Organ Systems Dependent on Nitric Oxide and the Potential for Nitric Oxide‐Targeted Therapies in Related Diseases

Abstract

Nitric oxide (NO) is a universal messenger molecule that plays diverse and essential physiologic roles in multiple organ systems, including the vasculature, bone, muscle, heart, kidney, liver, and central nervous system. NO is produced by 3 known isoforms—endothelial, neuronal, and inducible NO synthase—each of which perform distinct functions. Impairment of NO bioactivity may be an important factor in the pathogenesis of a wide range of conditions, including preeclampsia, osteoporosis, nephropathy, liver disease, and neurodegenerative diseases. Although increased levels of NO synthase or NO bioactivity have been associated with some of these disease states, research increasingly suggests that preservation or promotion of normal NO bioactivity may be beneficial in reducing the risks and perhaps reversing the underlying pathophysiology. Based on this rationale, studies investigating the use of NO‐donating or NO‐promoting agents in some of these diseases have produced positive results, at least to some degree, in either animal or human studies. Further investigation of NO‐targeted therapies in these diverse diseases is clearly mandated.

Nitric oxide (NO) is a messenger molecule that performs diverse biologic functions. ¹ This simple molecule appears to occur in virtually every tissue, and impairment of NO bioactivity has been documented in a wide array of diseases. ¹ , ² There are 3 known isoforms of NO synthase (NOS), the enzyme necessary for the production of NO from its substrate, L‐arginine. ¹ , ² These isoforms include endothelial NOS (eNOS), found primarily in the endothelium of blood vessels and airway epithelium; neuronal NOS (nNOS), concentrated in nerve endings and in tissues of the central and peripheral nervous systems, as well as skeletal muscle; and inducible NOS (iNOS), expressed in a variety of tissues. ² The eNOS and nNOS isoforms are primarily constitutively expressed and dependent on increased intracellular calcium and calmodulin binding to generate NO, while iNOS is calciumindependent and primarily induced by the presence of inflammatory factors, including cytokines. ³ , ⁴ All 3 isoforms can be induced, although by varying stimuli, and all 3 can be constitutively expressed in some cells or tissues. ⁴

Because NO bioactivity plays important physiologic roles in diverse organs including the blood vessels, brain, lungs, liver, kidney, stomach, and the immune, musculoskeletal, and reproductive systems, impairment of NO bioactivity could be an important factor in a wide range of disease states. ¹ , ² The complexity of NO bioactivity, however, makes it difficult to identify its precise functions in many areas, and to target and modulate these actions or pathways therapeutically. ¹ Under conditions of oxidative stress, inflammation, and ischemia, NO may be produced at excessive levels and become dysfunctional or “uncoupled,” reacting with superoxides (O₂ ⁻) to produce peroxynitrite (ONOO⁻). ³ , ⁵ Despite this complexity, understanding of the roles of NO in a range of organ systems is rapidly increasing through extensive research. ⁴ This article explores the evidence suggesting a contribution from NO in a selected series of organ systems and the potential for therapeutically targeting NO in the treatment of diseases affecting these organ systems.

PREECLAMPSIA

A leading cause of maternal and fetal morbidity and mortality, preeclampsia complicates 5%–10% of pregnancies in the United States. ⁶ , ⁷ , ⁸ Preeclampsia is defined as the new onset of hypertension and proteinuria during pregnancy, usually occurring after the 20th week of gestation and ending with the delivery of baby and placenta. ⁹ Other clinical characteristics include edema, markedly elevated systemic vascular resistance, intravascular coagulation, reduced plasma volume, decreased perfusion of major organs, and endothelial dysfunction. ⁶ , ⁹ When life‐threatening convulsions follow, this condition is called eclampsia. ⁹ Major risks of preeclampsia include abruptio placentae, thrombocytopenia, pulmonary edema, aspiration pneumonia, acute renal failure, cardiovascular (CV) complications, and death. ⁸ , ¹⁰ An epidemiologic analysis of US birth data found the mortality rate for preeclampsia/eclampsia was 6.4/10,000 cases at delivery. ¹⁰

Major preexisting risk factors for preeclampsia include a personal or family history of preeclampsia, nulliparity, chronic hypertension, antiphospholipid syndrome, thrombophilias, nephropathy, age older than 35 years, diabetes, high body mass index, and other manifestations of the metabolic syndrome such as dyslipidemia and insulin resistance. ¹¹ , ¹² Significantly, women with CV risks are at increased risk for preeclampsia, and those with a history of preeclampsia are at increased risk for postpregnancy CV morbidity and mortality, compared with women with a history of normal pregnancy, suggesting that preeclampsia and CV disease share common pathogenic mechanisms. ⁹ , ¹² , ¹³ , ¹⁴ Because endothelial dysfunction and impaired NO bioactivity are now recognized as key mechanisms of CV disease, the role of these factors in the pathophysiology of preeclampsia is being investigated. ¹⁵

Pathophysiology of Preeclampsia

The pathophysiology of preeclampsia is thought to represent a defective response to the physiologic demands of normal pregnancy. ⁶ During normal pregnancy, maternal cardiac output and blood volume increase by 40%–50%, renal plasma flow and glomerular filtration rate increase by 30%–40%, and peripheral vascular resistance decreases to accommodate these higher circulatory volumes. ⁶ Endotheliumdependent vasodilation and NO bioactivity are increased in normal pregnancy, consistent with the decrease in systemic vascular resistance. ⁶ , ¹⁶

A key function of these changes during normal pregnancy is to perfuse the placenta, and reduced perfusion of the placenta has been postulated as a major initial pathogenic mechanism of preeclampsia. ⁶ , ⁹ It is hypothesized that placental ischemia stimulates placental release of agents that interact with preexisting metabolic and CV risk factors, resulting in injury to endothelial cells and causing systemic endothelial dysfunction and impaired NO bioactivity. ⁹ , ¹⁵ This mechanism may contribute to preeclampsia, characterized by systemic vasoconstriction and increased intravascular coagulation and leading to reduced perfusion of other organs, including the heart, liver, kidney, and brain, with possible hemorrhage and necrosis. ⁶ , ⁹

Role of Impaired NO in Preeclampsia

Impaired NO bioactivity has been postulated as an important pathogenic factor in preeclampsia. ¹⁵ , ¹⁷ Endothelium‐dependent arterial vasodilation has been shown to be reduced and vascular impedance to be increased in preeclampsia compared with normal pregnancy. ¹⁸ , ¹⁹ , ²⁰ Clinical findings also suggest that reduced endothelial‐dependent vasodilation precedes preeclampsia. ²¹ Furthermore, women who develop preeclampsia have increased levels of platelet activation and increased plasma levels of adhesion molecules; these factors are inhibited by NO, suggesting an impairment of NO bioactivity. ¹⁵ Postpregnancy, women with a history of preeclampsia (3 months postpartum or later) have significantly reduced endothelium‐dependent vasodilation compared with women with a history of normal pregnancy. ⁷

The precise mechanism of endothelial dysfunction in preeclampsia is unclear, however, and may vary depending on the vascular bed studied. ²² While endothelium‐dependent vasodilation is reduced in the arteries, it is increased in the microcirculation of women with preeclampsia, compared with normal pregnant women, ²² and postpartum in women with a history of preeclampsia, compared with those with past normal pregnancy. ²³ The increased endothelium‐dependent vasodilation in the microcirculation of preeclamptic women may reflect increased sensitivity to NO in that vascular bed as a compensatory mechanism to offset impaired placental perfusion. ²²

Oxidative Stress Factors. Multiple studies have found that ONOO⁻, lipid peroxides, and thromboxane are increased in the serum and tissue samples of women with preeclampsia compared with women with normal pregnancy. ²⁴ , ²⁵ , ²⁶ Thromboxane and lipid peroxides are also toxic to the vasculature and impair endothelial function. ²⁵ Plasma levels of superoxide dismutase, which scavenges O₂ ⁻, are increased in normal pregnant women, perhaps as part of an inflammatory response, compared with nonpregnant women. ²⁴ Superoxide dismutase levels are decreased, however, in preeclampsia compared with normal pregnancy, and are similar to those in nonpregnant women. ²⁴ In addition, whereas normal pregnancy triggers significantly increased uptake of L‐arginine (the sole substrate of NO), L‐arginine uptake is not increased in women with preeclampsia. ²⁷ , ²⁸ These defective responses may thus result in oxidative stress and increased interaction of NOS with O₂ ⁻, production of ONOO⁻, and impaired NO bioactivity. ²⁷ Increased concentration of the vasoconstrictor endothelin‐1 has also been reported in arterial endothelial cells of preeclamptic women, perhaps indicating dysfunctional NO response levels. ²⁹

Potential Genetic Mechanisms. Altered genetic coding of the gene for eNOS may also be a pathogenic factor in preeclampsia. ³⁰ , ³¹ , ³²

Treatments Targeting NO

Since the pathway of impaired NO bioactivity in preeclampsia has not been clearly determined, few therapeutic interventions targeting NO for this condition have been investigated. Treatment with L‐arginine (2% in the drinking water), starting at day 10 of gestation, significantly reduced blood pressure (BP) in pregnant rats. ³³ In clinical studies, L‐arginine supplementation significantly reduced BP in women with preeclampsia, compared with baseline or placebo. ³⁴ , ³⁵ Supplementation with antioxidant vitamins C and E in women at high risk for preeclampsia was reported to significantly reduce the subsequent incidence of preeclampsia, compared with high‐risk women not given these supplements. ³⁶ Antioxidants may reduce BP by decreasing oxidative stress and promoting NO bioactivity. ³⁷ , ³⁸

In clinical trials, long‐term transdermal administration of the organic nitrate isosorbide dinitrate, an NO donor, reduced BP and pulsatility index, while improving fetoplacental circulation, in women with preeclampsia. ³⁹ Nitroglycerin has also significantly reduced BP and uterine or umbilical artery resistance and has improved fetoplacental circulation in women with preeclampsia. ⁴⁰ , ⁴¹ , ⁴²

RENAL DISEASE

Chronic kidney disease (CKD) is associated with high rates of morbidity and mortality, including increased risks for hypertension, CV disease, and end‐stage renal disease (ESRD). ⁴³ , ⁴⁴ The prevalence of CKD, generally defined as a glomerular filtration rate of <60 mL/min per 1.73 m², remained stable at about 12% of the US adult population between 1988 and 2000. ⁴⁵ The ESRD population in the United States almost doubled, however, from 196,000 in 1991 to 382,000 in 2000, with incidence growing from 53,000 to 93,000 annually over the same period. ⁴⁶ The US incidence and prevalence of ESRD are projected to further increase by 44% and 85%, respectively, between 2000 and 2015. ⁴⁶ A part of this increase undoubtedly is a result of fewer people dying at earlier ages of coronary heart disease and strokes as a result of hypertension and diabetes. Rates of hypertension among patients with elevated serum creatinine levels and with ESRD are 70% and 86%, respectively. ⁴⁷

Physiologic Roles of NO in the Kidney

NO influences renal vascular tone and BP, glomerular and medullary hemodynamics, and extracellular fluid volume. ⁴⁸ , ⁴⁹ In the kidney, NO primarily vasodilates the afferent arteriole in the cortical nephron and may also vasodilate both afferent and efferent arterioles in juxtamedullary nephrons. ⁵⁰ Renal release of renin, which is catalyzed to form angiotensin II, is increased in response to reduced renal perfusion pressure and decreased by elevated renal perfusion pressure; NO modulates renin release by the juxtaglomerular apparatus and tubuloglomerular feedback. ⁵¹ Increased renovascular resistance and hypertension, with potential for renal ischemia and injury, may in part represent the unopposed actions of angiotensin II and endothelin‐1 due to impaired NO bioactivity. ⁵⁰

Several experimental studies have demonstrated a renoprotective effect of NO. ⁵² , ⁵³ , ⁵⁴ Genetic evidence also suggests that NO is renoprotective. The Glu298Asp gene polymorphism of eNOS was significantly more common (P=.002) in patients with ESRD (n=185), especially those with both diabetes mellitus and ESRD, compared with healthy subjects (n=304), ⁵⁵ and the eNOS gene polymorphism G894→T may also be associated with ESRD. ⁵⁶

Impairment of NO in Renal Disease

Animal models also indicate that impairment of NO bioactivity is pathogenic in renal disease. ⁵¹ Inhibition of NO with NΩ‐nitro‐L‐arginine methyl ester (L‐NAME) accelerates progression of renal disease in nephrectomized rats ⁵⁷ , ⁵⁸ and decreases renal blood flow and sodium excretion, while increasing renal vascular resistance and renin secretion, in normal dogs. ⁵⁹

In clinical studies, patients with chronic renal failure have significantly reduced endothelium‐dependent vasodilation, compared with normal individuals, which is attributable to impaired NO bioactivity and independent of the presence of concomitant atherosclerosis or other established CV risk factors. ⁶⁰ , ⁶¹ Plasma levels of the endogenous NO inhibitor asymmetric dimethylarginine are increased in chronic renal failure, particularly among patients with concomitant atherosclerosis, and asymmetric dimethylarginine is a strong independent risk factor for renal and CV morbidity and mortality. ⁶² , ⁶³

While NO bioactivity is impaired in renal disease, studies have differed as to whether NOS production and expression are increased or decreased. ⁶⁴ , ⁶⁵ , ⁶⁶

Targeting NO in Renal Disease Multiple animal models of renal failure have found that administration of L‐arginine reduced renal inflammation and injury and improved function, ⁶⁷ , ⁶⁸ , ⁶⁹ and administration of the NO donor molsidomine or the organic nitrate sodium nitroprusside was also associated with decreases in kidney damage and improvement in renal function. ⁷⁰ , ⁷¹ , ⁷²

Agents that promote NO bioactivity have also demonstrated benefits in renal circulation and function. Vasodilating β‐blockers, including bopindolol, celiprolol, and nebivolol, cause vasodilation in the rat renal vasculature and reduce renal perfusion pressure in a dosedependent manner; these effects are abolished by L‐NAME, demonstrating that they were NO‐dependent. ⁷³ Moreover, nebivolol has demonstrated increases in glomerular filtration rate, renal plasma flow, urine flow, and urinary excretion of sodium and chloride, and reductions in plasma renin activity in rats. ⁷⁴ These effects are generally reversed by NO inhibition. Overall, these findings suggest that NO donors or NO‐promoting agents may provide significant renoprotection in various types of nephropathy, particularly in the early stages of disease.

NEURODEGENERATIVE DISEASES

More than 600 nervous system diseases have been identified, affecting approximately 50 million Americans each year. ⁷⁵ , ⁷⁶ Alzheimer's disease, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis, and Huntington's disease are some of the most common neurodegenerative diseases. ⁷⁵ , ⁷⁷ Health care costs for Alzheimer's disease alone exceed $100 billion in the United States, making it the third most costly disease after heart disease and cancer. ⁷⁸ Characterized by the gradual and progressive loss of neural cells, neurodegenerative diseases are often related to increasing age, and their prevalence is expected to increase with the aging of the US population. ⁷⁵

Functions of NO in the Brain

All 3 NOS isoforms—eNOS, nNOS, and iNOS—are expressed in the brain, each having distinct physiologic roles. ⁷⁷ Major actions of NO in the brain include neurotransmission of forms of synaptic plasticity associated with learning and memory, including long‐term depression in the cerebellum and striatum and long‐term potentiation in the hippocampus and cerebral cortex ⁷⁷ , ⁷⁹ as well as the cerebellum. ⁸⁰ NO bioactivity may also regulate defensive/flight reactions to danger, as demonstrated in animal experiments. ⁸¹ While nNOS is believed to be the primary neurotransmitting isoform in the brain, this hypothesis has not been confirmed. ⁷⁷ iNOS is produced by astroglial cells and neurons in response to release of proinflammatory factors, while eNOS influences blood flow in the cerebral vasculature and thus helps maintain sufficient cerebral perfusion. ⁷⁷ , ⁸²

Neuroprotector or Pathogenic Factor?

The role of NO bioactivity in neurodegenerative disease states, and strategies for modulating NO in the treatment of these disorders, are highly controversial areas. ⁷⁷ , ⁸² , ⁸³ Some researchers have focused attention on evidence that NO bioactivity, particularly of the iNOS isoform, is upregulated in conditions of neural injury and disease. ⁸³ , ⁸⁴ This overproduction of iNOS in the brain, induced by inflammation, can lead to increased reactivity of NO with O₂ ⁻ and production of reactive nitrogen species such as ONOO⁻. ⁸³ , ⁸⁴ These toxic factors inhibit components of the mitochondrial respiratory chain, leading to neural cellular deficiency and cell death in disorders such as Parkinson's disease, Alzheimer's disease, and multiple sclerosis. ⁸³ , ⁸⁴

Excessive levels of iNOS and reactive NO metabolites have been associated with multiple sclerosis and Parkinson's disease. ⁸⁴ , ⁸⁵ , ⁸⁶ Aberrant expression of nNOS has also been reported in amyotrophic lateral sclerosis and in Alzheimer's disease. ⁸⁷ , ⁸⁸ Such findings have led to suggestions that therapeutic use of NO inhibitors to suppress NO bioactivity may be neuroprotective. ⁸³ , ⁸⁴

An alternate view is that since NO bioactivity mediates normal cognitive processes, therapies that preserve or enhance normal NO bioactivity may be beneficial in neurodegenerative diseases. ⁸² , ⁸⁹ , ⁹⁰ An interesting rationale for this viewpoint is the hypothesis that Alzheimer's disease is primarily a vascular disorder with neurodegenerative consequences, rather than the reverse, with the initial pathogenic insult arising from a reduction in cerebral perfusion, leading to a critically attained threshold of cerebral hypoperfusion (CATCH). ⁸² , ⁹¹ While the etiology of Alzheimer's disease is still not well understood, inflammation, oxidative stress, and apoptosis are believed to play major roles in its pathogenesis. ⁹² These factors lead to the primary features of Alzheimer's disease, including brain deposition of senile plaques containing amyloid‐β, neurofibrillary tangles, and neuronal loss. ⁹² , ⁹³

The vascular theory of Alzheimer's disease is supported by findings that heart disease and CV risk factors such as diabetes, smoking, hypercholesterolemia, obesity, and hypertension, particularly when clustered, are associated with increased risk for dementia and Alzheimer's disease. ⁹⁴ , ⁹⁵ In addition, increased levels of amyloid‐β have been associated with obesity and insulin resistance. ⁹⁶ , ⁹⁷ Recent studies have also found that treatment with antihypertensive medications may lower the risk for Alzheimer's disease. ⁹⁸ The CATCH hypothesis proposes that inflammation and oxidative stress cause brain hypoperfusion in Alzheimer's disease that leads to impairment of NO bioactivity in the endothelia of the brain capillary structure; this results in a cascade of signaling defects among the immune, CV, and nervous systems and metabolic dysfunctions. ⁸² Because basal levels of NO mediate neural cell activity by keeping particular types of cells in a state of inhibition, dysfunction of NO bioactivity leads to an inappropriate hyperexcited state of these cells, promoting inflammation and oxidative stress that leads cyclically to further impairment of NO and progressive inflammation and neurodegeneration. ⁸² Studies in experimental models of Alzheimer's disease support the hypothesis that impairment of NO bioactivity signaling may be a significant component of the progression of Alzheimer's disease. ⁹⁰

Targeting of NO in Neurodegenerative Diseases

Efforts to therapeutically modulate NO bioactivity in neurodegenerative diseases are in preliminary stages, yet have yielded some intriguing findings. A study in a culture of untreated neuroblastoma cells found that morphine, an endogenous NO donor, inhibited the expression of amyloid‐β protein; this beneficial effect was blocked with administration of the NO inhibitor l‐NAME. ⁹¹

OSTEOPOROSIS

Osteoporosis is a common disease that results from a range of causes in various populations. ⁹⁹ Defined as a bone mineral density (BMD) T‐score of‐2.5 SD below the mean for young adult white women and characterized by microarchitectural deterioration and increased risk of fracture, osteoporosis affects an estimated 10 million people in the United States; another 18 million have low BMD, placing them at high risk for osteoporosis. ⁹⁹ An estimated 1.5 million osteoporotic fractures occur annually in the United States, at a cost of almost $18 billion. ¹⁰⁰

Primary osteoporosis is strongly associated with estrogen depletion and age‐related bone loss following menopause in women, older age in men, and failure to reach optimal bone mass during childhood and adolescence. ⁹⁹ Secondary osteoporosis is associated with medications such as glucocorticoids; inflammatory disorders involving the musculoskeletal, gastrointestinal, or pulmonary systems; chronic renal disease; and other conditions such as hypogonadism and celiac disease. ⁹⁹ , ¹⁰¹

Role of NO in Bone

Bone loss and osteoporosis result when the ongoing process of bone remodeling is unbalanced, with bone resorption exceeding bone formation. ¹⁰² Accumulating evidence suggests that NO influences the activity of both osteoclasts, which resorb bone, and osteoblasts, which participate in bone formation. ¹⁰³ iNOS appears to have very low basal presence in bone, being primarily expressed in response to inflammatory factors, while the constitutive expression of nNOS in bone may be similar to that of eNOS in some osteocytes. ¹⁰³ , ¹⁰⁴ , ¹⁰⁵ Osteoblasts are the main producers of NO in bone. ¹⁰⁶

Various animal models indicate that elevated levels of NO, as well as NO donors, inhibit bone resorption. ¹⁰⁷ , ¹⁰⁸ NO regulation of osteoblast function has been observed primarily in the postnatal or youthful stages of bone development. ¹⁰⁹

NO bioactivity appears to exert its greatest effects on bone physiology when its levels are markedly increased in response to stimuli that promote bone resorption or formation, and as a modulator or inhibitor of these stimuli. ¹⁰³ , ¹⁰⁴ , ¹⁰⁶ For example, NO appears to mediate the effects of prostaglandin in bone, ¹¹⁰ and inflammatory cytokines, which promote bone resorption, dramatically increase production of NO, predominantly from osteoblasts. ¹⁰⁶ These effects were largely reversed with administration of the NO antagonist N^G‐monomethyl‐l‐arginine. Paradoxically, in an animal model of inflammatory osteoporosis, increased activity of iNOS was observed and N^G‐monomethyl‐l‐arginine improved BMD, suggesting that iNOS may play a pathogenic role in inflammatory osteoporotic diseases such as rheumatoid arthritis. ¹¹¹

Mechanical strain on bone produced by body weight, gravity, and muscular contraction is a well known stimulus to bone formation ¹¹² and NO is a key mediator of the osteogenic response to mechanical load. ¹¹³ , ¹¹⁴ , ¹¹⁵ Mechanical loading induces rapid increases in NO release and bioactivity, and administration of NO antagonists reduce or completely block mechanically induced bone formation. ¹¹³ , ¹¹⁴ , ¹¹⁵

NO may also mediate the antiosteoporotic actions of estrogen, raloxifene, and statins. ¹¹⁶ , ¹¹⁷ The primary antiosteoporotic action of estrogen is inhibition of osteoclastic bone resorption, but it may also stimulate bone formation by promoting osteoblast activity. ¹¹⁸ Estrogen has also promoted NO bioactivity by inhibiting O₂ ⁻ production in cultured bovine aortic endothelial cells. ¹¹⁹ Statins have been shown to promote new bone formation ¹²⁰ and are associated with significantly reduced risk of fracture in individuals aged 50 years and older compared with nonuse of statins. ¹²¹ On the other hand, the Women's Health Initiative (WHI) Observational Study ¹²² in 93,716 postmenopausal women aged 50–79 years showed no significant differences in BMD levels between statin users and nonusers. Because statins reduce lipid‐related oxidative stress and promote endothelial function and NO bioactivity, ¹²³ their antiosteoporotic actions may be linked to effects on NO bioactivity. ¹¹⁶

Targeting NO for Osteoporosis Treatment

Therapies targeting NO bioactivity may significantly prevent or reverse bone loss in high‐risk populations. In animal models, the NO donor nitroglycerine prevented or reversed bone loss associated with estrogen deficiency and prevented corticosteroid‐induced bone loss, ¹²⁴ , ¹²⁵ and supplementation with L‐arginine prevented prednisolone‐induced inhibition of bone growth and increased bone resorption. ¹²⁶

A prospective study in 6201 elderly women enrolled in the Study of Osteoporotic Fractures ¹²⁷ showed that those taking nitrates daily had slightly greater hip BMD and the same heel BMD as non‐users of nitrates. Intermittent nitrate users, however, perhaps avoiding the tachyphylaxis associated with daily use, had substantially greater BMD at both the hip and heel. In a small randomized, placebo‐controlled study (n=16), daily nitroglycerin ointment treatment given to women within 4 weeks of undergoing oophorectomy was as effective as estrogen replacement therapy in preventing bone loss secondary to estrogen deficiency. ¹²⁸ A placebo‐controlled, randomized intervention study in 144 healthy postmenopausal women with T‐scores of 0 to ‐2.5 SD showed that daily treatment with oral isosorbide mononitrate for 3 months significantly decreased a urine marker of bone resorption and increased a serum marker of bone formation (Figure). ¹²⁹ A substantial reduction in markers of bone resorption and increased markers of bone formation was also observed in a similar study of isosorbide mononitrate treatment for 3 months in 50 postmenopausal women. ¹³⁰ Although very preliminary, these data suggest that NO‐targeted therapies may help prevent or reverse osteoporosis and clearly warrant further study.

LIVER DISEASE

The prevalence in the United States of elevated ala‐nine aminotransferase (>43 IU/L), a primary marker of liver dysfunction, is 8.9% according to an analysis of data from the National Health and Nutrition Examination Survey (NHANES). ¹³¹ Nonalcoholic fatty liver disease (NAFLD), which refers to a wide range of liver disorders, including steatosis and steatohepatitis, advanced fibrosis, and cirrhosis, is the most common cause of elevated liver enzymes among US adults. ¹³² An estimated 5% of US adults are believed to have NAFLD, including rates of 50%–75% of obese individuals and about 50% of those with diabetes. ¹³² , ¹³³ Hyperlipidemia and age 45 years or older are also important risk factors for NAFLD. ¹³² , ¹³³ Nonalcoholic steatohepatitis, one of the more pernicious forms of NAFLD, may progress to cirrhosis and liver‐related death in 25% and 10% of patients, respectively. ¹³³

NO in Liver Disease

Liver disease offers an example of the paradoxical roles that NO can play. While low physiologic levels of NO bioactivity appear to modulate hepatic vascular tone and portal pressure to ensure optimal liver perfusion and protection against thrombosis, oxidative stress, and inflammation, elevated levels of NO associated with prolonged or chronic ischemia and inflammation may become pathogenic in the setting of liver disease. ¹³⁴ , ¹³⁵ Early studies found that in regulating hepatic vascular tone and blood and plasma flow, NO was a major contributor to the hemodynamic abnormalities that characterize cirrhosis. ¹³⁶ These derangements include portal hypertension associated with increased hepatic vascular resistance; hyperdynamic circulation, characterized by increased total blood volume and cardiac output with decreased total systemic vascular resistance and arterial pressure; development of ascites; and increased renin‐angiotensin system activity. ¹³⁶ These changes are associated with increased blood volume in the splanchnic circulation and excessive systemic vasodilation, causing hypotension and renin‐angiotensin system activation. ¹³⁶ Morbidities associated with cirrhosis include renal dysfunction with excessive salt and water retention, variceal bleeding, hepatopulmonary syndrome, and increased CV fragility under stress. ¹³⁷

Animal models of cirrhosis have found marked increases in vascular expression of eNOS and nNOS and significantly reduced intrahepatic endothelium‐dependent vasodilation. ¹³⁸ , ¹³⁹ Moreover, NO was shown to be protective against ischemia‐reperfusion injury to the liver. ¹⁴⁰ , ¹⁴¹

In clinical studies, patients with cirrhosis had elevated synthesis and bioactivity of NO in the splanchnic vasculature and hepatic tissue and increased endothelium‐dependent vasodilation compared with normal subjects. ¹⁴² , ¹⁴³ Inhibition of NO with N^G‐monomethyl‐L‐arginine, however, showed no effect on portal hypertension in patients with cirrhosis, creating confusion as to the role of NO bioactivity in this disease. ¹³⁶

NO‐Targeted Therapies in Liver Disease

Interventions to enhance NO activity in liver disease have produced promising results. L‐arginine and several NO donors have demonstrated hepatoprotective benefits in rat models of liver injury. ¹⁴⁴ , ¹⁴⁵ , ¹⁴⁶ , ¹⁴⁷ Furthermore, an eNOS gene transfer to the cirrhotic rat liver demonstrated a marked decrease in portal hypertension. ¹⁴⁸ In multiple controlled clinical studies, isosorbide dinitrate reduced portal BP in patients with cirrhosis and portal hypertension, compared with placebo, either as monotherapy ¹⁴⁹ , ¹⁵⁰ , ¹⁵¹ or in addition to propranolol. ¹⁵² Isosorbide dinitrate has also helped prevent variceal bleeding in patients with cirrhosis, both as monotherapy and in combination with propranolol. ¹⁵³ , ¹⁵⁴ , ¹⁵⁵ These preliminary findings suggest that use of NO‐modulating therapies may be hepatoprotective, particularly in reducing portal hypertension.

CONCLUSIONS

The roles of NO bioactivity are varied and complex. At low basal levels, endogenous NO bioactivity appears to mediate and protect homeostasis of bone, liver, and kidney, as well as the central nervous and reproductive systems. When inflammation and disease occur, however, NO levels or bioactivity are often increased, perhaps as a protective response. iNOS is the isoform most responsive to the presence of inflammation and most likely to increase excessively. With the progression of inflammatory and pathogenic processes, increased NO bioactivity can become dysfunctional and contribute to disease, leading some researchers to believe that inhibition of NO could be therapeutic in some settings. Researchers are increasingly finding, however, that the very processes of inflammation, impaired circulation, or disordered bone metabolism that precede preeclampsia, liver and kidney disease, neurodegenerative diseases, and osteoporosis consistently involve impairment of NO bioactivity. Restoration of NO bioactivity is thus a rational therapeutic strategy, particularly as a preventive measure or as intervention at early stages of disease. Experimental treatments using NO‐donating or promoting agents have generally supported this rationale and provide a promising basis for further investigation.