.

In the Trial on Reversing Endothelial Dysfunction (TREND), treatment with the angiotensin‐converting enzyme inhibitor quinapril 40 mg daily for 6 months was shown to significantly improve coronary artery diameter responses to acetylcholine compared with placebo among normotensive patients (n=l OS) with coronary heart disease, but without heart failure, cardiomyopathy, or major lipid abnormalities. *p<0.0003 for quinapril vs. placebo at the 10⁻⁴ mol/L dose of acetylcholine. Reproduced with permission from Circulation. 1996;94:258–265. ¹⁶

Statins

Statins have also been shown to provide beneficial effects on endothelial function, independent of their effects on LDL cholesterol (LDL‐C). ¹⁹ In 49 patients with hypercholesterolemia, for example, treatment with the statin lovastatin plus the antioxidant therapy probucol (n=11) was shown to significantly improve coronary artery vasodilation in response to acetylcholine compared with treatment with the American Heart Association Step 1 diet (p<0.01). ²⁰ While lovastatin treatment alone did not significantly improve endothelial function compared with diet, its vasodilatory effect appeared to be potentiated by concomitant antioxidant therapy with probucol. Hypercholesterolemia is associated with endothelial dysfunction, and LDL‐C apheresis has been shown to augment endothelium‐dependent vasodilation, ¹⁹ , ²⁰ , ²¹ suggesting that statins could improve endothelial function via reduction of LDL‐C. However, further studies in human saphenous vein endothelial cell cultures showed that simvastatin and lovastatin upregulated eNOS expression by 3.8‐ and 3.6‐fold, respectively, and completely inhibited its down‐regulation by oxidized LDL‐C. ²² The statins appeared to achieve this effect by stabilizing the mRNA for NOS, thereby enhancing expression of NOS and NO release in cells through a direct effect on eNOS. ²² Statins that are relatively lipophilic appear to have a greater ability to affect NOS activity at the level of the cell membrane compared with statins that are more hydrophilic. ²³ Moreover, as previously noted, cholesterol enrichment in the endothelial cell membrane is associated with up‐regulation of caveolin‐1 and its excessive binding with eNOS, resulting in impaired NO bioactivity; lipid reduction with statins, therefore, has the potential to reduce this pathogenic mechanism associated with atherosclerosis. ¹¹

Calcium Channel Blockers

The CCB class of antihypertensive agents is heterogeneous and includes second‐ and third‐generation therapies with pleiotropic actions beyond their vasodilating effects through blockade of L‐type calcium channels. ²⁴ Various CCBs have demonstrated significant improvement in endothelium‐dependent vasodilation in animal models and in human subjects but the results of studies in different vascular beds have been mixed. ¹⁵ Some trial evidence suggests that CCBs improve endothelial function and restore NO bioactivity through antioxidant actions. ¹⁵ However, one study found that in six explanted human hearts with end‐stage heart failure, the CCB amlodipine significantly increased the level of nitrite in the coronary microvessels by 79% at the highest dose, compared with controls (p<0.01), and to a similar degree as the ACE inhibitor ramipril (Figure 3). ²⁵ The increased NO level observed with amlodipine was entirely abolished not only by the NOS inhibitor N(omega)‐nitro‐L‐arginine methyl ester, but also by HOE‐140, a bradykinin‐2 antagonist, and by dichloroisocoumarin, a serine protease inhibitor that blocks kallikrein activity. These findings, plus data from other experimental studies, suggest that amlodipine promotes the production and release of NO through activation of an angiotensin receptor followed by the generation of kinins, stimulation of the P₂‐kinin receptor, and activation of eNOS. ²⁴

Figure 3.

Administration of amlodipine (10 [−10] to 10 [−5] mol/L) significantly increased nitrite production in coronary microvessels from six explanted human hearts ivith end‐stage heart failure, obtained immediately at transplantation surgery. The increase in nitrite in response to the highest dose of amlodipine was 79%, which was similar to the 74% increase seen with ramipril. *p<0.01 vs. control. Adapted with permission from Am J Cardiol. 1999;84:27L–33L. ²⁵

Beta Blockers

The β‐blocker class is also highly heterogeneous with regard to pharmacologic properties and clinical effects and includes newer vasodilating agents as compared with the nonvasodilating, traditional β blockers. ²⁶ The vasodilating mechanism of nebivolol is the most well studied from among the newer β blockers. In one study, the effect of nebivolol on FBF response to acetylcholine, measured by venous occlusion plethysmography, was evaluated compared with that of the nonvasodilating, cardioselective β blocker atenolol in 12 hypertensive patients (mean ambulatory blood pressure [BP] of 154/97 mm Hg) (Figure 4). ²⁷ The patients were randomized in a double‐blind, crossover fashion to 8‐week treatment periods with once‐daily doses of nebivolol 5 mg plus bendrofluazide 2.5 mg or atenolol 50 mg plus bendrofluazide 2.5 mg (each treatment period was separated by a 2‐week placebo washout period). Although both treatments had similar effects on BP, the FBF response to acetylcholine was significantly increased with nebivolol/bendrofluazide, compared with atenolol/bendrofluazide (p<0.001 at the highest doses), which, along with placebo, had no effect on acetyl‐choline‐mediated vasodilation (Figure 4).

Figure 4.

The forearm blood flow (FBF) response to acetylcholine was measured by venous occlusion plethysmography in 12 hypertensive patients (mean ambulatory blood pressure of 154/97 mm Hg) randomized in a double‐blind, crossover fashion to 8‐week treatment periods with once‐daily doses of nebivolol 5 mg plus bendrofluazide 2.5 mg or atenolol 50 mg plus bendrofluazide 2.5 mg. Each treatment period was separated by a 2‐week placebo washout period. The endothelium‐dependent vasodilatory response to acetylcholine was significantly increased with nebivolol/bendrofluazide, compared with placebo (p<0.001), while atenolol/bendrofluazide had no effect on acetylcholine‐mediated vasodilation. In addition, the vasoconstrictive effect of the nitric oxide inhibitor N^G‐monomethyl‐L‐arginine (L‐NMMA) was significantly augmented with nebivolol/bendrofluazide, compared with only a small effect with atenolol/bendrofluazide, demonstrating a nitric oxide‐dependent mechanism of action for nebivolol (−26% vs. −54% change in FBF). *p<0.05; ^†p<0.001. Reproduced with permission from Circulation. 2001;104:511–514. ²⁷

The vasodilatory activity of nebivolol was further illustrated in a study comparing the effects of nebivolol and atenolol on systolic and diastolic left ventricular (LV) function in 25 patients with essential hypertension (diastolic BP a90 mm Hg). ²⁸ In this double‐blind, randomized, prospective trial, atenolol 100 mg once daily (n=13) and nebivolol 5 mg once daily (n=12) for 2 weeks achieved similar reductions in BP, but differed significantly in other hemodynamic parameters. Atenolol decreased heart rate to a greater extent than did nebivolol; however, nebivolol increased stroke volume, cardiac output, LV ejection fraction, and LV end‐diastolic volume to a greater extent than atenolol. In addition, nebivolol decreased peripheral vascular resistance and LV end‐systolic volume compared with increases in these parameters with atenolol. These findings suggest that nebivolol may lower BP and improve cardiac function through mechanisms other than, or in addition to, its highly selective β‐blockade.

Recent experimental investigations show that at the cellular level, nebivolol's high lipophilicity enables it to modulate mechanicosensitive receptors in the endothelium, which under physiologic conditions respond to shear stress by releasing NO. ²⁹ Nebivolol mimics the stimulatory action of shear stress on the mechanicosensitive receptors, thereby inducing release of adenosine triphosphate, which binds to purinergic receptors. The purinergic receptors stimulate the activation of calcium which, in turn, is an important cofactor in the activation of NOS. This mechanism of action can be blocked through inhibition of either adenosine triphosphate formation, purinergic receptors, or calcium mobilization, confirming that these components comprise the signal transduction pathway of nebivolol‐induced vasodilation. ²⁹

The direct effect of nebivolol on NO bioactivity was demonstrated in an experimental study using human umbilical vein and iliac artery endothelial cells isolated from age‐matched African‐American and white donors. ⁹ This study showed that at baseline, NO release was about five times slower, while release rates of O₂ ⁻ and ONOO⁻ were about two times and four times faster, respectively, in the cells from African‐American donors compared with those from whites. ⁹ Pretreatment of the cells with nebivolol for 10, 30, or 180 minutes significantly increased NO release in cells from both African‐American and white donors and disproportionately in African‐American compared with white cells, while atenolol had no effect (Figure 5). In addition, treatment of the cells with apocynin, a specific inhibitor of nicotinamide adenine dinucleotide phosphate oxidase, also significantly increased NO release, suggesting the benefit observed with nebivolol stemmed from inhibition of O₂ ⁻ formation that impairs NO bioactivity (Figure 5). Pretreatment of the cells of African‐American donors with nebivolol also brought the levels of NO release in response to calcium ionophore, and of O₂ and ONOO in those cells, to a level similar to that in the cells of white donors in response to calcium ionophore without nebivolol, thus abolishing the racial differences in NO bioactivity, primarily through inhibition of O₂ ⁻ production (Figure 6). These findings suggest that antihypertensive therapy with an NO agonist, such as nebivolol, not only reduces elevated BP but may help to reverse endothelial dysfunction and impaired NO bioactivity, which comprise a central pathogenic pathway linking multiple CV risk factors and manifestations of CVD. Thus, NO‐targeted therapies have the potential to treat not only a discrete risk factor or outcome, but also a basic mechanism underlying the development and progression of CVD.

Figure 5.

In human umbilical vein and iliac artery endothelial cells isolated from 12 healthy, young African‐American women and 12 age‐matched white women donors, release of nitric oxide (NO) at baseline, measured with nanosensors, was slower in the cells from African Americans compared with whites. However, pretreatment with nebivolol for 10, 30, or 180 minutes significantly increased NO release in both the African‐American and white cells, with disproportionately higher relative increases in the African‐American cells; atenolol had no effect on NO release, compared with controls. In addition, treatment of the cells with apocynin, a specific inhibitor of nicotinamide adenine dinucleotide phosphate oxidase, an oxidative stress enzyme, also significantly increased NO release, suggesting that the benefit observed with nebivolol stemmed from inhibition of superoxide formation that impairs NO bioactivity. L‐NAME=N^G‐nitro‐1‐arginine methyl ester. *p<0.001 vs. whites. Adapted from Circulation. 2005;112:3795–3801. ⁹

Figure 6.

In human umbilical vein and iliac artery endothelial cells isolated from 12 healthy, young African‐American women and 12 age‐matched white women donors, nitric oxide (NO) release was lower and release of superoxide (O₂⁻) and peroxynitrite (ONOO⁻) was higher in response to calcium ionophore in the African‐American compared with the white cells at baseline. After 180 minutes of pretreatment of the African‐American cells with nebivolol, the response of NO, O₂⁻, and ONOO⁻ in these cells approximated that of the white cells at baseline, showing that nebivolol could restore a favorable NO/oxidative stress balance in African‐American donor cells. Adapted from Circulation. 2005;112:3795–3801. ⁹

CONCLUSIONS

Endothelial dysfunction, characterized by impaired NO bioactivity, is a major underlying mechanism shared by multiple risk factors and manifestations of CVD. Oxidative stress that may stem from CV risk factors is a leading cause of impaired NO bioactivity and a major pathogenic mechanism in atherosclerosis. The presence of oxidized lipid crystalline domains contributes to the resistance of atherosclerotic arteries to antioxidant therapies, such as vitamin E, and indicates that other therapeutic pathways are necessary to treat atherosclerosis. Several pharmacologic therapies, including ACE inhibitors, CCBs, statins, and vasodilating p block ‐ers, have demonstrated the ability to improve NO bioactivity and endothelial function, independent of their BP or lipid‐lowering actions. In addition, NO‐agonist therapies may address the underlying mechanisms that place African Americans at higher risk for CVD than whites. Nebivolol, whose effects on NO bioactivity and endothelial function are the most well established of the vasodilating β blockers, has shown the ability to abolish racial differences in NO production and oxidative stress in isolated cells from African Americans compared with the cells of whites. Further research is needed to elucidate potential clinical consequences of these beneficial effects on endothelial function.