SYSTEMIC HYPERTENSION, ATHEROSCLEROSIS AND CORONARY ARTERY DISEASE Summary Risk factors The onset of essential hypertension (HT) is mostly beyond the third decade of life and the pathogenesis is multi-factorial. Factors that are or may be involved in the pathogenesis of HT are the following: Inflammation and obesity are leading risk factors for the development of essential HT. In obesity, many factors act together to promote vasoconstriction and sodium retention, including insulin resistance, salt sensitivity, activation of the sympathetic nervous system and inflammation. All together, these factors will lead to sustained hypertension. Genetic predisposition and renin-angiotensin system hyperactivity play also an important role. Excessive use of alcohol, cigarette smoking, polycythemia and nonsteroidal anti-inflammatory agents are exacerbating factors.

The inflammatory response is essential in the response to pathogens. It initiates metabolic changes to provide nutrients for the immune system, from host tissues. These changes include hyperlipidemia and increased gluconeogenesis. Insulin resistance and deregulation of lipid metabolism occur in obesity, type 2 diabetes and atherosclerosis. There is a strong association between indices of inflammation and abnormal lipid and carbohydrate metabolism, obesity and atherosclerosis.

Immunopathogenic mechanisms are involved in the pathogenesis of hypertensive disease, atherosclerosis and coronary artery disease. Alterations in both cellular and humoral immunity, changes in serum immunoglobulin levels, inherited abnormalities of the complement system and altered profile of pro- and anti-inflammatory cytokines have been identified in patients with essential HT. Atherosclerosis Atherosclerosis is a major complication of HT. Acute coronary syndromes are responsible for most of the morbidity caused by coronary atherosclerosis. Atherosclerosis is a progressive disease characterized by the accumulation of lipids and fibrous elements in the large arteries. Because high plasma concentrations of cholesterol, in particular LDLcholesterol, are one of the principal risk factors of atherosclerosis, the process of atherogenesis has been considered to consist largely of the accumulation of lipids within the artery wall. The insights of the scientific community in the pathophysiology of this important disease have evolved substantially over the past century. Atherosclerosis is now being increasingly recognised as an inflammatory disease. The cellular interactions in atherogenesis are fundamentally not different from those in chronic inflammatory/autoimmune-fibroproliferative diseases such as cirrhosis, rheumatoid arthritis, glomerulosclerosis, pulmonary fibrosis and chronic pancreatitis. Vascular inflammation produces atherosclerosis by the continuous recruitment of monocytes into the vessel wall and by contributing to an oxidant-rich inflammatory milieu that induces phenotypic changes in resident endothelial and smooth muscle cells. If the causative agent or agents are not removed or eliminated by the inflammatory response and the inflammation progresses, the response changes from a protective to an injurious response. Such constant or repetitive injury can stimulate each tissue to repair or wall off the damage by a fibroproliferative response, which, when excessive, diminishes the functional capacity of the tissue or organ and becomes part of the disease process. Pathogenesis: role of inflammation Functional abnormalities of the endothelium predate the development of anatomical changes of atherosclerosis. The first step in atherosclerosis is endothelial dysfunction. Possible causes of endothelial dysfunction leading to atherosclerosis include: Elevated and modified LDL, free radicals (oxidative stress) caused by cigarette smoking, hyperlipidemia, diabetes, insulin resistance, hypertension, obesity with a chronic and systemic pro-inflammatory stimulus due to the increased production of cytokines by the adipose tissue Genetic alterations Elevated plasma homocysteine concentrations. Infectious agents contribute directly through infection of the cells of the vessel wall and indirectly through long-distance effects from remote infection to the progression or exacerbation of atherogenesis. Chronic inflammatory disease: CRP is one of the most powerful and independent risk factor for coronary heart disease, and not only a non-specific but sensitive marker of the acute inflammatory response. Combinations of the above mentioned factors.

These potential contributors to endothelial injury initiate a compensatory response resulting in an inflammatory cascade that leads to activation of monocytes and lymphocytes in the arterial wall, contributing to smooth muscle cell proliferation and thickening of the arterial wall. Lymphocyte recruitment and expression of proinflammatory cytokines characterize early atherogenesis. Inflammatory pathways promote thrombosis, a late and dreaded complication of atherosclerosis responsible for myocardial infections and most strokes. If the inflammatory response does not effectively neutralize or remove offending agents, it can continue indefinitely. The immunologic responses that lead to the induction and progression of atherosclerosis are complex, and additional insight is required to clearly understand the relationship between different risk factors and immune components of atherogenesis in order to develop immunotherapeutic approaches. Although our knowledge on the intimate mechanisms of atherosclerotic disease are still incomplete, the control of the inflammatory response should be considered as a major therapeutic goal in combination with already established therapy options or preventive approaches. Therapeutic approaches and prevention Weight loss Since obesity is one of the most important risk factors for hypertension and cardiovascular diseases, weight loss is the primordial goal that needs to be achieved for the prevention and therapy of hypertension. Obesity is mediated through an increased secretion of pro-inflammatory cytokines by the adipose tissue. The pro-inflammatory response induces systemic vascular inflammation and insulin resistance. In turn, endothelial dysfunction generates a pro-inflammatory response, which potentiates the deleterious effect of cytokines on adipose tissue and lipid and carbohydrate metabolism. Thus, a vicious circle of inflammation, obesity-hypertension-atherosclerosis can be maintained in the artery by the presence of excess of adipose tissue. Reduction of body weight is associated with a reduction in markers of vascular inflammation and insulin resistance (Esposito et al., 2003). Although the cause-effect relationship of obesity and inflammation is not clearly established, the effects of hypocaloric diet and increased physical activity may be jeopardized by the vicious circle of inflammation. Therefore it should be recommended to eliminate or avoid any inflammatory stimulus, which may potentially interfere with glucose and lipid homeostasis. Since intestinal permeability is perturbed through the pro-inflammatory response related to obesity, multiple food hypersensitivities may occur as a consequence of abolition of oral tolerance. Preliminary results obtained by statistical evaluation of 200 patients records showed a beneficial effect of selective exclusion diet, based on IgG antibody reactivity against a panel of 264 food antigens, on obesity. The average weight loss in men was 8, and in women 6 kg respectively. The weight loss was sustained (>3 months) in patients who showed a high compliance and was accompanied by a reduction in blood pressure (BP) in a sub-collective of patients from whom data on BP were available. The advantage of the selective exclusion diet over alternative regimens is that there is no caloric restriction and it is adapted to the individual immunological profile. In consequence, the compliance is high, however patients need an extensive dietary counseling for the implementation of their individual diet. The high compliance may be also related to the reduction of the effects of chronic inflammatory response on insulin (glucose intolerance) and leptin resistance (abolition of satiety and weight-reducing action).

Inhibition of the inflammatory response The inhibition or reduction of the chronic (pro-) inflammatory response seems to be a key element in the prevention and therapy of atherosclerosis. The aim should not only be a local effect on the cytokine production and inflammatory response in atherosclerotic plaques but generalized in order to reduce the stimuli implicated in the early atherogenesis and finally in the plaque disruption which may be ultimately associated with myocardial infarction and death. Besides the direct effect on the inflammatory response involved in the different steps of atherogenesis from the beginning (endothelial dysfunction) to the end (plaque rupture), the inhibition of inflammation eliminates or reduces the impact of other independent risk factors for atherosclerosis (hypertension, diabetes, obesity and other chronic inflammatory diseases). In all these clinical conditions, there is a (pro)-inflammatory response with over-expression of cytokines, which in turn, activate the local vascular inflammation, thus creating a vicious circle. Inflammation an food hypersensitivity There is now growing evidence that chronic inflammation and food hypersensitivity may be closely related. Exclusion of nutrients, which elicit an IgG antibody response, from the alimentation has been proven beneficial in chronic inflammatory diseases. The prototype clinical entity related to food intolerance is celiac disease. Gluten-free diet prevents the progression of the disease. Exclusion diet has also been proven to ameliorate clinical symptoms in rheumatoid arthritis. According to the very promising results observed in a preliminary evaluation of patients who underwent selective food exclusion diet for different chronic diseases related to inflammation, we hypothesize that selective exclusion diet may prevent the progression of atherosclerosis through a direct local action and by eliminating or reducing systemic inflammation triggers, i. e. obesity, insulin resistance, and hypertension, which are risk factors for atherosclerosis. Exclusion diet based on the results of IgG antibody reactions against food antigens should therefore be considered as an additional therapeutic approach in the management of atherosclerosis, especially since at least a part of the benefit of established drugs is mediated via their anti-inflammatory activity.

Hypertension Definition and epidemiology Hypertension is diagnosed based upon elevations of either the systolic or diastolic blood pressure, and the objective of management is to achieve normalization of both. Arterial blood pressure (BP) is considered to be elevated if systolic and diastolic BP are higher than 160 and 95 mm Hg, respectively, on three readings on different occasions unless the elevations are severe or associated with symptoms. Values between 140/90 and 160/95 are considered as borderline. In more than 95% of the cases, hypertension (HT) is essential (primary), no cause can be established. A major hemodynamic abnormality in HT is increased peripheral resistance due to changes in vascular structure (reduced lumen diameter and arterial wall thickening) and function (increased vasoconstriction and/or decreased vasodilatation) (Touyz, 2003). The onset of essential HT is mostly beyond the third decade of life and the prevalence of HT in western countries is about 20%. The percentage of unsuspected or inadequately treated patients is of the same magnitude. Essential HT is frequently associated with metabolic disorders (syndrome X: Visceral fat accumulation, glucose intolerance respectively type II diabetes, hyperlipoproteinemia, low serum HDL-cholesterol, essential HT). They all have in common, insulin resistance of skeletal muscle tissue with consecutive hyperinsulinemia and development of premature atherosclerosis. Risk factors The dramatic increase in the prevalence of obesity is a global phenomenon associated with increased risk of the development of cardiovascular and renal disease. In obesity, many factors act together to promote vasoconstriction and sodium retention. All together, these factors will lead to sustained hypertension. Factors that are or may be involved in the pathogenesis of HT are the following: •

Nutrition: obesity and increased salt intake/sensitivity. HT develops in almost 60% of obese individuals (Sharma et al., 2001). Obesity is a leading risk factor for the development of essential HT. It has been estimated in the Framingheart study that for each 4.5 kg of weight gain, there is an accompanying increase of 4 mm Hg in systolic blood pressure in both men and women (Higgins et al., 1988). Several mechanisms have been implicated in the pathogenesis of obesity-associated HT, including insulin resistance, salt sensitivity, activation of the sympathetic nervous system (Mark et al., 1999).

•

Genetic predisposition plays an important role. Genetic factors may modify the influence of the blood pressure response to obesity (Mark et al., 1999). At present the number and definite role of genes implicated in HT are unknown. It appears that candidate gene loci that determine obesity play an important role in the inflammatory response. TNF receptor 2 (TNF-R2) may be a candidate gene for HT and other metabolic syndrome abnormalities (Glenn et al., 2000). The TNF-α gene locus contributes to the determination of obesity and obesity-associated HT (Pausova et al., 2000). The inducible nitric oxide synthase (NOS2A) gene may also play a role in human essential HT (Rutherford et al., 2001).

•

Renin-angiotensin system hyperactivity. Angiotensin II stimulates vascular smooth muscle cell growth, increases collagen deposition, increases contractility, decreases dilatation (Touyz, 2003) Angiotensin II induces oxidative stress in the

kidney. It stimulates the membrane NOX-1 oxidase, heme oxidase (HO-1) and activates NF-κB, a nuclear transcription factor pivotal to the production of inflammatory cytokines. These cytokines exert their effect, in part through oxidative mechanism (Griendling et al., 2000; Haugen et al., 2000; Klahr and Morrissey, 2000). •

Inflammation

•

Other endocrine disorders

•

Exacerbating factors: excessive use of alcohol, cigarette smoking, polycythemia and nonsteroidal anti-inflammatory agents.

•

Combinations of different factors

Immunopathogenic mechanisms Immunopathogenic mechanisms may be involved in the pathogenesis of hypertensive disease, atherosclerosis and coronary artery disease. Alterations in both cellular and humoral immunity, changes in serum immunoglobulin levels and inherited abnormalities of the complement system have been identified in patients with essential HT. In addition, many animal models of spontaneous HT have identifiable abnormalities in immune function that are associated with their hypertensive disease (Dzielak, 1992). Patients with essential HT show also an altered profile of pro- and anti-inflammatory cytokines (Peeters et al., 2001). The inflammatory response is essential in the response to pathogens. TNF-α, IL-1 and IL6 are key mediators of the response. They initiate metabolic changes to provide nutrients for the immune system, from host tissues. These changes include hyperlipidemia and increased gluconeogenesis. Insulin resistance and deregulation of lipid metabolism occur in obesity, type 2 diabetes and atherosclerosis. Population studies show a strong association between indices of inflammation and abnormal lipid and carbohydrate metabolism, obesity and atherosclerosis (for review see: Grimble, 2002). TNF-α is produced by cells of the immune system and by adipocytes. TNF-α results in insulin insensitivity. Production of TNF-α and leptin relates positively to adipose tissue mass. TNF-α and leptin show a broad range of activities and very probably act solely or together to promote hypertension and cardiovascular diseases associated with obesity and insulin resistance. Leptin resistance Central nervous system actions of leptin may contribute to increased sympathetic nervous system activity that is typically found in obesity. The predominant cardiovascular affect of chronic hyperleptinemia is a pressor effect mediated by increased sympathetic activity. Enhanced leptin-driven renal sympathetic out-flow, in combination with low atrial natriuretic peptide plasma levels possibly due to over-expression of the natriuretic peptide clearance receptor in adipocytes, may enhance sodium retention and volume expansion, both key features in the pathophysiology of obesity-associated hypertension (Engeli and Sharma, 2000). Leptin also induces depressor actions. Leptin acutely increases insulin sensitivity (Sivitz et al., 1997), endothelium derived nitric oxide (NO) production (Lembo et al., 1998) and angiogenesis (Sierra-Honigmann et al.,1998; Mark al., 2002). Although leptin possesses both depressor and pressor actions, the chronic effects of leptin appear to be predominantly pressor and human obesity appears to be associated with leptin resistance, since high circulating levels of leptin are observed in obese subjects. Recent studies indicate that leptin resistance may be selective, with preservation of adverse sympathetic effects despite the loss of the metabolic actions (satiety and weight-reducing action) of leptin (Shek et al., 1998). The melanocortin system, neuropeptide Y and corticotrophin-releasing factor have emerged as principal neuropeptide mediators of

leptin in the arcuate nucleus of the hypothalamus

(Rahmouni and Haynes, 2002; Rahmouni et al.,

2002).

Adipose tissue secretes in addition to TNF-α and leptin various biologically active adipocytokines including free fatty acids. Accumulation of visceral fat increases the portal free fatty acid concentrations, which contribute to insulin resistance and dslipidemia (Hotta and Matsuzawa, 2001). Free fatty acids, insulin and the adipose tissue-derived hormone, leptin, whose levels are increased in obesity, may act synergistically to stimulate sympathetic activity and vasoconstriction (Montani et al., 2002). Obesity-induced insulin resistance and endothelial dysfunction may operate as amplifiers of the vasoconstriction response. Increased renal tubular reabsorption of sodium may not only be the consequence of increased renal sympathetic nerve activity, but may be also the result of a direct effect of insulin, hyperactivity of the renin-angiotensin system and possibly by an alteration of intrarenal physical forces (Engeli and Sharma, 2001). Haemostatic factors The increased incidence of cardiovascular disease may be related to the elevated levels of haemostatic factors, like fibrinogen, factor VII, and plasminogen activator inhibitor 1 (PAI-1) observed in the plasma of obese patients (McGill et al., 1994; Potter van

Loon et al., 1993). PAI-1 is the primary inhibitor of plasminogen activation in vivo, and increased plasma PAI-1 compromises normal fibrin clearance mechanisms and promotes thrombosis, including myocardial infarction (Sprengers and Kluft, 1987; Carmeliet and Collen; 1997). PAI-1 is dramatically up-regulated in human obesity (McGill et al., 1994; Potter van Loon et al., 1993). Several observations implicate TNF-α in the increased expression of PAI-1 in obese adipose tissue. TNF-α expression is increased in adipose tissue of both human and rodents (Hotamisligil et al., 1993; 1995; Hofman et al., 1994; Kern et al., 1995). Insulin and transforming growth factor β (TGF-β) also have been implicated in the increased expression of PAI-1 in obesity, and TNF-α contributes to the increase in both of these mediators. Treatment of obese mice with neutralizing antibodies to TNF-α, or lacking p55 and p75 TNF receptors (TNFR), induces increased insulin sensitivity and reduces significantly plasma levels of PAI-1 and adipose tissue PAI-1 and TGF-β mRNAs (Samad et al., 1999). These observations provide direct evidence, that TNF-α is a common link between obesity, insulin resistance, PAI-1 and TGF-β, and thus establish a central role for TNF-α in a number of metabolic disorders, hypertension and cardiovascular diseases associated with obesity.

The fact that insulin and TGF-β are mediators with broad biological effects implies that the initial elevation of TNF-α in obesity is rapidly amplified through the activation of these and perhaps other genes. There are a number of potential pathophysiological consequences of these changes. For example, elevations in TGF-β not only stimulate PAI1, but also induce adipose tissue expression of tissue factor, the primary initiator of the coagulation cascade (Samad et al., 1998). TGF-β has also been implicated in atherosclerosis and fibrotic kidney disease (Border, 1994; Nabel et al., 1993; Pfeiffer and Schatz, 1995; Wang et al., 1997), two common complications of obesity. Insulin itself has been reported to stimulate PAI-1 and also influences smooth muscle function, blood pressure, and lipid metabolism. The chronic elevation of TNF-α in obesity thus may directly promote the development of the complex cardiovascular risk profile associated with this condition. Oxidative stress Enhanced vascular and endothelial reactive oxygen species (ROS) formation is leading to endothelial dysfunction, hypertension and atherosclerosis. It is becoming increasingly apparent that many signaling events that underlie abnormal vascular function in hypertension are influenced by changes in the intracellular redox status. Oxidative stress stimulates growth-signaling pathways, induces expression of pro-inflammatory genes, alters contraction-excitation coupling and impairs endothelial function (Touyz, 2003). Elevated leptin levels, an activated renin angiotensin system and oxidized LDL may play a prominent role for enhanced vascular oxidative stress. ROS avidly react with and inactivate nitric oxide (NO), also known as endothelium-derived relaxation factor (Beckman et al., 1996). ROS-mediated NO inactivation can contribute to hypertension and endothelial dysfunction by limiting the availability of biologically active NO (Vaziri et al., 2000). Increased amounts of ROS form with NO the potent peroxynitrite radical (OONO-). This radical interacts with proteins in the kidney that are important for normal glomerular and tubular function and reduces their activity (Szilvassy et al., 2001).

Complications Cardiac complications are the major causes of morbidity and mortality in essential HT. HT is may be asymptomatic for many years, typical early symptoms are: Headache (especially in the early morning) Vertigo, tinnitus, dyspnoea

nervosity,

precordial

pain,

palpitations,

exercise

Hypertensive cardiovascular disease with left ventricular hypertrophy with heart failure Hypertensive cerebrovascular hemorrhage) and dementia

disease

(cerebral

ischemia,

infarction,

Hypertensive renal disease with microalbuminuria in the early stages and nephrosclerosis with renal insufficiency as end stage disease Aortic dissection Atherosclerotic complications. Acute coronary syndromes are responsible for most of the morbidity caused by coronary atherosclerosis. Unstable angina and acute myocardial infarction are characterized by coronary thrombosis, usually caused by rupture or fissuring of a coronary plaque (Pasceri and Yeh, 1999). Malignant and accelerated hypertension: Any form of sustained hypertension may abruptly become accelerated, with resulting encephalopathy, nephropathy, retinopathy, heart failure or myocardial ischemia.

Atherosclerosis The response to injury Cardiovascular disease, currently the leading cause of death and illness in developed countries, will soon become the pre-eminent health problem worldwide (Murray et al., 1997). Atherosclerosis – a progressive disease characterized by the accumulation of lipids and fibrous elements in the large arteries – constitutes the single most important contributor to this growing burden of cardiovascular disease (Libby, 2002). Because high plasma concentrations of cholesterol, in particular LDL cholesterol, are one of the principal risk factors of atherosclerosis, the process of atherogenesis has been considered to consist largely of the accumulation of lipids within the artery wall. The insights of the scientific community in the pathophysiology of this important disease have evolved substantially over the past century. Atherosclerosis is now being increasingly recognized as an inflammatory disease (Ross, 1999). The cellular interactions in atherogenesis are fundamentally not different from those in chronic inflammatory/autominne-fibroproliferative diseases such as cirrhosis, rheumatoid arthritis, glomerulosclerosis, pulmonary fibrosis and chronic pancreatitis (Ross, 1999; Pasceri and Yeh, 1999). Vascular inflammation produces atherosclerosis by the continuous recruitment of monocytes into the vessel wall and by contributing to an oxidant-rich inflammatory milieu that induces phenotypic changes in resident endothelial and smooth muscle cells. If the causative agent or agents are not removed or eliminated by the inflammatory response and the inflammation progresses, the response changes from a protective to an injurious response. Such constant or repetitive injury can stimulate each tissue to repair or wall off the damage by a fibroproliferative response, which, when excessive, diminishes the functional capacity of the tissue or organ and becomes part of the disease process (Ross, 1999). Vascular inflammations: an independent risk factor It is now well established that vascular inflammation is an independent risk factor for the development of atherosclerosis (Brasier et al., 2002). Atherosclerosis does not result simply from the accumulation of lipids, but inflammation constitutes the missing link between hypercholesterolaemia and atherosclerosis. Atherosclerosis is a disease characterized by inflammation that usually begins at an early age in the absence of lipid accumulation, with fatty streaks composed of lipid-laden (foam) cells developing at later stages (Pockley, 2002). Its development and progression appear to be a balance between proinflammatory and regulatory immune responses. Studies done in many laboratories around the world over the past several years have shown an association between markers of inflammation (C-reactive protein (CRP), modified low-density lipoprotein (LDL), homocysteine, TNF and thermogenicity) and coronary atherosclerosis with an exacerbation of the inflammatory process during acute myocardial ischemia (Mehta et al., 1998; Farmer and Torre-Amione, 2002). CRP is a better predictor for the risk of cardiovascular events than LDL (Ridker et al., 2000, 2002). Hypertension alone and other traditional risk factors, such as smoking, dyslipdemia and type 2 diabetes constitute a high risk for early atherosclerosis but do not explain the presence of coronary atherosclerosis in a large proportion of patients, since inflammatory markers (TNF-α and IL-6) are increased in hypertensives despite good blood pressure control (Furumoto et al., 2002; Woodman et al., 2002).

About 50% of individuals with proven artery disease have “average” levels of cholesterol (Braunwald, 1997). Several epidemiological studies have provided evidence that serum markers of inflammation are associated with conventional risk factors for atherosclerotic diseases (for review see: Elkind et al., 2002). C-reactive protein (CRP) and fibrinogen are associated with these outcome events, but TNF-α, TNF-receptors, IL-6, soluble intercellular adhesion molecule-1 and E-selectin have been associated as well (Bruunsgaard et al., 1999; Elneihoum et al., 1997; Blann et al., 1998, Elkind et al., 2002; Woodman et al., 2002). Strategies aimed at modifying these factors might provide a novel

approach to prevention of stroke and other vascular diseases. Endothelial dysfunction Functional abnormalities of the endothelium predate the development of anatomical changes of atherosclerosis. The first step in atherosclerosis is endothelial dysfunction. Possible causes of endothelial dysfunction leading to atherosclerosis include: Elevated and modified LDL. LDL, which may be modified by oxidation in the arterial wall by cell-associated lipoxygenase and/or myeloperoxidase, glycation (in diabetes), aggregation, association with proteoglycans, or incorporation into immune complexes is a major cause of injury to the endothelium and underlying smooth muscle (Steinberg et al., 1997; Khoo et al., 1988, 1992; Navab et al., 1996; Morel et al., 1983; Griendling and Alexander, 1997, Mertens and Holvoet, 2001). Oxidized LDL induces atherosclerosis by stimulating monocyte infiltration and smooth muscle cell migration and proliferation. It contributes to atherothrombosis by inducing endothelial cell apoptosis and thus plaque erosion, by impairing the anticoagulant balance in endothelium, stimulating tissue factor production by smooth muscle cells, and inducing apoptosis in macrophages (Mertens and Holvoet, 2001). Free radicals (oxidative stress) caused by cigarette smoking, hyperlipidemia, diabetes and hypertension.

Hypertension. Concentrations of angiotensin II, which is a potent vasoconstrictor, are often elevated in patients with hypertension. It contributes to atherogenesis by stimulating the growth of smooth muscle (Chobanian et al., 1996) and increases smooth-muscle lipoxygenase activity, which can increase inflammation and the oxidation of LDL. Angiotensin II has also pro-inflammatory actions, increasing the formation of hydrogen peroxide and free radicals such as superoxide anion and hydroxyl radicals in plasma (Griendling and Alexander, 1997; Lacy et al., 1998; Swei et al., 1997). These substances reduce the formation of nitric oxide (NO) by the endothelium, increase leukocyte adhesion, pre-activate monocytes and increase peripheral resistance (Swei et al., 1997; Vanhoutte and Boulanger, 1995; Dörffel et al., 1999). Angiotensin II also induces inflammatory cytokines (IL-6) and adhesion molecules. IL-6 induces synthesis of angiotensinogen in the liver (Brasier et al., 2002). Enhanced angiotensinogen production, in turn, supplies more substrate to the activated vascular renin-angiotensin system, where locally produced angiotensin II, synergizes with oxidized lipid to perpetuate atherosclerotic vascular inflammation. Through these actions, angiotensin II augments vascular inflammation, induces endothelial dysfunction, and, in so doing, enhances the atherogenic process. Insulin resistance, type II diabetes. Diabetes-associated hyperglycemia produces intracellular oxidant stress that can lead to vascular dysfunction. Hyperglycemia activates the transcription factor NF-kB and promotes leukocyte adhesion to the endothelium through up-regulation of cell surface expression of adhesion molecules. NF-κB activation through hyperglycemia in vascular muscle cells increases smooth muscle cell proliferation, leading to increased intimal wall thickness (Collins and Cybulsky, 2001). Several acute phase reactants (α1-acid glycoprotein, serum amyloid A, fibrinogen) and IL-6, complement factor C3 and plasminogen activator inhibitor 1 (PAI-1) are elevated in diabetic patients (Lin et al., 2001). All these parameters are elevated at base line among patients at risk for future coronary occlusion (Lin et al., 2001). Obesity with a chronic and systemic pro-inflammatory stimulus due to the increased production of cytokines by the adipose tissue. Genetic alterations Elevated plasma homocysteine concentrations. Patients with slightly increased homocysteine levels have an increased risk of symptomatic atherosclerosis (Verhoef and Stampfer, 1995). Homocysteine has proinflammatory effects on both endothelial and smooth muscle cells. Homocysteine is toxic to the endothelium, is prothrombotic, increases collagen production and decreases the availability of nitric oxide (Harker et al., 1976; Hajjar, 1993; Majors et al., 1997; Upchurch et al., 1997). Infectious agents: Chlamydia pneumoniae (Kalayoglu et al., 2002) and herpesviruses, especially Cytomegalovirus (CMV) (Libby et al., 1997). C. pneumoniae has been found in atherosclerotic plaques and induces foam cell formation and elicits T-cell mediated immune responses, and T cells specific for C. pneumoniae have been isolated from atheromatous plaques. Heat shock protein (Hsp) 60 from C. pneumoniae induces macrophage production of TNF-α and matrix metalloproteinase activity (for review see Pockley, 2002). CMV infection generates intracellular reactive oxygen intermediates, activates NF-κB and induces the formation of intercellular gaps within the endothelium (Scholz et al., 1999; Speir et al., 1997; Hiscott et al., 2001). Virally induced NF-κB may increase the expression of cytokines and ICAM-1 (Knight et al., 1999). Additionally, herpesviruses can act as prothrombotic agents by activating the coagulation cascade (Nicholson and Hajjar, 1998). Although there is a correlation between the incidence of

atherosclerosis and the presence of C. pneumoniae and CMV, there is no direct evidence that infectious agents can initiate atherosclerosis (Ross, 1999; Kalayoglu et al., 2002). It is however possible, that they contribute directly through infection of the cells of the vessel wall and indirectly through long-distance effects from remote infection to the progression or exacerbation of atherogenesis. Chronic inflammatory disease (rheumatoid arthritis and systemic lupus erythematosus; Kaplan and McCune, 2003). The increased mortality observed in individuals with rheumatoid arthritis is a consequence of an increase in cardiovascular and cerebrovascular disease secondary to atherosclerosis (Wolf et al., 1994; Symmons et al., 1998). The immune dysregulation and systemic inflammation, which are present very early during the natural history of the disease, play an important role in the development of accelerated atherosclerosis (Goodson et al., 2002; Del Rincon et al., 2001; Hurlimann et al., 2002). The inflammatory and immunological responses in atherosclerosis and rheumatoid arthritis, the prototype of autoimmune disease, share numerous similarities (Pasceri and Yeh, 1999) and early events in rheumatoid arthritis could potentially initiate inflammatory processes in the artery wall:

o

Activated T cells are present in atherosclerotic plaques as well as in rheumatoid synovium (van der Wal et al., 1998).

o

Collagen degeneration, which is an essential element in the pathogenesis of rheumatoid arthritis, plays also an important role in the destabilization of plaques (van der Wal et al., 1994).

o

In both diseases, there is an abundance of cytokine-secreting inflammatory cells in the diseased tissue and acute-phase reactants are present in the synovium/plaques and in the peripheral blood. The release of inflammatory mediators (TNF-α, IL-1, adhesion molecules, growth factors and matrix metalloproteinases), further contributes to the inflammatory response. Such mediators also contribute to migration and proliferation of endothelial and smooth-muscle cells, collagen breakdown, platelet aggregation, release of oxygen free radicals and loss of endothelial nitric oxide (Greaves and Channon, 2002).

o

An imbalance between TH1 and TH2 immune response is present in rheumatoid arthritis and in patients with unstable angina (Liuzzo et al., 1999).

An autoimmune reaction against Hsp60, expressed by endothelial cells after pretreatment with TNF-α and IL-1, may be one of the initiating events in atherogenesis (Wick et al., 1995). Given the high phylogenetic similarity between microbial and mammalian forms of these molecules, hsps might act as potentially harmful autoantigens (Kaufmann, 1990). CRP is one of the most powerful and independent risk factor for coronary heart disease, and not only a nonspecific but sensitive marker of the acute inflammatory response (Edward et al., 2003). CRP is a direct participant in atherogenesis since it induces adhesion molecule expression in human endothelial cells and the chemokine, monocyte chemotactic protein production (Pasceri et al., 1999, 2000, 2001). CRP also induces the secretion of IL-6 and endothelin-1 and decreases the expression and bioavailability of endothelial nitric oxide synthase in human endothelial cells (Verma et al., 2002; Venupogal, 2002). CRP activates macrophages to express cytokine and tissue factor and enhances uptake of LDL (Zwaka et al., 2001). CRP also amplifies the pro-inflammatory effects of several other mediators, including endotoxins (Yeh et al., 2001; Nakagomi et al.,

2000).

The acute phase reaction is associated with elevated levels of fibrinogen, with autocrine and paracrine activation of monocytes by IL-6. IL-6 itself is a powerful inducer of the hepatic acute phase response. IL-6 decreases lipoprotein lipase (LPL) activity and monomeric LPL levels in plasma, which increases macrophage uptake of lipids (Yudkin et al., 2000). IL-6 stimulates the hypothalamicpituitary-adrenergic axis (HPA-axis), activation of which is associated with central obesity, HT and insulin resistance.

Combinations of the above mentioned factors

Inflammatory cascade These potential contributors to endothelial injury, initiate a compensatory response resulting in an inflammatory cascade that leads to activation of monocytes and lymphocytes in the arterial wall, contributing to smooth muscle cell proliferation and thickening of the arterial wall (Elkind et al., 2002; Ross, 1999). Lymphocyte recruitment and expression of pro-inflammatory cytokines characterize early atherogenesis. Inflammatory pathways promote thrombosis, a late and dreaded complication of atherosclerosis responsible for myocardial infections and most strokes (Libby, 2002). If the inflammatory response does not effectively neutralize or remove offending agents, it can continue indefinitely (Ross, 1999). In doing so, the inflammatory response stimulates migration and proliferation of smooth-muscle cells that become intermixed with the area of inflammation to form an intermediate lesion. If these responses continue unabated, they can thicken the artery wall, which compensates by gradual dilation, the lumen remains unaltered, a phenomenon called “remodeling” (Glagov et al., 1987). Leucocyte adhesion Under normal circumstances, the endothelial layer in contact with flowing blood resists firm adhesion of leucocytes (Libby, 2002, De Souza et al., 1997). Leukocyte adhesion may be initiated by the increased expression of vascular cell adhesion molecule-1 (VCAM-1), which binds particularly monocytes and T lymphocytes. VCAM-1 induction may be observed under different circumstances: •

Inflammation instigated by modified lipoprotein particles accumulating in the arterial intima in response to hyperlipidemia. Oxidized phospholipids and short chain aldehydes rising from lipoprotein oxidation induce transcription of VCAM-1, which is mediated in part by nuclear factor-kB (NF-κB) (Collins and Cybulsky, 2001).

•

Pro-inflammatory [TH1] cytokines (IL-1 and TNF-α) induce VCAM-1 expression in endothelial cells. IL-1 and TNF-α are present in athersclerotic lesions and/or in the peripheral blood. The presence of circulating pro-inflammatory cytokines is observed under various clinical conditions (infections, auto-immune diseases, allergy and hypersensitivity reactions) associated with inflammation.

•

CRP at concentrations ≥ 5 mg/ml, has significant pro-inflammatory effects in coronary artery endothelial cells, inducing high levels of expression of intercellular adhesion molecule (ICAM-1), VCAM-1, and E-selectin.

Leucocyte infiltration Activation of monocytes and T-cells leads to up-regulation of receptors on their surfaces, such as the mucin-like molecules that bind selectins, integrins that bind adhesion molecules of the immunoglobulin superfamily and receptors that bind chemoattractant

molecules (Springer et al., 1996). These ligand-receptor interactions further activate mononuclear cells, induce cell proliferation, and help define and localize the inflammatory response at the sites of lesions (Ross, 1999). Once adherent to the endothelial cell, leucocytes enter the intima by diapedesis between endothelial cells at their junctions. Chemoattractant cytokines are capable of recruiting leukocytes into the arterial intima. These include: (Gu et al., 1998).

•

Monocyte chemoattractant protein-1 (MCP-1)

•

IL-8

•

Atheroma overexpresses other chemokines that may contribute to lymphocyte recruitment, including three IFN-γ inducible CXC chemokines (IFN-inducible protein 10 (IP-10), monokine induced by IFN-γ (Mig) and IFN-inducible T-cell αchemoattractant (I.-TAC)) (Mach et al., 1999).

(Boisvert et al., 1998)

Foam cell formation Once resident in the arterial intima, monocytes acquire the morphological characteristics of macrophages, undergoing a series of morphological changes that lead ultimately to foam cell formation. The monocytes increase expression of scavenger receptors from modified lipoproteins such as the scavenger receptor A (SRA) and CD36, and than internalize modified lipoproteins (LDL), such that cholesteryl esters accumulate in cytoplasmic droplets (Libby, 2002). These lipid-laden macrophages, known as foam cells characterize the early atherosclerotic lesion. Candidate activators of the steps that stimulate the transition of the monocyte to the foam-cell are: •

Macrophage colony-stimulating factor (M-CSF) increases SRA expression, production of cytokines and growth factors by these cells. Human atherosclerotic plaques over-express M-CSF (Clinton et al., 1992).

•

Granulocyte-macrophage colony-stimulating factor (GM-CSF) may also promote inflammation in the atheroma. GM-CSF aids the survival of a population of mononuclear phagocytes that contain myeloperoxidase, an enzyme that gives rise to the pro-oxidant HOCl, a potential source of oxidative stress and inflammation in the human atherosclerotic plaque (Sugiyama et al., 2001). A recent case-observation study showed that myeloperoxidase deficient individuals show a lower risk for cardiovascular disease than control persons (Kutter et al., 2000). Conversely, elevated leukocyte- and blood-myeloperoxidase levels are associated with the presence of coronary artery disease (Zhang et al., 2001).

•

Modified LDL is chemotactic for other monocytes and can up-regulate the expression of genes for M-CSF and monocyte chemotactic protein derived from endothelial cells (Quinn et al., 1987; Rajavashisth et al., 1990; Leonard et al., 1990).

TNF-α, IL-1 and M-CSF increase binding of LDL to endothelium and smooth muscle and increase the transcription of the LDL-receptor gene (Stopeck et al., 1993). After binding to scavenger receptors, modified LDL initiates the induction of urokinase and inflammatory cytokines (IL-1) (Falcone et al., 1991; Palkama, 1991). Thus, a vicious circle of inflammation, modification of lipoproteins and further inflammation can be maintained in the artery by the presence of these lipids. Removal and sequestration of modified LDL are important parts of the initial, protective role of the macrophage in the inflammatory response and minimize the effects of

modified LDL on endothelial and smooth-muscle cells (Han et al., 1997; Diaz et al., 1997; Falcone Beside their scavenging activity, monocyte-derived macrophages are antigenpresenting cells, and they secrete cytokines (TNF-α, IL-1, IL-6 and TGF-β) chemokines, growth-regulating molecules (platelet-derived growth factor and insulin-like growth factor I), metalloproteinases and other hydrolytic enzymes (Ross, 1999; Yudkin et al., 2000).

et al., 1991).

Activated macrophages express class II HLA antigens that allow them to induce a cellmediated immune response (Raines et al., 1996). T-cell activation results in the production of IFN-γ, TNF-α and -β that amplify the inflammatory response (Hansson et al., 1989). Smooth muscle cells from the lesions also have class II HLA molecules on their surfaces. One possible antigen may be oxidized LDL, which can be produced by macrophages (Folcik et al., 1997). Heat-shock protein 60 may also contribute to autoimmunity (Ross, 1999). Atheroma disruption After formation of the fatty streak, the nascent atheroma typically evolves into a more complex lesion, which eventually leads to clinical manifestations. In accordance with more recent findings, growth of atheroma is not continuous but seems to occur in “bursts”. Current evidence suggests that physical disruption of plaques may trigger thrombosis and thus promote sudden expansion of atheromatous lesions (Davies, 1996). Three types of physical disruptions involving thrombosis and stimulation of smooth muscle growth and migration may occur (Virmani et al., 2001). Two processes related to inflammation may participate in endothelial desquamation. •

Endothelial cell death may result from local (or distant?) production of inflammatory mediators or cytotoxic attack by activated killer cells. Additionally, inflammatory mediators and oxidized lipoproteins can stimulate the expression and activation of matrix metalloproteinases specialized in degrading components of the sub-endothelial basement membrane (Rajavashisth et al., 1999).

•

In addition to secretion of growth factors from smooth muscle cells, inflammatory cells residing in the plaque produce angiogenic mediators: acidic and basic fibroblast growth factor and vascular endothelial growth factor (VEGF) (Brogi et al., 1993).

•

Macrophage accumulation may be associated with increased plasma concentrations of fibrinogen and CRP, two markers of inflammation thought to be early signs of atherosclerosis (Ridker et al., 1997; Haverkate et al., 1997; Toss et al., 1997).

The fracture of the fibrous cap of the plaque may be related to the production of proinflammatory cytokines, such as IFN-γ, that inhibit collagen formation by smooth muscle cells (Libby, 2002). Furthermore there is an over-expression of human interstitial collagenases in atheromatous plaques (Galis et al., 1994). After the limited proteolytic cleavage arising from the action of interstitial collagenases, gelatinases continue collagen catabolism. In vitro studies have shown that IL-1β, TNF-α and CD40 ligand increase the expression of gelatinases expression in mononuclear phagocytes, endothelial cells and smooth muscle cells (Saren et al., 1996). Furthermore, CD40 ligand induces the release of IL-1β by vascular cells potentially enhancing the inflammatory response (Schönbeck et al., 1997). The net result of inflammation, dissolution of the collagenous matrix of the fibrous cap, renders this structure weak, friable and susceptible to fracture when exposed to haemodynamic stresses (Libby, 2002). These changes may also be accompanied by the production of tissue-factor pro-coagulant and other haemostatic factors further increasing the possibility of thrombosis (Mach et al., 1997). Platelet adhesion and mural thrombosis are ubiquitous in the initiation and generation of the lesions of atherosclerosis (Ross, 1993). Platelets can adhere to

dysfunctional endothelium, exposed collagen, and macrophages. When activated, platelets release their granules, which contain cytokines and growth factors that, together with thrombin, may contribute to the migration and proliferation of smooth muscle cells and monocytes (Bombeli et al., 1998). Activation of platelets leads to the formation of free arachidonic acid, which can be transformed into prostaglandins, such as thromboxane A2, one of the most potent vasoconstricting and platelet-aggregating substances known, or into leukotrienes, which can amplify the inflammatory response (Ross, 1999). Conclusion In conclusion, the immunologic responses that lead to the induction and progression of atherosclerosis are complex, and additional insight is required to clearly understand the relationship between different risk factors and immune components of atherogenesis in order to develop immunotherapeutic approaches. Although our knowledge on the intimate mechanisms of atherosclerotic disease are still incomplete, the control of the inflammatory response should be considered as a major therapeutic goal in combination with already established therapy options or preventive approaches.

Therapeutic approaches and prevention Hypertension Since obesity is one of the most important risk factors for hypertension and cardiovascular diseases, weight loss is the primordial goal that needs to be achieved for the prevention and therapy of hypertension. Obesity is mediated through an increased secretion of pro-inflammatory cytokines by the adipose tissue. The pro-inflammatory response induces systemic vascular inflammation and insulin resistance. In turn, endothelial dysfunction generates a pro-inflammatory response which potentates the deleterious effect of cytokines on adipose tissue and lipid and carbohydrate metabolism. Thus, a vicious circle of inflammation, obesity-hypertension-atherosclerosis can be maintained in the artery by the presence of excess of adipose tissue. Reduction of body weight is associated with a reduction in markers of vascular inflammation and insulin resistance (Esposito et al., 2003). Although the cause-effect relationship of obesity and inflammation is not clearly established, the effects of hypocaloric diet and increased physical activity may be jeopardized by the vicious circle of inflammation. Therefore it should be recommended to eliminate or avoid any inflammatory stimulus, which may potentially interfere with glucose and lipid homeostasis. Since intestinal permeability is perturbed through the pro-inflammatory response related to obesity, multiple food hypersensitivities may occur as a consequence of abolition of oral tolerance. Preliminary results obtained by statistical evaluation of 200 patients records showed a beneficial effect of selective exclusion diet, based on IgG antibody reactivity against a panel of 264 food antigens, on obesity. The average weight loss in men was 8, and in women was 6 kg respectively. The weight loss was sustained (>3 months) in patients who showed a high compliance and was accompanied by a reduction in blood pressure (BP) in a sub-collective of patients from whom data on BP were available. The advantage of the selective exclusion diet over alternative regimens is that there is no caloric restriction and it is adapted to the individual immunological profile. In consequence, the compliance is high, however patients need an extensive dietary counseling for the implementation of their individual diet. The high compliance may be also related to the reduction of the effects of chronic inflammatory response on insulin (glucose intolerance) and leptin resistance (abolition of satiety and weight-reducing action). Atherosclerosis The important role of inflammation in the pathogenesis of atherosclerosis raises opportunities in the prevention and therapy of this disease. Anti-inflammatory drugs Beyond their recognized role for the prevention of recurrent myocardial infarction, different drug classes (aspirin, statins, angiotensin-converting enzyme (ACE) inhibitors and angiotensin-receptor blockers, fibric acid derivates) exert an anti-inflammatory action, which may in part be responsible for the clinical benefits (Ridker, 1999). Fibric acid derivates activate the peroxisome proliferator-activated receptor [alpha] and [gamma] (PPAR-α and PPAR-γ). PPAR-α agonism can limit cytokine-induced activation of inflammatory functions of vascular endothelial cells (expression of VCAM-1 in response to TNF-α and tissue factor gene expression in these cells (Marx et al., 1999; 2001; Neve et al., 2001). PPAR-γ exerts also an anti-atherosclerotic activity by decreasing pro-inflammatory functions of macrophages and smooth muscle cells (Ricote et al., 1998; Jiang et al., 1998; Itoh et al., 1999). Furthermore, infliximab, a monoclonal anti-TNF-α antibody ameliorates endothelial function in patients with rheumatoid arthritis (Hurliman et al., 2002). Certain

nutrients have the ability to diminish the endothelial dysfunction. Dietary long-chain unsaturated fatty acids of the omega 3 family are associated with diminished risk of atherosclerosis. They inhibit cytokine-stimulated expression of endothelial-leukocyte adhesion molecules and soluble cytokine production (TNF-α and IL-1) in the range of nutritionally achievable plasma concentrations (Collins and Cybulsky, 2001; Das, 2000). Antioxidant nutrients may inhibit early atherogenesis by inhibiting LDL oxidation or by decreasing cellular production and release of reactive oxygen species in endothelial cells. Antioxidant vitamins would diminish the cellular responses to oxidized LDL, reducing monocyte adhesion, foam cell formation and cytotoxicity to vascular cells. The inhibition or reduction of the chronic (pro-) inflammatory response seems to be a key element in the prevention and therapy of atherosclerosis. The aim should not only be a local effect on the cytokine production and inflammatory response in atherosclerotic plaques but generalized in order to reduce the stimuli implicated in the early atherogenesis and finally in the plaque disruption which may be ultimately associated with myocardial infarction and death. Besides the direct effect on the inflammatory response involved in the different steps of atherogenesis from the beginning (endothelial dysfunction) to the end (plaque rupture), the inhibition of inflammation eliminates or reduces the impact of other independent risk factors for atherosclerosis (hypertension, diabetes, obesity and other chronic inflammatory diseases). In all these clinical conditions, there is a (pro)-inflammatory response with over-expression cytokines, which in turn, activate the local vascular inflammation, thus creating a vicious circle. Chronic inflammation and food hypersensitivity There is now growing evidence that chronic inflammation and food hypersensitivity may be closely related. Exclusion of nutrients, which elicit an IgG antibody response, from the alimentation has been proven beneficial in chronic inflammatory diseases. The prototype clinical entity related to food intolerance is celiac disease. Gluten-free diet prevents the progression of the disease. Exclusion diet has also been proven to ameliorate clinical symptoms in rheumatoid arthritis. According to the very promising results observed in a preliminary evaluation of patients who underwent selective food exclusion diet for different chronic diseases related to inflammation, we hypothesize that selective exclusion diet may prevent the progression of atherosclerosis through a direct local action and by eliminating or reducing systemic inflammation triggers, i. e. obesity, insulin resistance, and hypertension, which are risk factors for atherosclerosis. Exclusion diet based on the results of IgG antibody reactions against food antigens should therefore be considered as an additional therapeutic approach in the management of atherosclerosis, especially since at least a part of the benefit of established drugs is mediated via their anti-inflammatory activity.

References Supplementary information is available (in french) under INTOLERANCE-HYPERSENSIBILITE ALIMENTAIRE: INTERET DU DOSAGE DES IGG ANTI-ANTIGENES ALIMENTAIRES PAR IMUPRO 300 on the URL http://www.labo.lu/fr/books/index.html

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Authors © Lieners - Thoma - Weber / LR-KLH Authors to contact for questions about Systemic hypertension, atherosclerosis and coronary artery disease: Prof. Dr. med. Bernard Weber Tel.: (352) 78 02 90 1 [email protected] Dipl.-Ing. Dr. Camille Lieners Tel.: (352) 78 02 90 1 [email protected] Layout: Msc. Biochem. John Thoma Tel.: (352) 78 02 90 1 [email protected] Laboratoires réunis Kutter-Lieners-Hastert Z.A.C. Langwies L-6131 Junglinster / Luxembourg Tel.: (352) 78 02 90 1 Fax: (352) 78 88 94 http://www.labo.lu