Cardiovascular disease (CVD) remains the number one killer of men and women worldwide. In 2015, there were an estimated 422.7 million cases of CVD and nearly 18 million of these people died of CVDs, with 85% being due to myocardial infarction and stroke. This represented 31% of all global deaths.¹ This is up 30% since 1990 when there were 12.59 million deaths due to CVD. The people who survive or live with CVD cost the United States alone over $200 billion annually. This total includes the cost of health care services, medications, and lost productivity. CVD will cost the United States over $1 trillion in medical expenses (direct costs) and lost productivity (indirect costs) by 2035 (unpublished report from American Heart Association [AHA]). This is simply unacceptable given that the scientific community knows without a doubt what causes CVD, the loss of production of nitric oxide (NO). CVD is mainly caused by endothelial dysfunction,² and one of the main functions of the endothelium is to produce NO.³ The functional loss of NO production by the vascular endothelium precedes the structural changes of arteriosclerosis and atherosclerosis by many years, sometimes decades.⁴ Therefore, clinicians need to recognize and appreciate endothelial NO function in their patients and restore and maintain NO production. The onset and progression of CVD cannot and will not be managed, treated, or prevented without first correcting NO production.

This chapter is designed and written to inform clinicians and health care providers on the mechanisms of vascular disease, risk factors and mechanisms of risks, effective methods of diagnosing endothelial dysfunction or NO deficiency, the earliest events in CVD, and evidence-based strategies for restoring vascular function to cure, treat, or prevent CVD. It is abundantly clear that treatment of CVD over the past 50 years has been unsuccessful by focusing on treating disease states rather than on prevention. Therefore, a new paradigm is prudent. Focusing on restoration of NO in healthy patients without CVD, patients at risk for CVD, or even patients with apparent CVD should be the first-line consideration by clinicians. This chapter will provide the clinician with all the tools to diagnose and recognize NO insufficiency, early vascular dysfunction, as well as safe and effective treatments and preventative measures for CVD.

The vascular endothelium is the organ system that maintains the integrity of the cardiovascular system. The endothelium is the single layer of cells lining various organs of the body, especially the blood vessels, heart, and lymphatic vessels. The endothelium is the largest endocrine organ and makes up over 14,000 square feet of surface area within the human body, enough cells to cover 6.5 tennis courts. The endothelium is responsible for a number of fundamental physiological functions. The endothelium serves a critical role as a barrier and primary sensor of physiological and chemical changes in the blood stream. Endothelial cells are highly specialized to detect diverse physical, chemical, and mechanical stimuli (blood pressure, pulsatile flow, shear stress). They are also involved in the regulation of blood flow through continuous modulation of vascular tone, primarily through the production of NO. When the endothelium is intact and functional, the cardiovascular system maintains its integrity and is protected from CVD. When the endothelium becomes dysfunctional, the cardiovascular system fails and CVD ensues. The manifestation of CVD, primarily atherosclerosis, is a reaction to injury and inflammation within the arterial wall only after the endothelium has lost its ability to maintain structure and function. The
integrity of the endothelium affects the structure and function of the other parts of the blood vessel including the intima and smooth muscle (see Figure 2.1). There are three finite responses by the vascular endothelium to the infinite number of insults it encounters all of which decrease NO production. These three finite responses are inflammation, oxidative stress, and immune dysfunction. One or all three of these are found and associated with all forms of CVD. However, they are more than associations and there is sufficient evidence that these are causal for CVD. Oxidative stress reduces vascular NO production.⁵ Inflammation, specifically microvascular inflammation, is due to insufficient NO production and the consequential sequelae of leukocyte adhesion, emigration, and immune dysfunction.⁶,⁷ The immune dysfunction that also occurs leads to a further dysregulation and production of NO. The inflammation that ensues also leads to further reduction in NO production creating a feed-forward mechanism exacerbating the vascular disease phenotype.⁸ In most cases, loss of NO production occurs as a result of oxidative stress and oxidation of tetrahydrobiopterin (BH4), which causes the endothelial nitric oxide synthase (eNOS) enzyme uncoupling.⁹

Loss of functional endothelial NO production, termed endothelial dysfunction, precedes the structural changes in the vasculature by many years, sometimes decades, and correlates with cardiovascular risks.² Aging and hypertension are established and validated cardiovascular risk factors.¹⁰,¹¹ The functional and structural vascular changes that lead to the complication of CVD are similar in older healthy subjects and younger adults who have had hypertension most of their life.¹² There are finite symptoms and conditions that result from vascular aging. These are shown in Table 2.1. The pathways of aging and vascular aging are identical, which allows the clinician to provide drugs, supplements, and lifestyle
changes and nutrition to improve or slow down the aging process. Furthermore, the vascular changes seen in essential hypertension appear to be an accelerated progressive form of vascular structure and function seen with normal aging.¹³ Young healthy individuals have normal and sufficient endothelial production of NO through L-arginine. However, as we age, we lose our ability to synthesize NO from L-arginine; this is termed endothelial dysfunction. Most of the work on the production of NO in cells, tissues, and humans agree that the bioavailability or the generation of NOS-derived NO decreases with aging. Increased oxidative stress through production of superoxide can scavenge NO thereby reducing its effective concentrations and signaling actions in cells.¹⁴ Aging also causes a decrease in the expression of the NOS enzyme.¹⁵,¹⁶ Concomitantly, there is an upregulation of arginase (an enzyme that degrades the natural substrate for NOS, L-arginine) in the blood vessels with age that also causes a reduction in NO production¹⁷ owing to a shuttling of l-arginine away from the NOS enzyme. Aging causes a gradual decline in NO production with a greater than 50% loss in endothelial function in some aged populations.¹⁸ Some studies show a more than 75% loss of NO in the coronary circulation in patients in their 70s and 80s compared with young, healthy 20-year-old persons.¹⁹ In fact, age may be the most significant predictor of endothelium-derived NO production²⁰ and loss of NO may be responsible for age-related disease, including CVD. These data clearly demonstrate that NO production from L-arginine declines as we get older. This is due to uncoupling of the NOS enzyme, which is then unable to convert L-arginine into NO. This process can be accelerated or decelerated depending on diet and lifestyle. The majority of studies reveal that loss of NO production was clearly evident by 40 years of age. However, the vasodilation to exogenous NO (endothelium-independent vasodilation) does not change over time with aging, illustrating that the body does not lose its ability to respond to NO, it only loses its ability to generate it with age. Vascular aging is characterized by progressive arterial stiffness, loss of arterial elasticity and arterial compliance, increase in pulse wave velocity and pulse pressure, and mechanosensitive gene expression from a myriad of structural and functional changes in the endothelium and vascular media and adventitia with altered gene expression. These include increased extracellular matrix, endothelial dysfunction loss of NO, increase cytokines and chemokines, altered vascular smooth muscle, altered adventitia, inflammation, loss of elasticity, and increased elastase and collagen along with calcium deposition.²¹

The control of blood pressure and the integrity of the cardiovascular system are critically dependent on NO, but there are also other vasoactive substances and mediators that affect the onset and progression of CVD. Another major player is angiotensin II. NO is a vasodilator, whereas angiotensin II is a vasoconstrictor. Angiotensin II is a hormone that binds to the angiotensin I receptor, which then mediates effects on the central nervous system to regulate renal sympathetic nerve activity, renal function, and, therefore, blood pressure. Angiotensin also stimulates the release of aldosterone from the adrenal cortex to promote sodium retention by the kidneys causing an increase in volume and subsequent increase in blood pressure. Maintaining the balance of NO with angiotensin II is vital to protecting the cardiovascular system. The loss of NO tips the balance of a healthy endothelium and cardiovascular system to one that becomes pro-oxidant, proinflammatory, proliferative, and constrictive, which becomes driven primarily by angiotensin II (Figure 2.2). These are hallmark conditions of CVD. These observations allow scientists and physicians to conclude that reduced production of NO occurs as we age and this creates the environment that is conducive to the onset and progression of CVD. The functional loss of NO leading to the structural changes in advanced vascular disease is illustrated in Figure 2.3.

There are a number of known risk factors for CVD. Risk factors are conditions or habits that make a person more likely to develop CVD and/or increase the chances that the existing disease will get worse. Known and important risk factors for CVD are high blood pressure (hypertension), hyperlipidemia (increased levels of certain lipoproteins), diabetes and prediabetes, smoking, being overweight or obese, physical inactivity, family history of early heart disease, unhealthy diet, periodontal disease, and age (55 years or older for women). There are others, but these are the primary risk factors. All of these risk factors are modifiable with the exception of family history and age. However, mechanistically the risks of increased age can be mitigated by NO, so realistically, family history is truly the only nonmodifiable risk factor. All of the known risk factors have a common denominator that is responsible for increased risk of CVD. That commonality is loss of NO production. It is known that if you can eliminate these known risk factors, your chances of developing CVD are much less and in fact perhaps even eliminated. We will discuss the pathophysiology of each risk factor and how to restore to normal physiology.

Hypertension, the number one modifiable risk factor for the development of CVD is often times not sufficiently
managed. In the United States, about 78 million (one of every three) people have high blood pressure or hypertension. Another one of three have prehypertension (CDC fact sheet). That puts over 150 million Americans at risk for heart disease. Despite major advances in understanding the pathophysiology of hypertension and availability of antihypertensive drugs, suboptimal blood pressure control is still the most important risk factor for cardiovascular mortality. According to the AHA 2015 Statistics Fact Sheet, of 75% of people who know they have hypertension and take medication for their high blood pressure, only about half are adequately managed. Because blood pressure remains elevated in approximately half of all treated hypertensive patients,²²,²³ new safe and cost-effective solutions are desperately needed. We now know from the Systolic Blood Pressure Intervention trial (SPRINT) that better management of blood pressure reduces all-cause mortality.²⁴ Additional drug therapy is not the solution. Loss of NO production and signaling is the cause of hypertension.²⁵,²⁶ Because hypertension is primarily responsible for structural changes in the vasculature that
increase the risk of myocardial infarction and stroke, a primary focus should be on normalizing blood pressure through the restoration of NO production.²⁷

Hyperlipidemia or an elevation in specific lipids is a known risk factor for CVD. Lipids refer to a number of fat particles or proteins, including cholesterol, lipoproteins, and triglycerides. Although routine lipid screening plays an important role in cardiovascular risk assessment, it does not provide a complete picture of your health. In fact, nearly 50% of all myocardial infarctions and strokes occur in patients with “normal” cholesterol levels.²⁸,²⁹,³⁰ This suggests that many people at risk are presumed low risk because they have normal or controlled cholesterol levels. Therefore, routine cholesterol tests may fail to fully identify people at risk for myocardial infarction and stroke. However, oxidized low-density lipoprotein (LDL) (OxLDL) is a more sensitive biomarker than total LDL. OxLDL detects the amount of protein damage due to the oxidative modification of the ApoB subunit on LDL cholesterol. This is part of the three finite responses. The oxidation of LDL cholesterol (LDL-C) inhibits NO production and is one of the first steps in the development of atherosclerosis.³¹ Briefly, LDL-C enters the artery wall where it becomes oxidized. OxLDL is then recognized by scavenger receptors on macrophages, which engulf OxLDL, resulting in foam cell formation, vascular inflammation, and the initiation of atherosclerosis. Providing an exogenous source of NO can reduce OxLDL, oxidative stress, and atherogenesis.³²,³³ Therefore, loss of the protective NO leads to oxidative stress, increased OxLDL, and increased progression of atherosclerosis, and providing an exogenous source of NO appears to inhibit oxidative stress, reduce OxLDL, and inhibit the progression of CVD.

Diabetes has become a major epidemic around the globe. Clinical diabetes mellitus is a syndrome of disordered metabolism with inappropriate hyperglycemia due either to an absolute deficiency of insulin secretion or a reduction in the biologic effectiveness of insulin or both. The prevalence of diagnosed and undiagnosed diabetes in the United States is 25.8 million, or 8.3% of the population, according to the 2011 National Diabetes Fact Sheet. In 2005 to 2008, 67% of adults aged 20 years or older with self-reported diabetes had blood pressure greater than or equal to 140/90 mm Hg or used prescription medications for hypertension. Type 2 diabetes mellitus accounts for 80% to 90% of diabetes cases in the United States and is associated with an increased risk for a number of life-threatening complications. These include heart disease and stroke, high blood pressure, blindness, kidney disease, nervous system disease, amputation, and complications of pregnancy and surgery. Probably not coincidental, all of the above-mentioned complications are associated with insufficient NO production.³⁴ Endothelial dysfunction with reduced NO generation and bioavailability plays a key role in the pathogenesis of diabetic vascular disease and complications and likely serves as the key link between metabolic disorders and CVD.³⁵ A number of previous experimental studies have demonstrated impaired endothelial function in animal models of diabetes mellitus.³⁶,³⁷,³⁸,³⁹ In addition, numerous clinical studies⁴⁰,⁴¹ have clearly documented severe endothelial dysfunction in people with diabetes mellitus. Polymorphisms in the eNOS gene have predictive value for the development of diabetic complications.⁴² The dysfunctional NO pathway in people with diabetes is thought to be the cause of the increased incidence of cardiovascular complications.⁴³ The potential mechanisms that may account for attenuated eNOS function and a reduction in endothelial NO synthesis in diabetics are numerous. The increases in circulating glucose, insulin, and cytokines that occur in type 2 diabetes have all been independently shown to impair eNOS enzyme activity in experimental studies.³⁸,³⁹ All of these conditions acting independently or in unison could render the eNOS enzyme dysfunctional (Figure 2.4). Mice without the genes that make NO become insulin resistant and diabetic.⁴⁴ This suggests that loss of NO may be causal for insulin-resistant diabetes. The physiological significance of impaired eNOS function and reductions in vascular NO bioavailability may serve to reduce blood flow to various organs in patients with diabetes mellitus as well as disrupt insulin-dependent glucose uptake. Restoring the functionality of NO-based signaling can improve glucose uptake and correct vascular dysfunction in diabetes.⁴⁵,⁴⁶

Smoking represents one of the most important preventable risk factors for the development of atherosclerosis. Vascular dysfunction induced by smoking is initiated by reduced NO production and/or bioavailability and further by the increased expression of adhesion molecules and subsequent endothelial dysfunction. For the past decades, it has been clear that smoking is an important (and modifiable) risk factor for CVDs; according to World Health Organization data, smoking is responsible for 10% of all CVD cases. Smoking reduces flow mediated dilatation (FMD) in systemic arteries in healthy young adults.⁴⁷ Several other experimental
studies suggested a link between proatherogenic cellular and molecular effects of cigarette smoke and initiation of CVD.⁴⁸ Smoking-induced increased adherence of platelets and macrophages provokes the development of a procoagulant and inflammatory environment.⁴⁹ In addition to direct physical damage to endothelial cells, smoking induces tissue remodeling and prothrombotic processes together with activation of systemic inflammatory signals, all of which contribute to atherogenic vessel wall changes. Restoring the function of eNOS enzyme through treatment of chronic smokers with BH4 (cofactor of endothelial NO-synthase) and/or vitamin C improved smoking-impaired vasodilatation.⁵⁰,⁵¹ Smoking-induced impairment of FMD can be improved by simultaneous consumption of red wine, probably because of its antioxidant properties,⁵² which will thereby improve NO production. Smoking cessation is the most effective measure for reversing damage that has already occurred and preventing fatal cardiovascular outcomes. At the very least, restoring NO production in smokers may mitigate many of the adverse effects of smoking.

Obesity (defined as a body mass index [BMI] of ≥30 kg/m²) is a risk factor for CVD. It is estimated that the relative risk of coronary heart disease in obesity is approximately 1.5 even after adjusting for all other traditional coronary heart disease risk factors that often comigrate with obesity (eg, hyperlipidemia, hypertension).⁵³ Increasing BMI actually predicts impaired endothelial NO function.⁵⁴ It is even more pronounced in patients with visceral obesity and insulin resistance.⁵⁵ The impact of and association between excess weight and endothelial dysfunction can present as early as childhood.⁵⁶ Obesity is thought to affect endothelial function predominately via comorbidities, such as insulin resistance and dyslipidemia. Obese individuals are resistant to the vasodilator actions of insulin.⁵⁷ Furthermore, hyperglycemia and hyperlipidemia, which are almost always present in obesity, are known to decrease NO-dependent vasodilation.⁵⁸,⁵⁹ Adipose tissue itself plays an important role in the release of vasoactive substances.⁶⁰ Adiponectin accumulates in the vessel wall where it has anti-inflammatory effects and increases NO production.⁶¹ Obesity is associated with relative adiponectin deficiency, which causes a loss of endogenous activator of NO production contributing to impaired endothelial function. Other cytokines such as interleukin 6 (IL-6) and fatty acids are thought to decrease NO production and so contribute to impaired endothelial function in obesity. Lastly, adipose-derived components of the renin-angiotensin system also contribute directly to fluid retention, vasoconstriction, and hypertension.⁶² Given that excess body weight now affects more than 300 million persons worldwide, prevention and treatment of obesity should be considered one of the cornerstones for the prevention of CVD or at least the physiological consequences of obesity such as loss of NO production should be corrected.