NO Insufficiency and the Manifestation of Disease
by Nathan S. Bryan, PhD
From the two previous articles on NO, hopefully you have begun to understand and appreciate its function within the body and how its complex production and regulation may be the root of many different diseases. In this third issue in the series, we will discuss specific diseases and how loss of NO function has been shown to be one of the earliest events in the disease process. Hopefully this will provide essential information on how to introduce new treatment regimens for some of your most complex patients, especially older patients where there is a recognized decline in endogenous NO production.
Consequences of NO insufficiency
Aging is considered the single largest risk factor related to cardiovascular-related diseases and deaths. Cardio-protection decreases with increasing age and is attributed to a decline in NO. The lack of NO production can lead to hypertension, atherosclerosis, peripheral artery disease, diabetes, heart failure and thrombosis leading to heart attack and stroke, the leading cause of death for all Americans. Remarkably, if we can recognize these conditions early on, all evidence points to the fact that they can all be prevented. We will now review a few of the most common conditions/symptoms your patients may present and its relationship to NO production.
Hypertension and NO
Hypertension, or high blood pressure, along with aging is a well-known cardiovascular risk factor that leads to functional and structural alterations in the heart and vasculature.1 Vascular changes associated with hypertension, such as endothelial dysfunction, are an accelerated form of the type of changes seen with aging.1 The presence of acetylcholine alongside a NO synthase inhibitor (L-NMMA) was tested for NO availability in the vasculature. A noteworthy finding is that after the age of 60 years old, the inhibiting effect of L-NMMA on response to acetylcholine was extremely weak, suggesting that NO availability is completely compromised in older populations.
These results indicate that essential hypertension is characterized by an age-related reduction of endothelial function by mechanisms that appear to be similar to those observed in older normotensive individuals. NO based therapies can reduce blood pressure. Transdermal nitroglycerin, which acts as a NO donor, has been shown to reduce blood pressure in patients with recent stroke.2 Dietary intervention with nitrite and nitrate has been shown to modestly reduce blood pressure in humans,3-6 showing remarkable efficacy using this natural dietary approach. In fact, we have suggested that the efficacy of the Dietary Approaches to Stop Hypertension (DASH) diet works through its ability to restore NO.7
Atherosclerosis and NO
Atherosclerosis is the major source of morbidity and mortality in the developed world. The magnitude of this problem is profound, as atherosclerosis claims more lives than all types of cancer combined and the economic costs are considerable.8 Reduced NO availability is a hallmark of atherosclerosis. The endothelium-derived NO plays a crucial role in regulating a wide spectrum of functions in the cardiovascular system, including vasorelaxation, inhibition of leukocyte-endothelial adhesion, vascular smooth muscle cell (SMC) migration and proliferation, as well as platelet aggregation.9
The concept of endothelial dysfunction arises from variations in blood flow observed in patients with atherosclerosis compared with healthy subjects. In healthy subjects, activation of eNOS causes vasodilation in both muscular conduit vessels and resistance arterioles. In contrast, in subjects with atherosclerosis, similar stimulation yields attenuated vasodilation in peripheral vessels and causes paradoxical vasoconstriction in coronary arteries, thus indicating a decrease in the production and/or bioavailability of NO.10-11 Interestingly, endothelial dysfunction can be demonstrated in patients with risk factors for atherosclerosis in the absence of atherosclerosis itself.12-13
These observations lend credence to the concept that endothelial dysfunction is integral to the development and progression of disease. Impaired endothelium may abnormally reduce vascular perfusion, produce factors that decrease plaque stability, and augment the thrombotic response to plaque rupture.14 There are a number of studies showing that insufficient NO production from the endothelium is associated with all major cardiovascular risk factors, such as hyperlipidemia, diabetes, hypertension, smoking and severity of atherosclerosis, and importantly also has a profound predictive value for the future atherosclerotic disease progression.15-18 Augmentation of NO or restoration of NOS function seems a logical means to inhibit atherosclerosis. Stokes et al. have demonstrated that supplementing nitrite, as a precursor to NO formation, in the drinking water inhibits the adhesion and emigration of leukocytes to the vascular endothelium, one of the earliest events of atherogenesis,19 suggesting this nitrate-nitrite-NO pathway may be useful in preventing chronic vascular disease.20
Thrombosis and NO
Thrombosis affects nearly 1 million patients in the United States annually. Out of those million, nearly 300,000 are reported as thrombosis related deaths. A majority of current treatment options are centered on preventative anticoagulants, such as warfarin but with risk of bleeding. NO inhibits platelet activation, adhesion and aggregation by influencing several signalling pathways, including activation of soluble guanylyl cyclase (sGC) and increasing intracellular cyclic GMP;21 inhibition of phosphatidylinositol-3 kinase;22 and inhibition of capacitative cation influx and agonist-dependent increases in intracellular calcium.23
These molecular events lead to an impairment of platelet activation, adhesion, secretion, fibrinogen-binding to glycoprotein lib/IIIa,24 and, ultimately, aggregation. In addition NO promotes platelet disaggregation. In conjunction with its vasorelaxing actions, these anti-platelet effects of NO maintain blood fluidity and tissue perfusion. A constitutive NO synthase has been found in both human platelets and megakaryocytic cells25 and this isoform is active.26
Using an NO-selective microelectrode adapted to a platelet aggregometer, Freedman et al. recently showed that this platelet-derived NO not only modestly modulates platelet activation to strong and weak agonists but, more importantly, markedly inhibits platelet recruitment to the growing platelet thrombus.27 NO, derived both from the endothelial cell and the platelet, modulates platelet activation, adhesion, and aggregate formation, thereby serving as an important deterrent to platelet-mediated arterial thrombosis.28 NO insufficiency, either through reduced production or oxidative inactivation, leads to thrombotic events.28 Efforts to restore the normal vascular redox balance and/or to restore normal NO availability may provide one therapeutic avenue for reducing platelet-dependent arterial thrombosis in patients. In fact, recent studies have shown that dietary nitrate in the form of beetroot juice can inhibit platelet aggregation,4 demonstrating this dietary approach to replete NO may provide first line of defense for NO insufficiency.
Alzheimer’s Disease and NO
The most feared disease of the geriatric population is Alzheimer’s disease (AD). In the United States, around 5.4 million people live with AD, a type of dementia.29 Patients with AD lose brain function, resulting in problems with language, perception and memory. AD can start before age 60 (early onset) or after age 60 (late onset). The risk for AD increases as a person ages—and that rising risk is being seen as the baby boomers start turning 65 years old. Out of every eight baby boomers, one will get AD after he or she turns 65 years old; at age 85, that risk grows to one in two. With the 65 and over population in the United States expected to double by 2030, there may be up to 16 million people with AD by 2050; there may be almost 1 million new AD cases diagnosed each year. Each year in the United States, more than 800,000 people die from this neurological disease. It is the sixth leading cause of death, with the number of deaths rising 66 percent from 2000 to 2008.
There is becoming a clear and convincing association with AD and NO. Decreased levels of nitrite and nitrate (NOx) has been detected in patients with different forms of dementia especially AD.30 The exact etiology of sporadic AD is unclear, but it is interesting that cardiovascular risk factors including hypertension, hypercholesterolemia, diabetes mellitus, aging, and sedentary lifestyle are associated with higher incidence of AD.31 The link between cardiovascular risk factors and AD has yet to be identified; however, a common feature is endothelial dysfunction, specifically, decreased bioavailability of NO.32 The pathogenesis of Alzheimer’s disease is closely associated with the accumulation of amyloid-b (Ab) peptides, which eventually form neuronal deposits known as senile plaques on the outside surface of the neurons33 and lead to neuron death. AD is characterized by progressive loss of neurons, cognitive decline, and two defining histopathologies: extracellular amyloid plaques and intracellular tangles composed primarily of Ab peptide and hyperphosphorylated tau, respectively.34
Furthermore, AD is often accompanied by cerebrovascular dysfunction, as well as amyloid deposition within the cerebral vessels, termed cerebral amyloid angiopathy.35 NO in the brain can be produced either by inducible NOS (iNOS) in microglia and astrocytes, or by constitutive NOS in neurons and endothelial cells (nNOS and eNOS). A large body of evidence suggests that the NO produced by neuronal and endothelial constitutive NOS is responsible for neuroprotection during Ab-induced cell death, while NO production in the case of iNOS activation plays a neurotoxic role due to the inflammatory response caused by the over generation of other reactive nitrogen species from NO.36 A decrease in neuronal NOS and an increase in hippocampal iNOS have been demonstrated in aged rats,37 suggesting the dual roles and complexity of NO signaling in the brain and during AD. In mice, higher levels of constitutive NO produced by NOS protects beta-amyloid transgenic mice from developing most typical human symptoms of AD.38 Treatment with NO donors and cGMP analogues suppresses cell death,39 and increasing intracellular cGMP levels prevents inflammatory responses in brain cells.40
It has also been shown that NO modulates expression and processing of amyloid beta precursor protein.41 However, an accumulation of Ab inhibits the NO signaling pathway and therefore may suppress the protective effects of endogenous NO in the brain. In postmortem temporal cortex from a series of AD patients there was reduced NO responsive sGC, providing the first evidence for a loss of NO responsive sGC activity in AD brain.42
Collectively, the literature demonstrates a critical role for NO in the development of AD. It appears that normal and sufficient NO production/availability can modulate and inhibit the expression and formation of Ab but once Ab becomes present it further compromises NO activity. This creates a perpetual system of NO insufficiency and a feed forward mechanism that may accelerate AD progression. The NO pathway may be an important therapeutic target in preventing and treating mild cognitive impairment, as well as AD. In fact, a high nitrate diet has been shown to increase regional cerebral blood flow to the frontal lobe in older patients.43
Diabetes
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 risk of death for those with diabetes is twice that of their non-diabetic age-matched counterparts. Persons with diabetes mellitus tend to suffer unduly from premature and severe coronary atherosclerosis and diabetes is a major independent risk factor for the development of coronary artery disease.44-45 In 2003–2004, 75 percent of adults with self-reported diabetes had blood pressure greater than or equal to 130/80 mmHg, or used prescription medications for hypertension. The cost of treating diabetes is staggering. It is estimated that it cost $116 billion for direct medical costs and $58 billion for indirect costs (disability, work loss, premature mortality).
Insulin mediated NO signaling
Insulin has vasodilator actions that depend on endothelium-derived NO.46 Part of this response and the key cardiovascular protective effects occur via a phosphatidylinositol 3’-kinase-protein kinase B-endothelial nitric oxide synthase (PI3K-Akt-eNOS)-dependent signalling mechanism in addition to its metabolic modulation, which renders insulin a potent organ protector in multiple clinical applications. The PI3-K pathway, which activates serine/threonine protein kinase Akt, enhances endothelial nitric oxide synthase (eNOS) phosphorylation and NO production.47
On a molecular level, metabolic insulin resistance results from impaired PI 3-kinase-dependent signaling in metabolic targets of insulin.48 The striking similarities between metabolic insulin signaling related to glucose uptake and insulin signaling related to vasodilation explain the parallel impairment of GLUT4 translocation in fat and muscles and endothelial NO production in the vessels, respectively, under insulin resistance conditions.49 The PI3/Akt pathway has been shown to promote NO production in healthy vascular endothelial cells and provide support that the vascular endothelium is a physiological target of insulin that couples regulation of glucose metabolism with NO production and hemodynamics.50 However, there is substantial evidence of impairment of this pathway in diabetes, either through disruption of Akt phosphorylation and expression,51 which then disrupts insulin secretion52 and/or eNOS phosphorylation and activation.47
Furthermore, mice lacking endothelial nitric oxide synthase develop insulin resistance, hyperlipidemia and hypertension mimicking the metabolic syndrome.53 There is a clear association between diabetes and disruption of endothelium derived NO through the insulin signaling pathway. Restoring this pathway will likely have positive effects in diabetes mellitus. Numerous clinical studies54-55 have clearly documented severe endothelial dysfunction in humans who suffer from diabetes mellitus. The dysfunctional NO pathway in diabetics is thought to be the cause of the increased incidence of cardiovascular complications. The increase in circulating glucose, insulin and cytokines that occurs in type 2 diabetes have all been independently shown to impair eNOS enzyme activity in experimental studies.56-57
Furthermore, advanced glycosylation products that are generated in the serum of diabetic patients can very readily quench any NO that is formed by the endothelium and this is thought to be a major mechanism responsible for defective endothelium-dependent vasodilation in diabetics.58-60
Another mechanism responsible for endothelial dysfunction in the setting of diabetes mellitus is mediated by vessel wall oxidant production. Generation of reactive oxygen species by smooth muscle cells and by the endothelium has been shown to inhibit eNOS and endothelial function.61 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. Therefore strategies to correct or prevent the progression and development of vascular disease in the diabetic and even non-diabetic population can dramatically improve their prognosis and quality of life and ease the burden on the health care system.
Erectile Dysfunction
For many years and for many patients, the first sign of cardiovascular disease was the manifestation of a heart attack. Symptoms included shortness of breath, heavy chest, pain in the chest or jaw, or radiating pain or numbness in the arm. The heart attack and subsequent symptoms indicated patients had developed overt coronary artery disease. At this stage of the disease, most preventative measures prove ineffective. This concerning reality directed scientific research to find subtle cardiovascular warning signs that could be recognized sooner, enabling corrective measures and even reversal of the disease. Cardiovascular medicine may have found its answer in erectile dysfunction (ED). We now know that sexual dysfunction is actually the first sign of insufficient NO production that sets the stage for the progression of heart disease.
Under normal healthy conditions, when stimulated for sexual activity, NO is produced in the penis that signals the blood vessels in the corpus cavernosum to dilate and accommodate more blood flow. It is engorgement from blood flow that causes and sustains an erection. Without sufficient NO production, there is poor circulation, thus no erection or a poor erection that is unsustainable. The same holds true for women. Without proper blood flow to the clitoris, women cannot have orgasms or enhanced sexual sensitivity. In fact, this is the signaling pathway affected by phosphodiesterase inhibitors (PD5) such as Viagra® and Cialis®. They work to prevent the degradation of the signal that NO turns on. However without NO, Viagra and Cialis will not work and that is the reason it is not effective in 100 percent of the population. Initially, these drugs were being developed for heart disease before it was realized that they worked in such a sexual way. However, this makes sense, since both are a result of endothelial dysfunction.
Conclusion
It appears that the inability to produce sufficient NO under the right preclinical conditions enhances the risk for a number of diseases that plague many Americans today. If true, then there exist an opportunity to intervene early during this process, implement strategies to restore NO homeostasis, and, perhaps, delay or prevent the onset and progression of certain diseases.
NO is essential for maintaining normal blood pressure, preventing adhesion of blood cells to the endothelium, and preventing platelet aggregation; it may, therefore, be argued that this single abnormality, the inability to generate NO, is the earliest step in the onset and progression of a number of diseases that plague us later in life, such as atherosclerosis, myocardial infarction, stroke, peripheral vascular disease and Alzheimer’s.
The first-ever salivary test for Nitric Oxide levels, which uses convenient test strips, is now available as an easy to use non-invasive colorimetric measure of total body NO availability. This can provide physicians and their patients a way to diagnose NO insufficiency and monitor NO levels as strategies are implemented to restore NO production. These strips can also give your patients the power to monitor their progress while giving the physicians a measure of compliance.
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