Hypertension Disease

By: Pharma Tips | Views: 3432 | Date: 17-Jun-2010

Blood pressure is the force with which blood pushes against the artery walls as it travels through the body.

Blood pressure is the force with which blood pushes against the artery walls as it travels through the body. Like air in a balloon, blood fills arteries to a certain capacity—and just as too much air pressure can cause damage to a balloon, too much blood pressure can harm healthy arteries. Blood pressure is measured by two methods—systolic pressure and diastolic pressure. Systolic pressure measures cardiac output and refers to the pressure in the arterial system at its highest. Diastolic pressure measures peripheral resistance and refers to arterial pressure at its lowest. Blood pressure is normally mea sured at the brachial artery with a sphygmomanometer (pressure cuff) in millimeters of mercury (mm Hg) and given as systolic over diastolic pressure.
A blood pressure reading thus appears as two methods. The upper method is the systolic pressure, which is the peak force of blood as the heart pumps it. The lower method the diastolic pressure, which is the pressure when the heart is filling or relaxing before the next beat. Normal blood pressure for an adult is 120/70 (on average), but normal for an individual varies with the height, weight, fitness level, age, and health of a person.

Hypertension Disease

What Is Hypertension?
Hypertension, or high blood pressure, is defined as a reading of 140/90 on three consecutive measurements at least six hours apart. The definition varies for pregnant women, where hypertension is defined as 140/90 on two consecutive measurements six hours apart. Consistently high blood pressure causes the heart to work harder than it should and can damage the coronary arteries, the brain, the kidneys, and the eyes. Hypertension is a major cause of stroke.


Category Systolic, mm Hg  Diastolic, mm Hg
Optimal <120 and <80
Normal <130 and <85
High normal 130–139 or 85–89
Stage 1 (mild) 140–159 or 90–99
Subgroup: borderline 140–149 or 90–94
Stage 2 (moderate) 160–179 or 100–109
Stage 3 (severe)  180
or  110
Isolated systolic hypertension  140
and <90
Subgroup: borderline 140–149 and <90

Types of Hypertension
Hypertension is classified as either primary (or essential) hypertension or secondary hypertension. Primary hypertension has no specific origin but is strongly associated with lifestyle. It is responsible for 90 to 95 percent of diagnosed hypertension and is treated with stress management, changes in diet, increased physical activity, and medication (if needed). Secondary hypertension is responsible for 5 to 10 percent of diagnosed hypertension. It is caused by a preexisting medical condition such as congestive heart failure, kidney failure, liver failure, or damage to the endocrine (hormone) system.
Primary Hypertension 
BP is a quantitative trait that is highly variable1 ; in population studies, BP has a normal distribution that is slightly skewed to the right. There is a strong positive and continuous correlation between BP and the risk of CVD (stroke, myocardial infarction, heart failure), renal disease, and mortality, even in the normotensive range. This correlation is more robust with systolic than with diastolic BP.2 There is no specific level of BP where cardiovascular and renal complications start to occur; thus the definition of hypertension is arbitrary but needed for practical reasons in patient assessment and treatment. 
The Sixth Report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure (JNC VI) defined and classified hypertension in adults, as shown in above The diagnosis of hypertension is made when the average of 2 or more diastolic BP measurements on at least 2 subsequent visits is  90 mm Hg or when the average of multiple systolic BP readings on 2 or more subsequent visits is consistently  140 mm Hg. Isolated systolic hypertension is defined as systolic BP  140 mm Hg and diastolic BP <90 mm Hg. Individuals with high normal BP tend to maintain pressures that are above average for the general population and are at greater risk for development of definite hypertension and cardiovascular events than the general population. With the use of these definitions, it is estimated that 43 million people in the United States have hypertension or are taking antihypertensive medication, which is  24% of the adult population. This proportion changes with (1) race, being higher in blacks (32.4%) and lower in whites (23.3%) and Mexican Americans (22.6%); (2) age, because in industrialized countries systolic BP rises throughout life, whereas diastolic BP rises until age 55 to 60 years and thus the greater increase in prevalence of hypertension among the elderly is mainly due to systolic hypertension; (3) geographic patterns, because hypertension is more prevalent in the southeastern United States; (4) gender, because hypertension is more prevalent in men (though menopause tends to abolish this difference); and (5) socioeconomic status, which is an indicator of lifestyle attributes and is inversely related to the prevalence, morbidity, and mortality rates of hypertension. Essential, primary, or idiopathic hypertension is defined as high BP in which secondary causes such as renovascular disease, renal failure, pheochromocytoma, aldosteronism, or other causes of secondary hypertension or mendelian forms (monogenic) are not present. Essential hypertension accounts for 95% of all cases of hypertension. Essential hypertension is a heterogeneous disorder, with different patients having different causal factors that lead to high BP. Essential hypertension needs to be separated into various syndromes because the causes of high BP in most patients presently classified as having 
Known Etiological Factors in Essential Hypertension 
Although it has frequently been indicated that the causes of essential hypertension are not known, this is only partially true because we have little information on genetic variations or genes that are overexpressed or underexpressed as well as the intermediary phenotypes that they regulate to cause high BP.4 A number of factors increase BP, including (1) obesity, (2) insulin resistance, (3) high alcohol intake, (4) high salt intake (in salt-sensitive patients), (5) aging and perhaps (6) sedentary lifestyle, (7) stress, (8) low potassium intake, and (9) low calcium intake.5 6 Furthermore, many of these factors are additive, such as obesity and alcohol intake. 

Secondary Hypertension
Secondary hypertension accounts for approximately 5-10% of all cases of hypertension, with the remaining being primary hypertension.  Secondary hypertension has an identifiable cause whereas primary hypertension has no known cause (i.e., idiopathic).
There are many known conditions that can cause secondary hypertension. Regardless of the cause, arterial pressure becomes elevated either due to an increase in cardiac output, an increase in systemic vascular resistance, or both. When cardiac output is elevated, it is generally due to either increased neurohumoral activation of the heart or increased blood volume.  
Patients with secondary hypertension are best treated by controlling or removing the underlying disease or pathology, although they may still require antihypertensive drugs.
Some causes for secondary hypertension are listed below:

Renal artery stenosis 
Chronic renal disease 
Primary hyperaldosteronism 
Sleep apnea 
Hyper- or hypothyroidism 
The pathophysiology of hypertension 
There is still much uncertainty about the pathophysiology of hypertension. A small number of patients (between 2% and 5%) have an underlying renal or adrenal disease as the cause for their raised blood pressure. In the remainder, however, no clear single identifiable cause is found and their condition is labelled "essential hypertension". A number of physiological mechanisms are involved in the maintenance of normal blood pressure, and their derangement may play a part in the development of essential hypertension. 

The relative frequency of primary and secondary hypertension
It is probable that a great many interrelated factors contribute to the raised blood pressure in hypertensive patients, and their relative roles may differ between individuals. Among the factors that have been intensively studied are salt intake, obesity and insulin resistance, the renin-angiotensin system, and the sympathetic nervous system. In the past few years, other factors have been evaluated, including genetics, endothelial dysfunction (as manifested by changes in endothelin and nitric oxide), low birth weight and intrauterine nutrition, and neurovascular anomalies. 

Physiological mechanisms involved in development of essential hypertension
Cardiac output
Peripheral resistance
Renin-angiotensin-aldosterone system
Autonomic nervous system
Other factors:
EDRF (endothelial derived relaxing factor) or nitric oxide
ANP (atrial natriuretic peptide)
• Cardiac output and peripheral resistance
Maintenance of a normal blood pressure is dependent on the balance between the cardiac output and peripheral vascular resistance. Most patients with essential hypertension have a normal cardiac output but a raised peripheral resistance. Peripheral resistance is determined not by large arteries or the capillaries but by small arterioles, the walls of which contain smooth muscle cells. Contraction of smooth muscle cells is thought to be related to a rise in intracellular calcium concentration, which may explain the vasodilatory effect of drugs that block the calcium channels. Prolonged smooth muscle constriction is thought to induce structural changes with thickening of the arteriolar vessel walls possibly mediated by angiotensin, leading to an irreversible rise in peripheral resistance. 

The heart, arteries, and arterioles in hypertension
Plasma renin in black and white hypertensive patients. Adapted from Freis ED, Materson BJ, Flamenbaum V. Comparison of propranolol or hydrochlorothiazide alone for treatment of hypertension. III. Evaluation of the renin-angiotensin system. Am J Med 1983;74:1029-41

Local versus systemic renin-angiotensin systems
It has been postulated that in very early hypertension the peripheral resistance is not raised and the elevation of the blood pressure is caused by a raised cardiac output, which is related to sympathetic overactivity. The subsequent rise in peripheral arteriolar resistance might therefore develop in a compensatory manner to prevent the raised pressure being transmitted to the capillary bed where it would substantially affect cell homeostasis. 

• Renin-angiotensin system
The renin-angiotensin system may be the most important of the endocrine systems that affect the control of blood pressure. Renin is secreted from the juxtaglomerular apparatus of the kidney in response to glomerular underperfusion or a reduced salt intake. It is also released in response to stimulation from the sympathetic nervous system. 
Renin is responsible for converting renin substrate (angiotensinogen) to angiotensin I, a physiologically inactive substance which is rapidly converted to angiotensin II in the lungs by angiotensin converting enzyme (ACE). Angiotensin II is a potent vasoconstrictor and thus causes a rise in blood pressure. In addition it stimulates the release of aldosterone from the zona glomerulosa of the adrenal gland, which results in a further rise in blood pressure related to sodium and water retention. The circulating renin-angiotensin system is not thought to be directly responsible for the rise in blood pressure in essential hypertension. In particular, many hypertensive patients have low levels of renin and angiotensin II (especially elderly and black people), and drugs that block the renin-angiotensin system are not particularly effective. There is, however, increasing evidence that there are important non-circulating "local" renin-angiotensin epicrine or paracrine systems, which also control blood pressure. Local renin systems have been reported in the kidney, the heart, and the arterial tree. They may have important roles in regulating regional blood flow. 
Renin-angiotensin system and effects on blood pressure and aldosterone release 
• Autonomic nervous system
Sympathetic nervous system stimulation can cause both arteriolar constriction and arteriolar dilatation. Thus the autonomic nervous system has an important role in maintaining a normal blood pressure. It is also important in the mediation of short term changes in blood pressure in response to stress and physical exercise. 

 The autonomic nervous system and its control of blood pressure. Reproduced with permission from Swales JD, Sever PS, Plart WS. Clinical atlas of hypertension. London: Gower Medical, 1991 

There is, however, little evidence to suggest that epinephrine (adrenaline) and norepinephrine (noradrenaline) have any clear role in the aetiology of hypertension. Nevertheless, their effects are important, not least because drugs that block the sympathetic nervous system do lower blood pressure and have a well established therapeutic role. 
It is probable that hypertension is related to an interaction between the autonomic nervous system and the renin-angiotensin system, together with other factors, including sodium, circulating volume, and some of the more recently described hormones.     

Endothelial dysfunction
Vascular endothelial cells play a key role in cardiovascular regulation by producing a number of potent local vasoactive agents, including the vasodilator molecule nitric oxide and the vasoconstrictor peptide endothelin. Dysfunction of the endothelium has been implicated in human essential hypertension. 
Modulation of endothelial function is an attractive therapeutic option in attempting to minimise some of the important complications of hypertension. Clinically effective antihypertensive therapy appears to restore impaired production of nitric oxide, but does not seem to restore the impaired endothelium dependent vascular relaxation or vascular response to endothelial agonists. This indicates that such endothelial dysfunction is primary and becomes irreversible once the hypertensive process has become established. 

The control of peripheral arteriolar resistance. 
 Vasoactive substances
Many other vasoactive systems and mechanisms affecting sodium transport and vascular tone are involved in the maintenance of a normal blood pressure. It is not clear, however, what part these play in the development of essential hypertension. Bradykinin is a potent vasodilator that is inactivated by angiotensin converting enzyme. Consequently, the ACE inhibitors may exert some of their effect by blocking bradykinin inactivation. 
Endothelin is a recently discovered, powerful, vascular, endothelial vasoconstrictor, which may produce a salt sensitive rise in blood pressure. It also activates local renin-angiotensin systems. Endothelial derived relaxant factor, now known to be nitric oxide, is produced by arterial and venous endothelium and diffuses through the vessel wall into the smooth muscle causing vasodilatation. 
Atrial natriuretic peptide is a hormone secreted from the atria of the heart in response to increased blood volume. Its effect is to increase sodium and water excretion from the kidney as a sort of natural diuretic. A defect in this system may cause fluid retention and hypertension. 
Sodium transport across vascular smooth muscle cell walls is also thought to influence blood pressure via its interrelation with calcium transport. Ouabain may be a naturally occurring steroid-like substance which is thought to interfere with cell sodium and calcium transport, giving rise to vasoconstriction. 

Patients with hypertension demonstrate abnormalities of vessel wall (endothelial dysfunction or damage), the blood constituents (abnormal levels of haemostatic factors, platelet activation, and fibrinolysis), and blood flow (rheology, viscosity, and flow reserve), suggesting that hypertension confers a prothrombotic or hypercoagulable state. These components appear to be related to target organ damage and long term prognosis, and some may be altered by antihypertensive treatment. 

Virchow's triad and the prothrombotic state in hypertension
Insulin sensitivity
Epidemiologically there is a clustering of several risk factors, including obesity, hypertension, glucose intolerance, diabetes mellitus, and hyperlipidaemia. This has led to the suggestion that these represent a single syndrome (metabolic syndrome X or Reaven's syndrome), with a final common pathway to cause raised blood pressure and vascular damage. Indeed some hypertensive patients who are not obese display resistance to insulin. There are many objections to this hypothesis, but it may explain why the hazards of cardiovascular risk are synergistic or multiplicative rather than just additive. 
Genetic factors
Although separate genes and genetic factors have been linked to the development of essential hypertension, multiple genes are most likely contribute to the development of the disorder in a particular individual. It is therefore extremely difficult to determine accurately the relative contributions of each of these genes. Nevertheless, hypertension is about twice as common in subjects who have one or two hypertensive parents, and many epidemiological studies suggest that genetic factors account for approximately 30% of the variation in blood pressure in various populations. This figure can be derived from comparisons of parents with their monozygotic and dizygotic twin children, as well as their other children, and with adopted children. Some familial concordance is, however, due to shared lifestyle (chiefly dietary) factors. 

Examples of specific genetic mutations causing hypertension
   Liddle's syndrome, a disorder associated with hypertension, low plasma renin and aldosterone levels, and hypokalaemia, all of which respond to amiloride, an inhibitor of the distal renal epithelial sodium channel
   Glucocorticoid-remediable aldosteronism, a disorder mimicking Conn's syndrome, in which there is a chimeric gene formed from portions of the 11 -hydroxylase gene and the aldosterone synthase gene. This defect results in hyperaldosteronism, which is responsive to dexamethasone and has a high incidence of stroke
   Congenital adrenal hyperplasia due to 11 -hydroxylase deficiency, a disorder that has been associated with 10 different mutations of the CYP11B1 gene
   Syndrome of apparent mineralocorticoid excess, arising from mutations in the gene encoding the kidney enzyme 11 -hydroxysteroid dehydrogenase; the defective enzyme allows normal circulating concentrations of cortisol (which are much higher than those of aldosterone) to activate the mineralocorticoid receptors
   Congenital adrenal hyperplasia due to 17 -hydroxylase deficiency, a disorder with hyporeninaemia hypoaldosteronism, absent secondary sexual characteristics, and hypokalaemia
   Gordon's syndrome (pseudo-hypoaldosteronism): familial hypertension with hyperkalaemia, possibly related to the long arm of chromosome 17
   Sporadic case reports of familial inheritance of phaeochromocytoma (multiple endocrine neoplasia, MEN-II syndrome), Cushing's syndrome, Conn's syndrome, renal artery stenosis due to fibromuscular dysplasia
Other associations
   The angiotensinogen gene may be related to hypertension
   The angiotensin converting enzyme gene may be related to left ventricular hypertrophy or hypertensive nephropathy
    -Adducin gene may be related to salt sensitive hypertension

Some specific genetic mutations can rarely cause hypertension. Experimental models of genetic hypertension have shown that the inherited tendency to hypertension resides primarily in the kidney. For example, animal and human studies show that a transplanted kidney from a hypertensive donor raises the blood pressure and increases the need for antihypertensive drugs in recipients coming from "normotensive" families. Conversely a kidney from a normotensive donor does not raise the blood pressure in the recipient. Increased plasma levels of angiotensinogen, the protein substrate acted on by renin to generate angiotensin I, have also been reported in hypertensive subjects and in children of hypertensive parents. 

 Renin and electrolytes in black and white people. He J, Klag MJ, Appel LJ, Charleston J, Whelton PK. The renin-angiotensin system and blood pressure; differences between blacks and whites. Am J Hypertens 1999;12:555-62 

Hypertension is rarely found in rural or "tribal" areas of Africa, but it is very common in African cities and in black populations in Britain and the United States. Whereas the rural/urban differences in Africa are clearly due to lifestyle and dietary factors, the finding that hypertension is commoner in black people compared with white people may have some genetic basis. There is some evidence from salt loading studies in medical students that black Americans are more susceptible to a given salt load than white Americans, and may be more sensitive to the beneficial effects of salt restriction. 

Intrauterine influences
There is increasing evidence that fetal influences, particularly birth weight, may be a determinant of blood pressure in adult life. For example, babies who are small at birth are more likely to have higher blood pressure during adolescence and to be hypertensive as adults. Babies who are small for their age are also more likely to have metabolic abnormalities that have been associated with the later development of hypertension and cardiovascular disease, such as insulin resistance, diabetes mellitus, hyperlipidaemia, and abdominal obesity (the "Barker hypothesis"). Insulin resistance almost certainly contributes to the increased prevalence of coronary disease seen in adults of low birth weight. 
It is possible, however, that genetic factors influence the Barker hypothesis. Mothers with above average blood pressure in pregnancy give birth to smaller babies who subsequently develop above average blood pressure themselves and eventually hypertension. It is entirely likely that the similarity of blood pressures in mother and child are genetic and, in a modern "healthy" society, unrelated to intrauterine undernutrition. 
Diastolic dysfunction
In hypertensive left ventricular hypertrophy, the ventricle cannot relax normally in diastole. Thus, to produce the necessary increase in ventricular input, especially during exercise, there is an increase in left atrial pressure rather than the normal reduction in ventricular pressure, which produces a suction effect as described above. This can lead to an increase in pulmonary capillary pressure that is sufficient to induce pulmonary congestion. The rise in atrial pressure can also lead to atrial fibrillation, and in hypertrophied ventricles dependent on atrial systole the loss of atrial transport can result in a significant reduction in stroke volume and pulmonary oedema. Exercise induced subendocardial ischaemia can also produce an "exaggerated" impairment of diastolic relaxation of the hypertrophied myocardium. 
Possible mechanisms to explain why low birthweight babies are more likely to develop hypertension in later life 
Pressure-volume curves demonstrating diastolic/systolic dysfunction
Molecular Mechanism Of Vascular Dysfunction In Hypertension
1. Role Of Endothelin In Molecular Mechanism Of Vascular Dysfunction In Hypertension
• Vascular function
The endothelium plays an important role in the regulation of vascular function by producing a large number of biologically active substances that participate in the regulation of vascular tone, cell growth, inflammation, and thrombosis/ haemostasis. Dysfunction of the vascular endothelium is an early finding in the development of cardiovascular disease and is closely related to clinical events in patients with atherosclerosis and hypertension [1]. Therefore, knowledge regarding the mechanisms behind the development of endothelial dysfunction and pharmacological strategies targeting endothelial dysfunction is of great importance. Endothelial dysfunction often refers to a situation of reduced bioavailability and consequently impaired vasodilator effect of endothelium derived relaxing factors such as nitric oxide (NO), prostcyclin or endothelium-derived hyperpolarizing factor. One additional important alteration in endothelial dysfunction is an increased production and biological activity of the potent vasoconstrictor and pro-inflammatory peptide endothelin(ET)-1. In the present review the pathogenic role of the altered expression and biological actions of ET-1 and its receptors in vascular dysfunction and the development of cardiovascular disease are summarized. In particular the changes of pathophysiological importance mediated by ET-1 in clinical studies and the possible mechanisms behind these changes are reviewed. These changes may be of significance for the . pulmonary arterial hypertension which is the currently approved indication for ET receptor antagonists.

• The family of ET peptides
Since the discovery of an endothelium-derived constricting factor in 1985 [2] and the complex description of ET performed  by Yanagisawa et al. in 1988 [3], three structurally
different ET isoforms [4] have been described (i.e. ET-1, ET-2, ET-3 as well as vasoactive intestinal constrictor) [4]. In addition, 31-residue ETs have been identified [5].Amongst the
three ET isopeptides, the 21-amino acid peptide ET-1 is regarded as the most prominent isoform in the cardiovascular system, accounting for the majority of pathobiological effects
exerted by ETs [6]. Mature ET-1 is formed from pre-pro-ET-1 via a 39-amino acid intermediate, big ET-1 [7]. Big ET-1 is processed to ET-1 by a family of ET converting enzymes (ECEs) and other enzymes such as chymases, non-ECE metalloproteinases and
endopeptidases [7,8]. Under physiological conditions, ET-1 is produced in small amounts mainly in endothelial cells, primarily acting as an autocrine/paracrine mediator. Under
pathophysiological conditions however, the production is stimulated in a large number of different cell types, including endothelial cells, vascular smooth muscle cells, cardiac
myocytes [9], and inflammatory cells such as macrophages [10] and leukocytes [11] (Fig. 1) 

• The receptors of ET peptides
The biological effects of ET-1 are transduced by two pharmacologically distinguishable receptor subtypes, ETA and ETB receptors, respectively [12]. In the vasculature, the ETA
receptor is mainly located on vascular smoothmuscle cells and mediates potent vasoconstriction (Fig. 1). ET-1 may also induce indirect vasoconstrictor effects due to the generation of endothelium-derived thromboxane A2 [13]. The ETB receptor is primarily located on endothelial cells, but may also be present on vascular smooth muscle cells. Stimulation of the endothelial ETB receptor results in release of NO and prostacyclin [14] which cause vasodilatation, whereas stimulation of the vascular smooth muscle cell ETB receptor results in vasoconstriction (Fig. 1). Thus, the net effect produced by ET-1 is determined on the receptor localisation and the balance between ETA and ETB receptors. Under physiological conditions, the net effect is vasoconstriction mediated by the ETA receptor, which is partly counteracted by ETB receptormediated release of NO. However, under certain pathophysiological conditions the response to ET receptor antagonists
may be changed, which will be discussed below.

The endogenous ET system and vascular dysfunction

•  Changes in vascular reactivity to ET-1
In healthy humans ET-1 increases mean arterial blood pressure, reduces heart rate, cardiac output and stroke volume and causes potent and long lasting vasoconstriction in the pulmonary [15], renal, splanchnic, myocardial [16], and skeletal muscle [17] vasculature. Haynes and Webb demonstrated that the selective ETA receptor antagonist  BQ123 evokes increases in forearm blood flow in healthy men [18]. ETB receptor antagonism may either alone or on a background of ETA receptor antagonism cause local
vasoconstriction in young healthy subjects [19]. These findings suggest that endogenous ET-1 has a physiological role in the maintenance of vascular tone in healthy humans. Several studies have demonstrated marked changes in the vascular reactivity to ET-1 in 

Fig. 1. Schematic figure of the arterialwall under healthy conditions (left) and in endothelial dysfunction (right). In healthy arteries the production of ET-1 is small and the bioavailability of NO is preserved. This means that the balance of effects favours vasorelaxation through increased signalling of cyclic GMP. In endothelial dysfunction there is increased expression of ET-1 in smooth muscle cells and macrophages (MØ). There is also increased expression of ETB receptors on smooth muscle cells mediating vasoconstriction. ET-1 may decrease endothelial NO synthase (eNOS) expression, thereby reducing NO production. Both the ETA and the ETB receptor on smooth muscle cells may mediate formation of superoxide (O2 − ) in endothelial dysfunction. Superoxide will decrease the biological activity of NO by forming peroxynitrate (ONOO−). Collectively the balance of effects is shifted towards more vasoconstriction, inflammation and oxidative stress in endothelial dysfunction.

 disease (Table 1). Increased vascular sensitivity to ET receptor stimulation is shown in patients with hypertension and atherosclerosis. Cardillo et al. found that the vasoconstrictor response to intra-arterial infusion of ET-1 in the forearm was enhanced in hypertensive as compared to normotensive individuals [20]. This response was mediated via activation of both ETA and the ETB receptors. In patients with atherosclerosis, the vasoconstrictor response to ET-1 was not different from that observed in age-matched controls [21]. On the other hand, the ETB receptor agonist sarafotoxin S6c produced more pronounced reduction in forearm blood flow in patients with atherosclerosis than in the control group, indicating an
upregulation of vasoconstrictor ETB receptors. Results from studies using receptor agonists may be difficult to interpret, however. Therefore, studies in which ET receptor antagonists were administered have been performed. Administration of the selective ETA receptor antagonist BQ123 increased forearm blood flow only in hypertensive patients but not in normotensive controls [20]. Obese hypertensives dilate more following ETA receptor blockade than non-obese hypertensive patients [22]. In addition, BQ123 induced a greater vasodilatation in hypertensives than in subjects with hypercholesterolemia or in smokers [23]. Cardillo et al. showed that BQ123 induced a significant increase in forearm blood flow in patients with hypercholesterolemia compared to normal subjects [24] supporting the notion that risk factors for cardiovascular disease stimulate the ET system in vivo. The increase in forearm vasodilatation in response to BQ123 was attenuated by inhibition of NO generation [19] indicating that the effect to a major part is dependent on  increased NO availability. A combination of ETA and the ETB receptor antagonists (BQ123 and BQ788) also evokes a more pronounced increase in forearm blood flow in patients with hypertension than in controls [20]. In accordance, Taddei et al. found that the dual ETA/ETB receptor antagonist TAK-044 produced a greater degree of vasodilatation in hypertensive than in normotensive patients [25]. Collectively these observations indicate that the increased vascular tone induced by ET-1 seems to be more pronounced in hypertension than in association with other risk factors for cardiovascular disease. The formation and activity of endogenous ET-1 has also been evaluated in patients with atherosclerosis (Table 1). Administration of big ET-1 by intra-brachial artery infusion resulted in more pronounced forearm vasoconstriction in patients with atherosclerosis than in age-matched controls [26]. This effect was accompanied by increased formation of ET-1 as well as presence of ECE immunoreactivity in atherosclerotic plaques in the radial artery, indicating increased ECE activity in patients with atherosclerosis. In another study, dual ETA/ETB receptor blockade evoked greater increase in forearm blood flow in patients with atherosclerosis than in controls indicating enhanced vasoconstrictor tonemediated by ET-1 [27]. Furthermore, the vasodilator response to dual ETA/ETB receptor blockade was greater than that induced by selective ETA  receptor blockade in patients with atherosclerosis, whereas the opposite was observed in control subjects. This suggests that antagonizing both receptors may be of greater value in achieving vasodilatation in patients with atherosclerosis. Kinlay et al. investigated the response to ETA receptor blockade in coronary arteries of patients with coronary artery disease by intracoronary infusion of BQ123. They found thatBQ123 caused coronary dilatation and that the dilator response was more pronounced in severely stenotic than in angiographically normal segments [28]. Collectively, these observations suggest that the importance of ET-1 for vascular tone becomes greater in severe atherosclerosis than under normal conditions. Patients with type 2 diabetes also seem to have increased vasoconstrictor activity induced by endogenous ET-1. Accordingly, administration of BQ123 resulted in a significant increase in forearm blood flow in patients with type 2 diabetes whereas it had no effect in age-matched controls [29]. There was no difference between selective ETA and dual receptor blockade in this patient group, however Furthermore, dual ETA/ETB receptor blockade with BQ123 and BQ788 elicited forearm vasodilatation in patients with atherosclerosis and type 2 diabetes mellitus (Settergren et al., 2007a, manuscript in preparation; [98]). It is also of interest to note that ETA receptor blockade increases nutritive skin capillary blood flow in patients with type 2 diabetes and microangiopathy, whereas no effect was observed in agematched controls (Settergren et al., 2007b, manuscript in preparation; [99]). Of further importance in type 2 diabetes, we recently showed that dual ETA/ETB receptor blockade improved insulin sensitivity more than selective ETA receptor blockade in obese patients with coronary artery disease and insulin resistance [30]. It is noticeable that dual ETA/ETB receptor antagonism seems to be more effective as vasodilators than selective ETA receptor antagonists in various cardiovascular disorders like hypertension [20] and atherosclerosis [27] (Table 1). Even though all studies have not compared the two different ET receptor blocking strategies, available literature suggests that it is probably of importance to block both receptors to fully antagonize the vasoconstrictor actions of ET-1 in cardiovascular disease.

• 2.2. Mechanisms behind changed vascular activity
One explanation behind the altered response to ET receptor blockade in cardiovascular disease states may be the upregulation of ET-1 expression as described above. Anotherpossible mechanismis related to changes in the expression and activity of the different receptor subtypes. An increased number of ETB receptors has been demonstrated in human atherosclerotic arteries [31]. The receptors were present on inflammatory cells (i.e. macrophages, T-lymphocytes) and vascular smooth muscle cells. Moreover, intimal smooth muscle cells close to foam cells showed increased expression of ET-1 and ETB receptors. The authors suggested that foamy macrophages and T-lymphocytes may modulate the switch from ETA to ETB receptors on vascular smooth muscle cells and that this switch may be of importance for the progression of atherosclerosis [31]. A recent study found both ETA and ETB receptor expression were increased in internal mammary arteries from patients with coronary artery disease [32]. Increased expression of ETB receptors in relation to ETA receptors has also been demonstrated in experimental models and patients with pulmonary arterial hypertension [33,34].The vasodilatation induced by ETA receptor antagonism in healthy humans was  reduced by 95% following inhibition of NO generation [19], whereas inhibition of prostanoid generation did not affect the response. This finding suggests that improvement of NO bioavailability plays an important role in the vasodilatation induced by ET receptor blockade. Both dual ETA/ETB and selective ETA receptor blockade increase endothelial NO synthase activity in hypercholesterolemic pigs [52]. Total and calcium-dependent NO synthase activity was significantly higher in aortic endothelial cells after dual ETA/ETB antagonism than in those after selective ETA blockade [52]. ET-1 impairs NO production and downregulates the expression of endothelial NO synthase in endothelial cells [53]. In addition, bosentan increased the expression of endothelial NO synthase in hearts subjected to ischaemia and reperfusion [54]. Thus, ET-1 may reduce NO bioavailability via interference with the expression and activity of endothelial NO synthase.  Another mechanism linking ET-1 to NO may be via formation of reactive oxygen species, which will result in decreased bioactivity of NO by virtue of formation of peroxynitrite (Fig. 1). The reactive oxygen species can, apart from interfering with NO, also inhibit other endotheliumdependent vasodilator pathways mediated through prostacyclin and endothelium-derived hyperpolarizing factor [55,56]. ET-1 increases superoxide production in the rat aorta in vitro, an effect that could be inhibited by the selective ETA receptor antagonist BQ123 [57]. ET-1 also stimulates NADPH oxidase-derived superoxide formation in hypertensive rats, an effect that could be inhibited by ETA receptor blockade [58]. ET-1 increased the expression of gp91phox, the ratelimiting subunit of NADPH oxidase [59], and augmented superoxide production in endothelial cells via the ETB receptor in human endothelial cells [60]. The stimulating effect of ET-1 on superoxide production may also be coupled to the NADPH oxidase subunit p22phox [61,62]. The stimulation of superoxide is linked to functional effects since ET-1 was demonstrated to  
Fig. 5. Molecular mechanisms of vascular dysfunction induced by ET-1 including pro-inflammatory and pro-atherosclerotic effects. Potential benefit in cardiovascular disease states may be mediated by altering these mechanisms through dual ETA/ETB receptor blockade and/or selective ETA receptor blockade. []=Reference
impair endothelium dependent relaxations of aorta from control and diabetic rats via a mechanism involving superoxide production, PI3-kinase activity and p22phox expression. Furthermore, chronic treatment with the dual ETA/ETB receptor antagonist J-104132 improved acetylcholine-mediated endothelium-dependent vasodilatation, reduced superoxide formation and prevented p22phox formation in diabetic rats [61]. These data are in agreement with in vivo observations in transgenic mice overexpressing ET-1 [63]. These mice exhibit endothelial dysfunction, increased NADPH oxidase activity, and increased expression of gp91phox. The endothelial dysfunction could be restored by vitamin C, supporting the role of increased oxidative stress [63]. Furthermore, vitamin C has been shown to inhibit the formation of reactive oxygen species induced by ET-1 in isolated smooth muscle cells [64]. In addition, the effects of ET-1 on coronary vasoconstriction may be more pronounced in states of reduced bioavailability of the eNOS-co-factor tetrahydrobiopterin (BH4) [65]. Recent data demonstrate that ET-1 mediates superoxide production and vasoconstriction through activation of NADPH oxidase and uncoupled NOS
in the rat aorta [66]. The uncoupling of NOS means that NOS generates superoxide instead of NO in states of BH4 deficiency. Interestingly, these effects could be inhibited by BH4 and by dual ET receptor blockade, but not by selective ETA receptor blockade [66]. ET-1 may also promote BH4 deficiency in a rat model of hypertension via an ETAmediated NADPH oxidase pathway which contributes to impaired endothelium-dependent relaxation [67]. These data suggest that increased oxidative stress induced by ET-1 in the vessel wall is an important factor leading to endothelial dysfunction and enhanced susceptibility to atherosclerosis. ET-1 has also been demonstrated to be associated with increased oxidative stress and endothelial dysfunction in humans. ET-1 stimulates superoxide formation and impairs endothelium-dependent vasodilatation in human venous bypass conduits from patients with diabetes [68]. Importantly, recent data suggest that the impairment in endothelium-dependent vasodilatation in vivo induced by ET-1 in healthy humans can be prevented by administration of the anti-oxidant vitamin C (Fig. 2) [37]. Taken together, these findings suggest that ET-1 may increase oxidative stress through induction of reactive oxygen species. Furthermore, ET receptor antagonists may be a therapeutic option that results in increased NO bioavailability and decreased levels of reactive oxygen species, thereby improving endothelial function in various cardiovascular disease states. 

Pro-inflammatory and pro-atherosclerotic effects
Apart from its direct vasomotor activity, ET-1 has been implicated in inflammatory processes within the vascular wall (Fig. 5). Specifically, ET-1 in subnanomolar concentrations has been demonstrated to activate macrophages, resulting in the release of pro-inflammatory and chemotactic mediators, including tumor necrosis factor (TNF)-α, interleukin (IL)-1, IL-6 and IL-8[69–71] which are of importance in the atherosclerotic process [72]. Cardiac overexpression of ET-1 in mice is associated with an inflammatory response involving increased activation of the pro-inflammatory transcription factor NF-κB and expression of several proinflammatory cytokines including TNF-α, IL-1 and IL-6 [73]. Interestingly, significant prolongation of survival was observed only with a dual ETA/ETB antagonist, but not with a selective ETA antagonist [73]. In turn, transcription factors and pro-inflammatory cytokines such as NF-κB, TNF-α, and IL-6 stimulate ET-1 production [74]. ET-1 enhances the expression of adhesion molecules on TNF-α stimulated vascular endothelial cells [75] and stimulates aggregation of polymorphonuclear neutrophils [76]. Conversely, ET receptor blockade attenuates the accumulation of neutrophils and myeloperoxidase activity in the ischemic myocardium [77]. IL-6 has been implicated in the development of atherosclerosis [72] and endothelial dysfunction in humans [78]. As noted above, ET-1 stimulates IL-6 release in vitro [71] and in vivo [37]. The release of IL-6 induced by ET-1 from human vascular smooth muscle involves activation of NF-κB [71]. Possibly, release of IL-6 may further increase oxidative stress as suggested by the in vitro observation that IL-6 induces production of reactive oxygen species [79]. C-reactive protein (CRP) has emerged as a predictor and possible mediator of atherosclerotic cardiovascular disease. Verma et al. demonstrated that CRP stimulated the expression of adhesion molecules and monocyte chemoattractant protein-1 in endothelial cells [80]. Interestingly, this effect was inhibited by bosentan as well as an antibody against IL-6 suggesting involvement of ET-1 and IL-6 in the pro-inflammatory effect of CRP. Hypercholesterolemia is associated with impaired endothelium-dependent vasodilation and elevated plasma and  tissue ET-1 concentrations, which may account for the vasomotor dysfunction under this condition [81]. In support of this notion, inhibition of either ETA or both ETA and ETB receptors restores endothelium-dependent vasodilation and NO production in hypercholesterolemic pigs [82]. The normalized NO production results from increased activity of NO synthase. The effect of dual ETA/ETB blockade was significantly higher than that of selective ETA antagonism [52]. Statin therapy may further improve the beneficial effects of ET antagonism on NO-mediated vasodilation in hypercholesterolemia [83,84]. There also seems to exist important interactions between oxidized low-density lipoprotein (LDL) and ET-1 which may be of importance in atherogenesis. ET-1 augments the uptake of oxidized LDL [85], whereas oxidized and native LDL in turn stimulates the production of ET-1 [86]. Interestingly, statins have been demonstrated to decrease the expression of pre-pro ET-1 mRNA in endothelial cells [87] and the vasoconstrictor response to ET-1 in vitro [88]. In addition, ET-1 stimulates uptake of oxidized LDL in endothelial cells via an ETB receptor-mediated effect [89]. ET-1 is known to be elevated in both type 2 diabetes and by high LDL cholesterol 14 F. Böhm, J. Pernow / Cardiovascular Research 76 (2007) 8–18 [8,90]. Previous in vitro studies indicate that lipid-lowering treatment suppresses the expression of ET-1 in endothelial cells [91] thereby attenuating the negative effect of ET-1 on endothelial function. Therefore we have recently evaluated the effect of dual ET receptor blockade before and after treatment with simvastatin 80 mg od or simvastatin 10 mg plus the cholesterol absorption inhibitor ezetimibe 10mg od in patients with dysglyceamia and coronary artery disease (Settergrenet al., 2007a, manuscript in preparation; [98]). We observed a significant vasodilator effect and improvement in endothelium-dependant vasodilatation which was unaffected by aggressive cholesterol lowering, suggesting that is not through interaction with the ET system that statins exert their effect on endothelial function. On the other hand, this indicates that ET receptor blockade may exert beneficial effects on top of aggressive lipid-lowering therapy. ET-1 may also stimulate activation and accumulation of macrophages (Fig. 1). Kowala et al. [92] demonstrated that an ETA receptor antagonist inhibited monocyte infiltration and development of fatty streak in hypercholesterolemic hamsters. A dual ETA/ETB receptor antagonist reduced foam cell formation in macrophages exposed to oxidized LDL [93]. In the same study, the ET receptor antagonist significantly inhibited the development of atherosclerosis in LDL receptor knock out mice. These observations are in support of the previous observation that selective ETA receptor blockade attenuates the development of atherosclerotic lesions in apolipoprotein E knockout mice [8,38].Taken together, these data clearly suggest that ET receptor blockade exerts anti-atherogenic effects. 

Selective ETA vs. dual ETA/ETB receptor blockade
The changes in ET receptor expression in the vascular wall in pathological states described above may imply that blockade of both receptors is preferable to selective ETA receptor blockade in order to fully antagonize the effects of ET-1. The changes in receptor expression are paralleled by more pronounced functional effects of dual ETA/ETB receptor blockade in comparison with selective ETA receptor blockade in clinical studies. Thus, dual ETA/ETB receptor antagonism seems to induce more effective vasodilation than selective ETA receptor antagonism in various cardiovascular disorders (Table 1). Additional biological effects beyond direct vascular effects of potential importance during pathophysiological conditions such as superoxide production, stimulation of pro-inflammatory cytokines and LDL uptake as well as insulin resistance seem to be mediated via the ETB receptor in addition to the ETA receptor (Fig. 5). Even though most studies have not compared the two different ET receptor blocking strategies, available literature suggests that it may be preferable to block both receptors to fully antagonize the pathophysiological actions of ET-1 in cardiovascular disease. On the other hand, blockade of ETB receptors will reduce clearance of ET-1 [94] and thereby increase circulating levels of ET-1. Furthermore, the ETB receptor may exert beneficial effects by releasing NO from endothelial cells. An additional beneficial effect mediated by the ETB receptor is the stimulation of renal sodium and water excretion [95].Accordingly, it has been demonstrated that only selective ETA receptor blockade increases renal blood flowand improves renal function in patients with renal failure [96]. On the other hand, dual ETA/ETB receptor blockade, but not selective dual ETA receptor blockade, increased renal blood flow in patients with coronary artery disease and type 2 diabetes but with normal renal function [30]. These apparently conflicting results illustrate the need for carefully designed larger randomised clinical studies to clarify the potentially beneficial clinical effects of dual ETA/ETB receptor blockade over selective ETA receptor blockade in different patient  groups.Moreover, since the expression of ET receptors differs between healthy subjects and patients with cardiovascular disease as well as between various types and states of cardiovascular disease, it is of importance to characterize the response to receptor blockade in each population. 

2. Role Of COX-2 In Molecular Mechanism Of Vascular Dysfunction In Hypertension           
 Cyclooxyenase (COX)-2 selective inhibitors were developed to create a new class of nonsteroidal anti-inflammatory drugs (NSAIDs) with properties similar to those of nonselective NSAIDS but without their potential COX-1–mediated gastrointestinal toxicities.1,2 Studies of the various COX-2 selective inhibitors have shown that they are in  fact associated with a significantly lower risk of upper and lower gastrointestinal complications than traditional NSAIDs, except in patients who are taking concomitant low-doses of aspirin. Recent evidence also suggests that some doses of the COX-2 selective inhibitors, and perhaps some traditional NSAIDs as well, are associated with an increased risk of adverse cardiovascular (CV) events. Reports of a higher incidence of myocardial infarction (MI) among patients with arthritis taking high doses of the COX-2 selective inhibitor rofecoxib compared with those taking the NSAID naproxen2– 4 have had heightened concerns since 2001 regarding selective COX-2 inhibitor safety. In addition, in
early 2005, elevated CV event rates were reported in patients with spontaneous adenomatous polyps who were taking high doses of celecoxib compared with placebo5 and in patients who received parenteral parecoxib followed by oral valdecoxib versus placebo immediately after coronary artery bypass graft surgery.6 This article represents a compilation of the data concerning the effects of both nonselective and selective NSAIDs on blood pressure (BP), particularly in patients with hypertension and/or on antihypertensive agents. Subsequently, the impact that the COX inhibitors have on CV events from several recent clinical trials for the treatment of arthritis or for cancer prevention, as well as from selected large observational studies, is discussed. 

Effects of COX Inhibitors on the Gastrointestinal Tract 
The development of the selective COX-2 inhibitors was based on concerns associated with the effects of COX-1 inhibition on the upper gastrointestinal tract. The gastrointestinaladverse effects of aspirin and traditional NSAIDs are well defined and include development of gastric or duodenal ulcers, hospitalizations because of gastrointestinal bleeding complications, perforated ulcers or gastric obstruction, and  gastrointestinal-related deaths.21,22 Lower rates of these complications during the past decade have been attributed to the use of lower nonselective NSAID doses, concomitant use of proton pump inhibitors, and the introduction of COX-2 selective inhibitors, which are fundamentally COX-1–sparing drugs.23 The gastrointestinal toxicity of traditional NSAIDs is
attributable in part to nonselective inhibition of both COX-1 and COX-2 isoenzymes involved in PG synthesis.24 Data from large-scale clinical trials have confirmed that COX-2
inhibitors are associated with substantial reductions in gastrointestinal risk in the majority of patients who do not use aspirin. Clinical trials demonstrate that COX-2 inhibitors are associated with a reduction in risk of gastrointestinal adverse events, including endoscopic ulcers, equivalent to that achieved by adding proton pump inhibitor therapy to traditional NSAID therapy.25,26 Regardless of the dose of the COX-2 selective inhibitor, endoscopic findings for these agents are not significantly different from those observed for placebo.26
The VIoxx Gastrointestinal Outcomes Research (VIGOR)2 Study was the first large-scale trial to provide evidence that COX-2 selective inhibitors minimize the risk of upper gastrointestinal adverse effects in older (age _50 years) patients with rheumatoid arthritis.2 Over 9 months of follow-up, rofecoxib 50 mg once daily and naproxen 500 mg twice daily showed equivalent efficacy; however, the incidence of confirmed upper gastrointestinal adverse events per 100 patientyears in the rofecoxib group was less than half of that observed in the naproxen group. Of interest, a posthoc analysis of the trial indicated that _40% of the serious events occurred in the lower gastrointestinal tract; these events were also reduced by more than half in patients who received rofecoxib.27 It has been of concern that there is no evidence that proton pump inhibitors decrease the incidence of lower gastrointestinal tract complications in patients receiving NSAIDs. The CeLecoxib Arthritis Safety Study (CLASS) provided additional evidence that COX-2 inhibitors reduce the risk of gastrointestinal events in adults with osteoarthritis or rheumatoid arthritis.1 Patients enrolled in CLASS were randomly assigned to receive celecoxib 400 mg twice daily versus ibuprofen 800 mg thrice daily or versus diclofenac 75 mg twice daily and were permitted to take low-dose aspirin (_325 mg daily) if indicated for CV prophylaxis. During the 6-month treatment period, the annualized incidence of upper gastrointestinal complications alone and in combination with symptomatic ulcers was nearly twice as high among patients who received the nonselective NSAIDs as among those who received celecoxib. In addition to minimizing ulcers and their complications, studies typically show that the COX-2 inhibitors are better tolerated than traditional NSAIDs.28 Of importance, however, is that the subgroup of patients who were taking chronic low-dose aspirin (21% of the patients at doses of 81 to 325 mg daily) failed to show a significant  reduction in gastrointestinal complications for celecoxib relative to the nonselective NSAIDs in the CLASS trial. Similar findings have occurred with endoscopic and gastrointestinal outcome studies with the newer COX-2 inhibitors etoricoxib, lumiracoxib, and valdecoxib.29–32 

COX Inhibitors in Patients With Hypertension
Coadministration of NSAIDs or COX-2 selective inhibitors with antihypertensive agents is quite common.33 Metaanalyses of the NSAIDs from the early 1990s showed that many agents within the class (eg, ibuprofen, indomethacin, and naproxen) could increase mean arterial pressure by as much as 5 to 6 mm Hg in hypertensive patients.34,35 As reported by Grover et al,36 increases in BP by NSAIDs of this magnitude are of sufficient magnitude to be of clinical concern. Sustained BP elevations in the elderly are associated with increases in the risk of both ischemic and hemorrhagic stroke, congestive heart failure, and ischemic cardiac events.37–39 In the VALUE Study, differences of _4 mmHg in systolic BP control in an older population of hypertensive patients randomly assigned to 2 treatment groups (valsartan or amlodipine) resulted in a clinically and statistically significant relative increase in cardiac events of _40% in the less well-controlled group (valsartan recipients) during the first 6  months of the trial.39 Thus, it has become of clinical relevance to study the effects of the NSAIDs and COX-2 selective inhibitors on BP destabilization in patients with both treated and untreated hypertension.33

3. Role Of Cytokines In Molecular Mechanism Of Vascular Dysfunction In Hypertension            
ARTERIAL BLOOD PRESSURE is maintained within normal ranges by changes in cardiac output and peripheral resistance. Peripheral resistance and cardiac output are regulated by the peripheral and central nervous systems, as well as by many humoral factors. Peripheral resistance is primarily determined by the distal part of the arterial vasculature, which consists of the small resistance arteries and the arterioles (8). Disturbances in peripheral resistance are basic contributors to different cardiovascular pathologies. Therefore, alterations in the neurohumoral and mechanical systems that regulate the tone of resistance arteries may play an important role in the pathogenesis and progression of cardiovascular disease. The tone of resistance arteries depends on a complex interplay between endothelial and smooth muscle cells. Endothelial cells contribute to the regulation of vascular tone by releasing several vasoactive factors, some of which, such as nitric oxide (NO), endothelium-derived hyperpolarizing factor, and prostacyclin, are potent vasodilators; others, such as the peptide endothelin (ET)-1, prostanoids, such as thromboxane A2 (TxA2), prostaglandin H2, radical superoxide anion (O2 . ) and components of the renin-angiotensin system, are vasoconstrictors. The most studied aspect of endothelial function is the regulation of vascular tone, and the association between endothelial dysfunction and vascular disease is well established. The term endothelial dysfunction refers to an imbalance in the production or bioavailability of endothelial vasodilator mediators, and such an imbalance may promote vasospasm and thrombosis and has been implicated in many cardiovascular disorders. Inflammation can be the result of infection, trauma, ischemia, or immunologic processes. There is increasing evidence suggesting a link between infection or inflammation and the risk of cardiovascular disease (14, 37, 49). Because the inflammatory response is associated with cytokine release, cytokines may have an important role in the vascular injury induced by inflammation. Cytokines are soluble proteins or large peptides produced by leukocytes and other cell types, and they act as chemical communicators between cells. Some cytokines are involved in the effector phase of the inflammatory response and include proinflammatory cytokines, the most important being TNF-_, IL-1_, and IL-6, as well as anti-inflammatory cytokines, such as TNF-_, IL-4, IL-10, and IL-13. TNF-_ is a multifunctional circulating cytokine derived from endothelial and smooth muscle cells as well as macrophages. IL-1_ and IL-6 are cytokines with a broad range of humoral and cellular immune effects related to inflammation, host defense, and tissue injury. Cytokines usually act synergistically on the initiation of the inflammatory cascade, leading to the expression of further factors (37). Thus cytokines can induce the expression of cytokine receptors and other cytokines, thereby constituting an amplification cascade. In addition, cytokines also induce the expression of several enzymes, such as inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2), which will, in turn, produce mediators with actions at the vascular level that contribute to the inflammatory response. During the onset of sepsis, an early appearance of cytokines in the serum is well established in rodents, whereas such patterns are less evident in humans (40). The change in cytokine levels that occurs during sepsis has been characterized in studies in which the endotoxin from gram-negative bacteria, lipopolysaccharide (LPS), is administered. TNF-_, IL-1_, and IL-6 are often generated in response to LPS by a mechanism that is regulated by nuclear factor-_B (NF-_B) (37), which also reg ulates the transcription of many other mediators implicated in the inflammatory response (37). Nevertheless, as well as triggering a generalized response that involves increase of the above-mentioned cytokines, LPS increases a large number of other mediators and, in later stages of sepsis, anti-inflammatory cytokines (37, 40). A balance between the effects of the pro- and anti-inflammatory cytokines is thought to determine the outcome of this and other cardiovascular diseases, in the short or the long term. The purpose of this review is to provide an insight into the effect of cytokines, mainly TNF-_, IL-1_, and IL-6, on the vascular reactivity of resistance arteries. As pointed out by Christensen and Mulvany (8), relatively large “feed arteries” offer substantial resistance. Thus, in agreement with these authors, we will use the term resistance arteries for arteries with an internal diameter of _400 _m. First, we will describe the effects of cytokines on vascular reactivity. Next, we will focus mainly on the effects of cytokines on endotheliumdependent vasodilator responses to assess the putative role of cytokines on endothelial dysfunction. Finally, we will summarize the most important changes in resistance arterial function in some vascular pathology with elevated cytokine levels.

Cytokines induce genes that synthesize other peptides in the cytokine family and several mediators, such as prostanoids, leukotrienes, NO, bradykinin (BK), reactive oxygen species, and platelet-activating factor, all of which can affect vascular function. The vascular response appears to be related to the balance between all the vasoactive factors released under the influence of cytokines, and regional differences in release and  responsiveness to these factors appear to contribute to the dilator or constrictor response observed within a specific vascular bed. The effects of cytokines on vascular reactivity also
seem to rely on the exposure time, which, in turn, will influence mediator release. The possible mediators involved in the changed vascular reactivity in resistance arteries induced by cytokines are summarized in Fig. 1. Short-term exposure to cytokines. Cytokines could exert rapid vasoactive effects in blood vessels, likely by acting on the endothelial and smooth muscle receptors of the vessels. Receptors for TNF-_ and IL-1_ have been found on endothelial and smooth muscle cells (23, 47). Iversen et al. (23), using
distal ends of human internal mammary arteries, reported that elevated concentrations of TNF-_, IL-6, and IL-10 contracted the arteries but failed to relax norepinephrine-precontracted vessels. The observed vasoconstrictor effects were mediated by ETA receptors and were endothelium dependent. In contrast, in isolated human resistance arteries, IL-1_ and TNF-_ (alone or in combination), at a concentration similar to that found   pathophysiologically, did not exert any direct vascular effects on precontracted vessels (38).
In rat skeletal muscle arterioles, 2 min of incubation with TNF-_ had no direct vasomotor effect. However, pretreatment with endotoxin allowed the TNF-_ to cause arteriolar dilation,
possibly through a mechanism involving COX and NOS (17). In these arterioles, 1 h of incubation with IL-1_ and IL-6 produced a potent vasodilator effect in vivo, but not in vitro,
suggesting that cytokine interaction with parenchymal or intravascular factors elicits arteriolar relaxation (32). On the other hand, IL-10, an anti-inflammatory cytokine, did not
affect vascular responses to phe nylephrine or ACh, although it  did prevent the loss of vascular tone in skeletal muscle arterioles exposed to the endotoxin for 1 h (43). Bronchial vascular resistance in sheep decreased after 20 min of infusion with TNF-_ but increased and remained elevated 2 h after the start of infusion. The observed increase in bronchial vascular resistance was due to a secondary release of ET-1 (50). In agreement, exposing cultured endothelial cells to TNF-_ enhanced ET-1 secretion (10). On the other hand,  
Fig. 1. Mechanisms involved in effects of cytokines on vascular reactivity. Cytokines can induce synthesis of
endothelin (ET)-1, which induces vasoconstriction. Cytokines also induce expression of several enzymes that
release mediators that will relax [PGI2, PGE2, nitric oxide (NO)] or contract [PGH2, thromboxane A2 (TxA2)] arteries. Cytokines can also increase production of O2 . , which, in turn, will reduce bioavailability of endothelial NO and, thus, endothelium-dependent relaxation. Production of ONOO_ due to simultaneous increase of O2 . and NO from inducible NO synthase (iNOS) can decrease endothelial NO synthase (eNOS)
expression and/or activity. Stimulatory and inhibitory effects are shown as solid and dashed lines, espectively.
COX-2, cyclooxygenase-2; EC, endothelial cell; ETA, ET type A receptor; ETB, ET type B receptor; PGI2, prostacyclin; SMC; smooth muscle cell; EC, endothelial cell; XO, xanthine oxidase.

ET-1 could induce cytokine release via NF-_B activation (49). Vasoconstriction and increase in ET-1 circulating levels have also been observed in rat coronary vessels after 15 min of TNF-_ infusion (28). In human forearm resistance artery, 1 h of exposure to TNF-_ raised basal vascular resistance by increasing basal bioavailability of the vasoconstrictor prostanoids and reducing the basal bioavailability of NO (33). In rat aorta, 1 h of exposure to IL-1_ enhanced the vasoconstrictor responses to angiotensin II by a mechanism that involves prostaglandin H2/TxA2 (48). In agreement, serotonin-mediated vasoconstriction was enhanced by an increase in TxA2 production from COX-2 in rat middle cerebral arteries after 1 h of incubation with LPS (20). Long-term exposure to cytokines. Proinflammatory cytokines play an important role in the systemic inflammatory response and secondary tissue damage in patients with sepsis (37). Endotoxins, in addition to other bacterial molecules, trigger a generalized response that involves the generation of the cytokines. Furthermore, long-term exposure to TNF-_ and IL-1_ induces a hyporeactivity to vasopressor agents that seems to be related to the large decrease in systemic resistance usually found in sepsis (1). In cerebral arterioles, application of TNF-_ induced a progressive dilation, with a maximum increase in diameter at 4 h that was inhibited by aminoguanidine and dexamethasone, suggesting an important role for NO from iNOS in this vasodilation (5). Cytokines can also enhance the vasoconstrictor responses mediated by different agonists. Thus segments from human temporal artery incubated in organ culture for 48 h with TNF-_ or IL-1_ enhanced the vasoconstrictor response mediated by
ETB receptors (51). Similarly, subchronic treatment for 3 days with IL-1_ and IL-6 potentiated the ET-1- and norepinephrineinduced perfusion pressure without modifying perivascular nerve stimulation-evoked contraction in isolated rat mesenteric vascular bed. This increased contraction could be related to the impairment of endothelium-induced relaxation observed in the same study (12). In rat middle cerebral arteries, LPS increased
vasoconstriction to seroton in from the 1st to the 4th h of incubation, whereas after 5 h the contraction to serotonin returned to the control value (20). An increased production of TxA2 from COX-2, O2 . , and H2O2 seems to enhance vasoconstriction to serotonin during the first hours of LPS exposure, and this would be counteracted by the increased iNOS and superoxide dismutase expression at 5 h (20). These results are interesting, because the use of pharmacological agents to inhibit the synthesis of NO by iNOS has been proposed in
patients with septic shock. The presence of these inhibitors could unmask cytokine enhancement of the vasoconstriction induced by such important vasoactive agents as angiotensin II, ET-1, and serotonin. All these results seem to suggest that changes in vascular reactivity as a result of short-term contact of vessels with cytokines are mainly due to the effects of the cytokines; however, the participation of mediators such as ET-1 or those derived from COX-2 expression, which is rapidly upregulated when exposed to cytokines (21), should not be excluded. However, the long-term effects of cytokines on vascular reactivity do involve the release of other mediators, such as NO from iNOS and prostanoids from COX-2. 

Endothelial dysfunction and elevated levels of proinflammatory cytokines are observed in several cardiovascular diseases, such as congestive heart failure (CHF), atherosclerosis, septic shock, diabetes, and hypertension, and in aging (9, 14, 30, 37, 42, 49). Under the influence of cytokines, the endotheliumdependent dilatation can be impaired and the endothelium may lose its ability to respond to circulating hormones or autacoids. This effect may favor a predisposition to vessel spasm, thrombosis, or atherogenesis. The influence of cytokines on endothelium-dependent relaxation has been analyzed in conductance and resistance arteries from humans and animals. In healthy volunteers, a mild systemic inflammatory response impairs endotheliumdependent dilatation to ACh and BK but does not influence endothelium-independent relaxation to nitroglycerin in resistance and conduit vessels (22). Similarly, brief exposure of human forearm resistance artery to TNF-_ impairs vasodilatation to ACh (33, 39), probably through an increased basal bioavailability of vasoconstrictor prostanoids and reduced NO bioavailability (33). In isolated rat mesenteric resistance arteries, the effects of cytokines on endotheliumdependent relaxation have been studied at different exposure times. Thus, De Salvatore et al. (12) found that subchronic (3 days) in vivo treatment with IL-1_ and IL-6 impaired the reduction of perfusion pressure induced by ACh. The incubation of these arteries in organ culture for 14 h with IL-1_ almost abolished ACh-mediated relaxation, at least partly through increased O2 . production in endothelial and smooth muscle cells (24). In contrast, when the incubation time was only 30 min, IL-1_ had no effect on ACh-mediated relaxation, whereas TNF-_ impaired the NOdependent component of endothelium-dependent relaxation in response to ACh and BK without modifying responses to sodium nitroprusside (52). This effect may be attributable to
the ability of TNF-_ to increase he levels of O2 . , thereby inactivating NO (52). In fact, TNF-_ has been seen to stimulate NADPH oxidase to generate sustained amounts of O2 . in vascular smooth muscle cells (34). In agreement, in vitro LPS treatment (1–5 h) of rat middle cerebral arteries reduced the BK-induced endothelium-dependent relaxation by mechanisms that include production of NO from iNOS and release of O2 . , generated in part from COX-2 (21). Cytokines may affect endothelial function in resistance arteries through a number of signaling mechanisms. Thus TNF-_ is able to impair the stability of endothelial NO synthase mRNA (14). In addition, cytokines and LPS may also induce iNOS expression in vascular smooth muscle and endothelial cells (6, 20), and this would account for an excessive basal NO increase that might participate in the impairment of endothelium-
dependent relaxation observed in the presence of LPS and cytokines. The nitration of protein tyrosine residues by peroxynitrite, due to the simultaneous generation of NO from iNOS and O2 . , could inhibit the enzymes involved in endothelium- dependent relaxation. It has been reported that a high concentration of NO, such as would be produced after iNOS, could downregulate endothelial NO synthase and soluble guanylate cyclase activity (7, 35).

4. Role Of Kallikrein-kinin In Molecular Mechanism Of Vascular Dysfunction In Hypertension           

THE MECHANISM FOR ACTIVATION of the plasma kallikreinkinin system (KKS) has been elusive. Although it is well known that the plasma KKS becomes activated when exposed to a negatively charged surface, hence its name the “contact system,” a comprehensive physiological, negatively charged surface has never been discovered. We observe that when the proteins of the plasma KKS assemble on endothelial cells or their matrix on a multiprotein receptor complex, the zymogen plasma prekallikrein (PK) becomes activated to
plasma kallikrein (81, 90, 91). Our efforts to identify an endothelial cell-associated plasma PK activator recognized that the enzyme prolylcarboxypeptidase (lysosomal  carboxypeptidase, angiotensinase C, PRCP, PCP) has this property (88, 118). Inasmuch as PRCP had only been previously proposed as a degrading enzyme of ANG II, the recognition that it also functions as a PK activator indicates a new interaction between KKS and the renin-angiotensin system (RAS) (99, 116). This interaction, along with the many other communications between these two systems, has led us to formulate a new hypothesis for the physiological activity of the plasma KKS. The plasma KKS serves as the physiological counterbalance to the RAS (116). The purpose of this review is to describe the intimacy and profundity of the interaction between these two systems. These places of interaction serve as foci to examine this hypothesis in the future in both in vitro and in vivo models. 

Figure 1 presents a schema on the interactions between the KKS and the RAS. The assembly of high molecular weight kininogen (HK) and PK on endothelial cells results in PRCP activation of PK to plasma kallikrein (118, 119). Plasma kallikrein has several substrates in these systems. It autodigests HK or, at lower affinity, low molecular weight kininogen (LK), to liberate bradykinin (BK). The residual, cleaved HK (HKa) participates in its many activities such as cysteine protease inhibition, anti-angiogenesis, and antiproliferation of cell growth (23, 63, 51). Alternatively, BK, HK, LK, and tissue kallikrein are proangiogenic (Table 1) (24, 39). Not shown on Fig. 1, plasma kallikrein also converts factor XII to its activated forms and favorably activates single-chain urokinase activation to two-chain urokinase (90, 107). Plasma and tissue kallikreins have also been recognized to be one of the activators of prorenin to renin, an older observation whose physiological significance is questioned (Fig. 1). Renin, an aspartyl protease, activates angio- 

Fig. 1. Interaction of the plasma kallikrein/kinin system (KKS) with the renin-angiotensin system (RAS). HK, high molecular weight kininogen; PK, prekallikrein; PRCP, prolylcarboxypeptidase; HKa, plasma kallikrein-cleaved, high molecular weight kininogen free of bradykinin; ACE, ANG I converting enzyme; ACE2, angiotensin converting enzyme 2; tPA, tissue plasminogen activator; PAI-1, plasminogen activator inhibitor 1; PGI2, prostaglandin I2 or prostacylin. tensinogen to ANG I. Angiotensin converting enzyme (ACE) has the bifunctional activity of being one of the degrading peptidases (kininase II) of BK and converting the inactive 10-amino acid ANG I to the biologically active 8-amino acid peptide ANG II [ANG-(1–8)]. ACE is another regulatory juncture point between these two systems (Fig. 1). Liberated BK stimulates vasodilation, nitric oxide (NO) formation, tissue plasminogen activator (tPA) liberation, prostacyclin formation, and superoxide formation (61, 62, 102, 126). BK also results in lowering of blood pressure. BK and its ACE breakdown product BK-(1–5) inhibit thrombin-induced platelet activation (55). ANG II counterbalances some of the activities of BK. Although ANG II can stimulate superoxide and NO formation like BK (30), it induces local vasoconstriction and contributes to elevation of  blood pressure. ANG II also directly stimulates tissue factor production and plasminogen activator inhibitor 1 release (97, 138). PRCP degrades ANG II to form angiotensin-(1–7) along with ACE2 and, possibly, neutral endopeptidase 24.11 (45, 139). Previously angiotensin-(1–7) was believed to be an inactive breakdown product of ANG II, but it too has been recognized to have biologic activities that result in vasodilation and blood pressure lowering (134). Last, stimulation of the BK B2 receptor (BKB2R) and ANG II receptor result in vasodilation and NO and prostacyclin formation, whereas stimulation of the ANG I receptor results in vasoconstriction and blood pressure elevation (Fig. 1). Thus there appears to be many interaction points and a number of counterbalancing influences of each of these systems on the other in health and inflammatory diseases (10). It is the intent of this review to examine each of these  interactions in more detail and ascertain their relative importance as determined by in vitro and in vivo studies.In particular, the role of ACE in activating ANG I and inactivating BK will be discussed. The contribution of plasma kallikrein to prorenin activation will be reexamined in light of a physiological mechanism for PK activation. The summating biologic effects of BK and angiotensin-(1–7) will be studied. The role of PRCP in ANG II degradation and plasma PK activation will be presented. The modifying influence of angiotensin receptors 1 and 2 on these systems also will be reviewed. Finally the counterbalancing effect of each of these systems on thrombosis, fibrinolysis, and angiogenesis will be introduced. 

The recognition that angiotensin-(1–7) has biologic activity embellishes the knowledge of  

Fig. 2. Degradation pathways for ANG I and bradykinins (BKs) by the angiotensin converting enzymes. desArg9BK, des-Arg9-BK; ATII, ANG II; BK 1–7, BK-(1–7); BK 1–5, BK-(1–5); BKB2R, BK B2 receptor; BKB1R, BK B1 receptor; AT1R, ANG II receptor 1; AT2R, ANG II receptor 2; Ang(1–7)R ?, hypothesized angiotensin-(1–7) receptor. It is important to appreciate that ANG II can stimulate both the angiotensin 1 and 2 receptors. Likewise, angiotensin-(1–7) can stimulate both angiotensin receptors and may have its own receptor, Ang(1–7)R, that has yet to be physically identified.
the interaction between the RAS with the KKS and itself (109, 134). Angiotensin-(1–7) is produced by ACE2 (Km _ 2 _M) or prolylcarboxypeptidase (Km _ 200 _M) degradation of  ANG II (99, 139) (Fig. 3). Neprilysin (endopeptidase24.11) and thimet oligopeptidase (endopeptidase 24.15) also can produce angiotensin-(1–7) from the breakdown of ANG I (45, 47, 51) (Fig. 3). All of these enzymes are directly or indirectly involved in BK metabolism. 

Fig. 3. Detailed ANG I degradation pathways. EP24.11, endopeptidase 24.11, neprilysin; EP24.15, endopeptidase 24.15; thimet oligopeptidase. ANG II (99, 139) (Fig. 3). Neprilysin (endopeptidase 24.11) and thimet oligopeptidase (endopeptidase 24.15) also can produce angiotensin-(1–7) from the breakdown of ANG I (45, 47, 51) (Fig. 3). All of these enzymes are directly or indirectly involved in BK metabolism.

There may be other angiotensinases as well, because CE2 is only found in the heart, kidney, and testis, and angiotensin-(1–7) is found ubiquitously throughout the vasculature. Once formed, angiotensin-(1–7) exerts its effects by binding to the angiotensin receptor 1 in some cases to antagonize ANG II and, in other cases, to the angiotensin receptor 2 (60, 110) (Fig. 2). Angiotensin-(1–7) may also have its own receptor (47, 67, 75) (Fig. 2). Angiotensin-(1–7) also is degraded by ACE (Fig. 3). The interactions between angiotensin-(1–7) and the KKS have been best studied in the kidney (111). In essence, there are two kinds of interactions between BK and angiotensin-(1–7): potentiation of BK by angiotensin-(1–7) and mediation of the vascular activity of angiotensin-(1–7) by BK (111). Angiotensin-(1–7) potentiates the hypotensive effect and vasodilation action of BK in the normotensive or hypertensive rat and in rat mesenteric vessels, respectively. Evidence that angiotensin-(1–7) action is mediated by BK is provided by the observation that HOE140 blocks some angiotensin-(1–7)-mediated activity (111). Angiotensin-(1–7) by stimulating the angiotensin 2 receptor may stimulate BK release (136) Angiotensin-(1–7) has been described as the most pleotropic metabolite of ANG I, manifesting actions often the opposite of those described for ANG II (47). It dilates canine coronary arteries through kinins and NO (11). Angiotensin-(1–7) augments BK by locally acting as a synergistic modulator of kinin-induced vasodilation by inhibiting ACE and releasing NO (103). These investigations in isolated aortic rings were confirmed by animal studies in rats. Angiotensin-(1–7) decreases blood pressure in the rat, and this effect is mediated by the BKB2R and is unaffected by angiotensin  receptor 1 and 2 antagonists (1). Angiotensin-(1–7) is both a substrate and inhibitor of ACE (29). It potentiates arachidonic acid release by an ACE-resistant BK analog acting on BKB2Rs (29). Vasodilation and NO formation induced by angiotensin-(1–7) result from indirect potentiation of BK as an agonist of the BKB2R (29). Angiotensin-(1–7) along with angiotensin-(1–9) also may potentiate the effects of BK by inducing cross talk between ACE and the BKB2R (84). Because ACE inhibitors block desensitization of the BKB2R, angiotensin-(1–7) functions as an ACE inhibitor blocking the ACE COOH domain (29, 132). In doing so, angiotensin-(1–7) acts synergistically with NH2 domain-specific ACE inhibitors (29, 132). Both angiotensin-(1–9) and angiotensin-(1–7) potentiate BK’s action on the BKB2R to elevate arachidonic acid and NO release to occur at lower concentrations (0.01–0.1 _M) than the IC50 (1.2 _M) for ACE inhibition (29, 68). This finding indicates that angiotensin-(1–7) potentates BK by another mechanism independent of ACE inhibition. ACE inhibition results in reduced ANG II vasoconstriction and increased angiotensin-(1–7) in plasma and tissue, resulting in vasodilation (108). In human internal mammary arteries, contractions induced by ANG I and II and a non-ACE-specific substrate, Pro11,D-Ala12- ANG I, are antagonized by angiotensin-(1–7) (108). Topical application of BK or angiotensin-(1–7) induces vasodilation in exposed rat mesenteric vessels, and this phenomenon is abolished by the BKB2R antagonist HOE140 or the angiotensin-(1–7) antagonist A-779 (100). This result suggests that each of these biologically active peptides is mediating this activity through its own receptor system. This assessment is especially important for angiotensin-(1–7), because its own receptor has yet to be identified. The potentiation of BK-induced vasodilation by angiotensin-(1–7) is a receptor-mediated phenomenon that is dependent on  cyclooxygenase-related products and NO release (100). Angiotensin-(1–7) significantly increases formation of cGMP and NG-nitro-L-arginine methyl ester (L-NAME),  the NO synthase inhibitor, and a selective soluble guanylate cyclase inhibitor blocks the angiotensin-(1–7)-induced relaxations in canine middle cerebral arteries (48). Finally, angiotensin-(1–7) causes afferent rabbit arteriole dilatation and this effect is mediated by NO and not cyclooxygenase products, suggesting a role for kinins (106). Thus angiotensin-(1–7) influences BK by inhibiting ACE, stimulating the BKB2R, and possibly  stimulating its own receptor that may cross talk with the BK receptors (see below). However, there are some data in animals and humans suggesting that the KKS does not counterbalance  the RAS. Widdop et al. (141) found that angiotensin-(1–7) failed to enhance the hypotensive effects of BK in the spontaneously hypertensive (SHR) and Wister-Kyoto rats. Furthermore, angiotensin-(1–7) infusion for 7 days has a variable effect of blood pressure in SHR (141). Angiotensin-(1–7) infusion in the forearm of patients with heart failure treated with an ACE inhibitor did not lower blood pressure nor potentiate the  vasodilating effects of BK (28). This latter study should not be considered definitive because the model is in a limited population of patients heavily pretreated with medication. However, these animal and human studies question the importance of angiotensin-(1–7) as a clinically significant vasodilator. More animal and human models are needed to clarify the physiological role of angiotensin-(1–7).

Although the RAS and KKS are recognized as important modulators of vascular biology, blood pressure regulation, and vascular inflammation (10), they have also been examined for their influence on thrombosis, fibrinolysis, and angiogenesis. Therapeutic manipulations
of the RAS and KKS appear to result in risk alteration for arterial thrombosis. Treatment with ACE inhibitors or AT1 receptor antagonists results in an _15–20% decrease in risk for myocardial infarction and stroke (27, 130). Furthermore, ACE inhibitor treatment before thrombolytic therapy reduces an early increase in plasma plasminogen activator inhibitor 1 (PAI-1) levels in acute myocardial infarction (140). The experimental basis for these clinical results will be reviewed. 

5. Role of reactive oxygen species in Molecular Mechanism Of Vascular Dysfunction In Hypertension           
The endothelium plays a critical part in the regulation of vascular function through elaboration of paracrine factors that maintain vascular tone, inhibit platelet and inflammatory cell adhesion, promote fibrinolysis, and limit vascular proliferation.1 Endothelial dysfunction refers to a pathophysiological disease state in which homoeostatic functions of endothelial cells are perturbed promoting vasospasm, thrombosis, intimal growth, inflammation, and plaque rupture leading to tissue ischaemia, atherothrombosis, and infarction.2 Impaired endothelial function is associated with atherothrombotic risk factors and atherothrombotic disease, is pathophysiologically linked to acute cardiovascular syndromes, and provides prognostic information with regard to increased cardiovascular risk.3 4.
              A central feature of impaired endothelial function in the presence of cardiac risk factors and under pathological conditions is impairment in endothelium-derived nitric oxide (EDNO) bioactivity.5 Nitric oxide is produced in endothelial cells from the conversion of L-arginine to L-citrulline through the tightly regulated activity of (endothelial) nitric oxide synthase. EDNO regulates vascular tone through a dilator action on vascular smooth muscle cells that depends on soluble guanylyl cyclase activation and consequent increase in intracellular cyclic 3’5’- guanosine monophosphate.1 Additional antiatherogenic functions of EDNO relate to inhibition of platelet activity, leucocyte adhesion, and vascular smooth muscle cell proliferation. Reduced nitric oxide synthesis or inactivation appears to be a common functional disturbance in the presence of cardiac risk factors and atherothrombosis.6 Other abnormalities in endothelial function relate, in part, to increased expression of adhesion molecules supporting inflammatory cell recruitment to the vessel wall; enhanced release of constrictor agents such as angiotensin-II that promote vascular growth and alter vascular tone; and loss of antithrombotic function through reduced production of prostacyclin and fibrinolytic factors.
              Mechanisms underlying impaired endothelial function in various disease states such as hypertension, diabetes mellitus, hypercholesterolaemia, and atherosclerosis are likely multifactorial. There is growing evidence that oxidative stress (defined as an imbalance between endogenous oxidants and antioxidants in favour of the former) contributes to mechanisms of vascular dysfunction.7 These observations fit well with the recognition that increased oxidative stress may be central to the atherogenic process.8 In this review,  we will discuss the role of oxidative stress in endothelial dysfunction and its contribution to vascular disease, and discuss potential therapeutic antioxidant strategies.

Mammalian cells produce energy by reducing molecular oxygen to water during aerobic respiration. During this process, intermediates referred to as reactive oxygen species are generated that include superoxide anion, hydroxyl radicals, and hydrogen peroxide (fig 1). Under homoeostatic conditions, these molecules likely play a regulatory part in cellular function, and antioxidant defences modulate their steady state balance. Owing to their highly biologically reactive properties, reactive oxygen species have the potential to interact with proteins, lipids, and DNA, and their excessive production has been implicated in the pathogenesis of various disease states including aging, reperfusion injury, dementia, and atherosclerosis.7
            A dominant mechanism of impaired vascular nitric oxide bioavailability relates to its oxidative inactivation by superoxide. Superoxide anion rapidly reacts with nitric oxide and eliminates its biological activity.9 There is considerable evidence that vascular production of superoxide is increased in hypercholesterolaemia, diabetes mellitus, hypertension, and cigarette use.10 11 Arterial tissue isolated from rabbits fed a hypercholesterolaemic diet releases increased amounts of superoxide anion that is associated with impaired EDNO dependent dilation.10 In patients, the finding that an infusion of recombinant human superoxide dismutase (SOD)  mproves acetylcholine-mediated coronary dilation further  supports the importance of increased superoxide anionproduction as a mechanism of endothelial dysfunction.12 Inhibiting endogenous copper-zinc SOD in normal vascular tissue decreases nitric oxide action.13
Figure 1 Generation of reactive oxygen species. Molecular oxygen (O2 ) reacts with an impaired electron (e− ) to form the superoxide anion (O2.-). Superoxide is converted to hydrogen peroxide (H2O2) by the enzyme superoxide dismutase. Hydrogen peroxide undergoes spontaneous conversion to the highly reactive hydroxyl radical (.OH). Alternatively, it can be detoxified via either glutathione peroxidase or catalase to water (H2O) and oxygen (GSH, reduced glutathione; GSSG, oxidised glutathione).

               In addition to abrogating the antiatherogenic effects of nitric oxide, the combination of superoxide anion with nitric oxide generates peroxynitrite, a highly reactive intermediate that fuels lipid peroxidation, generation of reactive aldehydes and nitrogen oxides, and protein nitration supporting proatherogenic modification of low density lipoprotein (LDL). The “oxidative modification hypothesis of atherosclerosis” refers to the central role of oxidised LDL (ox-LDL) in the atherosclerotic process and provides the basis for a mechanistic link between hypercholesterolaemia and vascular disease.14 This hypothesis proposes that LDL initially localises in the vascular subendothelial space and is subsequently oxidatively modified by resident vascular cells. Although mechanisms of LDL oxidation in vivo are incompletely understood, endothelial cells, vascular smooth muscle cells, and monocytes are collectively able to oxidise LDL. Macrophages within the vessel wall internalise ox-LDL via scavenger receptors, and develop into lipid-rich “foam cells”. In contrast to regulated uptake of native (unoxidised) LDL by apo B/E receptors, the incorporation of ox-LDL into foam cells through scavenger receptor pathways is not subject to negative feedback regulation. Thus, progressive lipid accumulation within lipid laden macrophages occurs in an unchecked manner, and is believed to represent a dominant mechanism of subintimal fatty streak evolution that characterises the earliest manifestations of atherosclerosis.15 Evidence that LDL oxidation occurs in vivo is supported by studies demonstrating that antibodies against ox-LDL react with atherosclerotic lesions but not normal arteries, and their titres correlate with extent of atherosclerosis.14 In addition to fuelling lipid accumulation in foam cells, ox-LDL contributes to vascular dysfunction and atherosclerotic plaque formation by additional mechanisms. Ox-LDL stimulates expression of proinflammatory signals including monocyte chemotactic protein-1 and intercellular adhesion molecule-1 that facilitate monocyte recruitment and adhesion to the vessel wall.16 Further, ox-LDL directly inactivates nitric oxide, is cytotoxic to endothelial cells,17 stimulates vascular smoothmuscle cell proliferation, and upregulates tissue factor and plasminogen activator inhibitor-1 expression that have the potential to support atherothrombosis.18 
                   In addition to LDL oxidation, reaction of reactive oxygen species with cell membrane bound fatty acids can promote a vicious cycle of continued oxidative damage, resulting in alterations in cell membrane permeability and functional impairment in cellular transport and signalling. For example, superoxide anion may combine with transition metal ions to form hydroxyl radical and hydrogen peroxide, which are also relevant to the molecular underpinnings of cellular dysfunction.19 


                    A variety of enzymatic and non-enzymatic sources of reactive oxygen species exist in blood vessels.20 The primary biochemical source of reactive oxygen species in the vasculature, particularly of superoxide, appears to be the membraneassociated  nicotinamide dinucleotide (phosphate) (NADH/NAD(P)H) oxidase enzyme complex.21 This system catalyses the reduction of molecular oxygen using NAD(P)H as an electron donor, generating superoxide. The function of this enzyme complex is most easily understood in the context of the activated neutrophil, wherein it generates large amounts of toxic superoxide anion and other reactive oxygen derivatives important in bactericidal function. 

Figure 2 Balance of oxidative stress and nitric oxide action on vascular function (H2O2, hydrogen peroxide; NAD(P)H, nicotinamide dinucleotide (phosphate); PDGF, platelet derived growth factor; O2 .-superoxide anion; .OH, hydroxyl radical; TNF-α, tumour necrosis factor-α).

                 NADH/NAD(P)H oxidases are also functional in membranes of vascular endothelial and smooth muscle cells, and fibroblasts providing a constitutive source of superoxide anion. Various cytokines and hormones relevant to the pathogenesis of vascular disease and reduced nitric oxide bioavailability (fig 2) including angiotensin II, thrombin, tumour necrosis factor-α, and platelet derived growth factor upregulate vascular NADH/NAD(P)H oxidase activity and superoxide production.21 NADH/NAD(P)H oxidase activity plays an important part in  angiotensin II-mediated hypertension. Administration of
angiotensin II to rats raises blood pressure and increases vascular superoxide production, and this effect is dependent on activation of membrane-associated oxidases.11 Impaired arterial relaxation to acetylcholine and increased production of superoxide anion are also features of angiotensin II-induced, but not catecholamine-induced, hypertension.22 The increase in superoxide production and impairment in vessel relaxation during angiotensin II infusion is prevented by concurrent Figure 1 Generation of reactive oxygen species. Molecular oxygen (O2 ) reacts with an impaired electron (e− ) to form the superoxide anion (O2 .-). Superoxide is converted to hydrogen peroxide (H2O2) by the enzyme superoxide dismutase. Hydrogen peroxide undergoes spontaneous conversion to the highly reactive hydroxyl radical (.OH). Alternatively, it can be detoxified via either glutathione peroxidase or catalase to water (H2O) and oxygen (GSH, reduced glutathione; GSSG, oxidised glutathione). Figure 2 Balance of oxidative stress and nitric oxide action on vascular function (H2O2, hydrogen peroxide; NAD(P)H, nicotinamide dinucleotide (phosphate); PDGF, platelet derived growth factor; O2, superoxide anion; .OH, hydroxyl radical; TNF-α, tumour necrosis factor-α). administration of losartan, suggesting that activation of this  oxidase system occurs by an angiotensin II receptor dependent mechanism. Increased NADH/NAD(P)H oxidase activity may also be important in other cardiovascular diseases. Superoxide is increased in aortic tissue of cholesterol fed rabbits and in blood vessels of patients with coronary artery disease, hypercholesterolaemia, and diabetes mellitus.23 There is increased expression of angiotensin converting enzyme (ACE) in atherosclerotic plaques that serve as a source of local angiotensin II production. Shoulder regions of coronary lesions are characterised by more abundant NADH/NAD(P)H oxidase dependent superoxide activity that may be relevant to plaque inflammation and propensity for rupture.24 The link between angiotensin II, ACE activity, and superoxide anion production underscores the general importance of the renin-angiotensin system in cardiovascular disease. 
                 Another source of vascular superoxide is the xanthine oxidoreductase enzyme system that catalyses the oxidation of hypoxanthine to xanthine during purine metabolism.20 Early stages of atherosclerosis are associated with increased superoxide anion production by endothelial cells, and inhibition of xanthine oxidase activity with oxypurinol improves impaired vasodilation in hypercholesterolaemic patients.25 A third potential source of vascular reactive oxygen species production is endothelial nitric oxide synthase (eNOS). eNOS is a cytochrome P450 reductase-like enzyme that requires cofactors  including tetrahydrobiopterin, flavin nucleotides, and NAD(P)H for transfer of electrons to a guanidino nitrogen of L-arginine to form nitric oxide. L-arginine and tetrahydrobiopterin deficiency are associated with uncoupling of the L-arginine-nitric oxide pathway resulting in decreased formation of nitric oxide, and increased eNOS-mediated generation of superoxide (and peroxynitrite). Tetrahydrobiopterin repletion improves endothelial function in chronic smokers,26 and augments nitric oxide bioactivity in hypercholesterolaemic humans.27 Additional intracellular sources of reactive oxygen species include mitochondrial respiration, cyclo-oxygenases, lipoxygenases, and cytochrome P450 mono-oxygenase, but the relative contribution and clinical relevance of these enzymatic sources remain incompletely understood. 
                    The biological activity of reactive oxygen species depends upon their relative balance in relation to intracellular antioxidant defences. For example, SOD catalyses the metabolism of superoxide to hydrogen peroxide. Hydrogen peroxide may combine with transition metal ions to generate hydroxyl radical intermediates, or be detoxified to water by glutathione peroxidase or catalase (fig 1). Other intracellular antioxidants such as ascorbic acid, α-tocopherol, and glucose-6-phosphate dehydrogenase also play an important part in the regulation of intracellular redox status. Ascorbic acid supplementation improves EDNO-dependent dilation in patients with coronary artery disease and diabetes mellitus.28 29 A balance of nitric oxide-mediated versus oxidant-mediated signals may determine the ambient phenotypic behaviour of endothelial cells. In disease states, increased production or activity of reactive oxygen species overwhelms endogenous antioxidant protection, tipping the scale towards atherothrombogenesis (fig 2).
There is growing evidence to suggest that reactive oxygen species, particularly superoxide and hydrogen peroxide, participate  in vascular cell signalling and proatherogenic geneexpression by modulation of redox-sensitive transcription and transduction pathways.30 In the setting of increased oxidative stress, endothelial cells lose their protective phenotype, and express proinflammatory molecules. These molecules include vascular cell adhesion molecule-1, intercellular adhesion molecule-1, and monocyte chemotactic protein-1, all of which facilitate endothelial-leucocyte interactions and initiate early stages of atherosclerosis. Expression of inflammatory signals is, in part, controlled by a redox-sensitive transcriptional regulatory protein nuclear factor kappa B (NF-κB).31 NF-κB is also important in proliferative signals involved in vascular smooth muscle cell growth, vascular remodelling, and atherogenesis. Cultured cells overexpressing catalase exhibit suppressed activation of NF-κB in response to tumour necrosis factor-α,30 while those overexpressing SOD exhibit intracellular accumulation of hydrogen peroxide and NF-κB activation. Further evidence for the involvement of reactive oxygen species in NF-κB activity is provided by studies demonstrating its inhibition by antioxidants such as N-acetylcysteine and pyrrolidine dithiocarbamate. Oxidant species also play a regulatory part in other aspects of intracellular signalling. Mitogen activated protein kinase (MAPK) and tyrosine kinases consist of key regulatory proteins that control cellular response to growth and stress signals.19 30 In vascular cells, growth factors and angiotensin II are powerful activators of extracellular signal-regulated kinase and p38 MAPK that stimulate smooth muscle cell proliferation and fibroblast migration through mechanisms that involve hydrogen peroxide. These proliferative responses drive neointimal growth and likely play a part in atheroma development and restenosis. Stimulation of vascular smooth muscle cells by the mitogen platelet derived growth factor increases intracellular production of hydrogen peroxide and tyrosine phosphorylation.32 This process is abrogated by enhancing intracellular concentrations of free radical-scavenging enzymes such as catalase, and by the antioxidant N-acetylcysteine. Reactive oxygen species modulate both Akt kinase and caspase activity, which play a part in endothelial cell proliferation and activation of apoptotic signals leading to endothelial cell loss, respectively.33 Reactive oxygen species also modulate collagen matrix metabolism through activation of proteolytic matrix metalloproteinases that play an important part in plaque behaviour and stability.34 Matrix metalloproteinase expression is increased in shoulder regions of atherosclerotic plaques where its increased activity may increase the propensity for plaque rupture.35 Atherectomy specimens from patients with unstable coronary syndromes exhibit increased expression of reactive oxygen species compared with individuals with stable angina, supporting a mechanistic role of reactive oxygen species in plaque composition and behaviour.36 N-acetylcysteine prevents matrix metalloproteinases-9 expression and activation in hypercholesterolaemic rabbits, implicating a potential role for antioxidant treatment in modulating plaque stability.37  

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