Department of Medicine IV, Division of Cardiology, J. W. Goethe University, Frankfurt am Main, Germany
Initial step in atherogenesisimpairment of endothelial function
Prior to the development of atherosclerotic plaques, endothelial vasodilator function is impaired early in the process of atherogenesis. The endothelium is not only a single cell layered mechanical barrier between the blood and vessel wall, but regulates various important functions of the vasculature such as vasomotion and, therefore, blood flow regulation, as well as haemostasis and wall proliferation processes.
To control vasomotor tone, the endothelium releases a variety of substances, such as prostacyclin, hyperpolarizing factor, endothelin and, most importantly, nitric oxide (NO) [1]. Physiologically, exercise is an important mechanical endothelial stimulus mediated by shear stress due to increased blood flow: the flow-dependent dilation of pre-capillary resistance as well as conductance vessels allows blood flow to increase according to metabolic demand. Whereas increased blood flow stimulates the endothelium mechanically, other stimuli such as catecholamines, bradykinin or platelet-released serotonin stimulate specific receptors on endothelial cells. If the integrity of the endothelium is intact, these stimuli lead to vasodilation. However, in case of endothelial dysfunction, a direct vasoconstrictor action of these stimuli on the vascular smooth muscle cells outweighs the endothelium-dependent vasodilator effect and leads to paradoxical vasoconstriction. Hypercholesterolaemia, but also other cardiovascular risk factors, are associated with endothelial dysfunction (for review see [2]). In addition, endothelium-dependent coronary vasodilator function progressively deteriorates with increasing extent of atherosclerotic disease.
Nitric oxide
When the endothelium is stimulated by shear stress due to increased blood flow, the endothelial NO synthase (NOS III=eNOS) is activated via a non-receptor-dependent pathway by AKT-dependent phosphorylation of the NO synthase [3] and produces NO from its precursor L-arginine. Abluminally secreted NO activates the soluble guanylyl cyclase of smooth muscle cells which leads to flow-dependent vasodilation [4]. In contrast, bradykinin or platelet-derived serotonin stimulate the endothelium via receptors leading to G-protein mediated activation of the NO synthase.
NO bioactivity might be reduced by a variety of different mechanisms.
NO inactivation due to oxidative stress
In the early stages of atherosclerosis, LDL cholesterol accumulates in the vessel wall and is oxidized [5]. Oxygen-derived free radicals are capable of inactivating NO rapidly. Thereby peroxinitrite (ONOO-) is formed, which no longer has the vasodilator and vasoprotective functions of NO [6].
Besides hypercholesterolaemia, nearly all classical cardiovascular risk factors, such as smoking or hypertension, are also associated with oxidative stress in the vessel wall [2]. Sources of superoxide anions are xanthine oxidase, an uncoupling of the NO synthase (switch to production of superoxide radicals) and, especially, NADH/NADPH oxidase [6], which produces superoxide anions in macrophages after stimulation with angiotensin II, a key mediator of oxidative stress in the vascular wall [7]. In the early stages of coronary artery disease, reactive oxygen species are released from the endothelium [8]; however, later in the atherosclerotic process, when atherosclerotic wall thickening is present, macrophages which release superoxide anions accumulate in the intima, and the pro-oxidative process extends to the vessel wall (Figure 1). The redox equilibrium between NO and oxidative stress has a profound impact on expression of genes in the vessel wall related to progression and vulnerability of atherosclerotic lesions as well as on parameters of inflammation and cell apoptosis [9,10]. Oxidation is essential for life; however, it is deleterious for the vasculature if oxidative processes are out of control, as seen in endothelial dysfunction and subsequent atherosclerosis [11].
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Reduced NO production
In addition, NO bioactivity may be reduced through other mechanisms beyond breakdown by reactive oxygen species such as reduced NO production by the endothelial NO synthase; e.g. in hypercholesterolaemia there is an interaction between oxidatively modified LDL and certain receptor-mediated signal transduction processes which leads to NO synthesis. Especially lysophosphatidylcholine, a component of oxidized LDL, selectively impairs pertussis toxin sensitive Gi protein-dependent signal transduction pathways which are used by acetylcholine and serotonin [12]. Additionally, elevated concentrations of NO synthase inhibitors, such as asymmetric dimethyl-arginine (ADMA) [13] or L-glutamine, as well as reduced levels of cofactors of NO synthase, such as tetrahydrobiopterin, may contribute to reduced NO production [12]. Furthermore, in the case of insufficient availability of tetrahydrobiopterin, endothelial NO synthase can paradoxically switch towards production of superoxide anions (uncoupling).
NO is not equal to NO
Experimental findings demonstrating that enhanced NO production also may be associated with risk factors, such as especially hypercholesterolaemia [14], seem to contradict the above mentioned pathophysiological mechanisms of reduced NO bioactivity in endothelial dysfunction. However, in these experiments NO bioactivity was reduced as well because of enhanced inactivation from reactive oxygen species. The discrepant findings with respect to NO production in the vasculature might be explained by the fact that not only the amount but also the source of NO is important for biological action. Experimental findings indicate that in atherosclerosis the activity of endothelial NO synthase is reduced. However, total NO production might be enhanced [15,16], since NO is not only produced by endothelial NO synthase (eNOS=NOS III) but also through neuronal NO synthase (nNOS=NOS I) and, more importantly, by inducible NO synthase (iNOS=NOS II) in macrophages and other cell types in the atherosclerotic plaque.
Basic mechanisms of endothelial activation in atherosclerosis
Alteration of the endothelium not only results in dysfunction of the vasodilator capacity of vessels, but also induces a variety of active processes (=endothelial activation) which have a major impact on the vascular milieu participating in inflammatory processes, proliferation and apoptosis of vascular cells.
Inflammation
Reduced NO bioactivity or increased oxidative stress lead to tyrosine nitration of proteins in the vessel wall [1] and activate NF-B, a transcriptional protein increasing proliferation of smooth muscle and other cells [6,10]. Furthermore, reduced NO bioactivity together with enhanced oxidative stress stimulates production of cytokines such as interleukins, TNF
, MCP-1 or interferon, thereby attracting monocytes. Induction of adhesion molecules such as VCAM or ICAM promotes migration of monocytes into the vessel wall, where they differentiate into macrophages (Figure 2
). Accumulation of macrophages and production of cytokines is a characteristic feature of the inflammatory process. Thus, C-reactive protein (CRP), an unspecific marker of inflammation, is related to endothelial dysfunction in the forearm microcirculation of patients with coronary artery disease [17], due to enhanced oxidative stress and reduced NO bioavailability [18].
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Proliferation
The redox equilibrium between NO and reactive oxygen species directly interacts with redox-sensitive expression of genes related to vascular wall proliferation processes [6]. Whereas NO reduces proliferation of vascular smooth muscle cells and migration of monocytes, endothelin-1 as well as angiotensin II directly exert proatherosclerotic effects on the vasculature. Another hint illustrating the physiological relevance of intact endothelial NO bioactivity comes from experimental findings in knock-out mice. Mice lacking the endothelial NO synthase (NOS III) demonstrate a reduced endothelium-dependent dilation, compared with wild type mice, as well as an exaggerated intimal proliferation after external irritation of the vascular wall [26].
An important process in response to plaque development is vascular remodelling, which describes an adaptive increase in vessel size in early atherosclerosis to compensate for luminal encroachment by the growing plaque. The remodelling process, first described by Glagov et al. [27], is thought to be the consequence of increased blood flow at the site of the growing plaque stimulating the endothelium and releasing NO [2830]. Chronically decreased blood flow is associated with a reduction of vessel calibre, whereas an increase of blood flow and thereby of shear stress on the vascular wall will lead to vessel enlargement [28]. Recently, it has been proposed, based on studies in knock-out mice, that NO derived from inducible NO synthase (NOS II) is responsible for vascular growth, initiating remodelling, whereas endothelial NO synthase (NOS III) is involved in the inhibition of wall thickening [31]. However, the initially compensatory, adaptive mechanism of vascular remodelling might turn into a deleterious situation with increased plaque vulnerability in very advanced stages of the disease [32].
Apoptosis
The redox equilibrium between NO and reactive oxygen species also controls apoptosis, or programmed cell death (suicide), which may alter the various physiological functions of the endothelial cell layer. Cardiovascular risk factors influence the balance of vascular cells. Thus, e.g. oxidized LDL or angiotensin II promote apoptosis of endothelial cells [33]; in contrast, shear stress, endothelial NO and antioxidant vitamins inhibit it [34,35].
Endothelial activation and clinical manifestations of atherosclerosis
As described above, endothelial activation, associated with risk factors such as hypercholesterolaemia, is the consequence of a redox imbalance between NO and reactive oxygen species, resulting in low-grade inflammation within the vascular wall. Macrophages attach to the inflamed endothelium and migrate into the vessel wall where they produce further oxidative stress and degenerate to foam cellsthus forming an atherosclerotic plaque (Figure 2). Initially the lipid core of the plaque is covered by a thick fibrous cap. However, especially at the border zone of plaquesadjacent to normal vessel wallmacrophages produce metalloproteinases, enzymes which degrade collagen of the fibrous cap, thus thinning the cap and making the plaque vulnerable to rupture [36,37]. As a result, the same principles seen in endothelial activation, namely oxidative stress and inflammation are also involved in these mechanisms of plaque destabilization.
Cardiovascular events such as acute coronary syndromes (unstable angina or myocardial infarction) as well as ischaemic cerebral infarction are characterized by rupture or erosion of a vulnerable plaque and subsequent thrombosis [36,37]. Even though in this advanced stage of the disease the main pathogenic processes take place within the vascular wall beneath the endothelial cell layer, the activated endothelium on the surface still plays an important role in modulating the manifestation of the disease [38] (Figure 3).
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Paradoxical vasoconstriction associated with endothelial dysfunction is likely to be a trigger for the rupture of vulnerable plaques. Clinical studies have shown enhanced paradoxical vasoconstriction in culprit lesions of patients with unstable angina pectoris, compared with stable angina pectoris [39], indicating endothelial vasodilator dysfunction at these sites.
The integrity of the endothelium determines the extent of thrombus formation after plaque rupture, since the normal endothelium is endowed with a variety of antithrombotic mechanisms: these include antiplatelet activity of NO, prostacyclin, ADPase/CD39, anticoagulant activity of heparin, protein C and protein S, as well as fibrinolytic activity of tPA. In contrast, the activated endothelium has a prothrombotic phenotype induced by CRP, adhesion molecules or PAI. Apoptotic endothelial microparticles, released during an acute coronary syndrome, also exert profound prothrombotic effects [40]. Since most of the plaque ruptures are clinically silent, increased thrombogenicity of the blooddue to endothelial activationmight be the systemic prerequisite for thrombus formation in response to plaque rupture or erosion. In addition to this inflammation of the blood, local inflammation within the plaque, characterized by macrophages which produce the thrombogenic tissue factor, is another determinant of thrombus formation. Mediators derived from activated platelets, such as serotonin and thrombin, may aggravate the paradoxical vasoconstriction due to impaired endothelial function in this setting [41].
Prognostic relevance of endothelial vasodilator dysfunction
As described above, there is ample experimental evidence that endothelial activation is involved in plaque growth and impacts on the clinical manifestation of the acute coronary syndrome, suggesting that endothelial activation is a connector between functional alterations (e.g. endothelial vasodilator dysfunction) and progression of atherosclerotic disease.
To prove this hypothesis, we have recently performed a clinical study in patients in whom endothelial function of the epicardial coronary arteries had been tested. The follow-up was 10 years. Endothelial dysfunction was a strong and independent predictor of cardiovascular event rates during long-term follow-up [42]. Beginning after about 2 years of follow-up, patients with a paradoxical vasoconstriction to acetylcholine (indicating endothelial dysfunction) had a progressively worse outcome than patients with intact endothelial vasodilation of epicardial arteries. In addition to the pharmacological test, using acetylcholine, physiologically more relevant tests (such as flow-dependent dilation and sympathetic activation by cold pressor testing) were associated with adverse long-term prognosis as well. Of importance, the prognostic value of endothelial dysfunction was independent of the presence of other cardiovascular risk factors, indicating that assessment of coronary endothelial vasodilator function is a diagnostically useful index which integrates the stress imposed on the vasculature by the classical cardiovascular risk factors.
In addition, blunted endothelium-independent vasodilation in response to exogenous NO (nitroglycerin) was related to an adverse long-term prognosis as well. It is intriguing to speculate that oxidative stress within the vessel wall reduced both endogenous and exogenous NO in patients with adverse prognosis. Alternatively, an alteration in the signal transduction cascade downstream of NO might be present in these patients, such as a reduced sensitivity of guanylyl cyclase [43].
In addition, the integrity of the coronary microcirculation seems to be important for progression of epicardial disease, since the increment in blood flow determines the magnitude of epicardial shear stress thereby and NO production. Indeed, Al Suwaidi et al. [44] have recently shown that impaired acetylcholine-induced blood-flow regulation was predictive of cardiovascular events. Due to its invasive nature, assessing endothelium-dependent vasodilation of coronary arteries will also in future be limited to research protocols. Recently, Heitzer et al. [45] demonstrated that endothelial dysfunction associated with oxidative stress, as measured by less invasive forearm plethysmography, also predicted cardiovascular events in patients with coronary artery disease.
Treatment of endothelial activation
Experimentally, improvement of endothelial dysfunction precedes regression of atherosclerosis, suggesting that therapeutic strategies which improve endothelial vasodilator function might also improve prognosis [46]. Whereas cardioprotective therapies such as statins or angiotensin-converting enzyme (ACE)-inhibitors have indeed been demonstrated to prevent cardiovascular events, doubt persists with respect to other strategies such as oestrogens.
Statins
Several large-scale clinical trials in primary (WOSCOP, AFCAPS/TEXCAPS) or secondary prevention (LIPID, CARE, 4S (for review see [47]), recently the Heart Protection Study) as well as trials addressing the early phase of an acute coronary syndrome (MIRACL) have provided additional evidence that statins reduce cardiovascular events. Non-pharmacological as well as pharmacological lipid-lowering therapies with different agents improve endothelium-dependent arterial vasodilation. However, therapy with statins, which primarily inhibit the lipid-lowering HMG-CoA reductase, has an exceptional position among the pharmacological strategies to reduce cholesterol levels: besides lowering cholesterol, statins have additional pleiotropic effects on the process of atherosclerosis. Inhibition of the HMG-CoA reductase not only interferes with cholesterol biosynthesis but inhibits also the production of additional mevalonate products such as isoprenoids, which influence cell proliferation processes (Figure 4). In addition, it has been shown that statins directly activate endothelial NO synthase [48]. Clinical studies demonstrated an additional benefit of statin therapy on endothelial dysfunction even after the maximal lipid lowering effect has been reached, supporting the notion that statins have beneficial effects on the vasculature independent of cholesterol lowering [49]. Besides improving endothelium-dependent vasodilation, statins also reduce serum CRP as a marker of inflammation [50] and act on the coagulation system [51], indicating that further parameters of endothelial activation are influenced favourably. In addition, improvement of endothelial vasodilator function results in anti-ischaemic effects, as demonstrated by ST segment analysis during 48 h Holter monitoring [52]. In a retrospective analysis of the CARE trial, in patients with moderate LDL cholesterol levels, markers of inflammation selectively identified those patients who benefited most from statin therapy [53]. Thus, pleiotropic effects of statins, including improvement of endothelial activation, contribute to improvement of prognosis with lipid-lowering therapy in patients with hypercholesterolaemia.
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ACE-inhibitors and angiotensin blockers
Angiotensin-converting enzyme [54] as well as angiotensin II have been detected in atherosclerotic plaques. Expression of angiotensin II is associated with expression of inflammatory cytokines such as interleukin 6 and macrophages [55]. Thus, the renin angiotensin system aggravates the inflammatory process in the vessel wall. The infusion of ACE-inhibitors acutely improves endothelium-dependent vasodilation in the human forearm. Apart from reduction of angiotensin II, inhibition of the breakdown of bradykinin seems to be important [56]. Bradykinin stimulates endothelial NO synthesis. Chronic ACE-inhibition improves coronary endothelial vasodilator dysfunction (TREND) [57] and increases myocardial blood-flow [58]. In addition, ACE-inhibition has beneficial effects on fibrinolysis, coagulation and the inflammatory process [59,60]. The HOPE study [61] demonstrated that during a 4-year follow-up administration of the ACE-inhibitor ramipril (titrated to 10 mg/day), cardiovascular events were reduced by more than 20%. Since this effect was not explained by blood pressure lowering, it is intriguing to speculate that improvement of endothelial activation might have contributed to the results. Like ACE-inhibitors, angiotensin II receptor blockers are also able to improve endothelial dysfunction. Correspondingly, a recent study (LIFE) showed a decrease in strokes in hypertensive patients treated with losartan compared with atenolol [62] as well as other cardiovascular events in hypertensive diabetics [63]. However, so far no direct comparison is available to define whether angiotensin receptor blockers are as effective as ACE-inhibitors to prevent cardiovascular events.
Vitamins and hormones
Improvement of endothelium-dependent vasodilation might serve as an index to estimate the ability of a drug to reduce cardiovascular events. Nevertheless, improvement of endothelial dysfunction per se is insufficient to evaluate its therapeutic potential, since additional effects of a drug might override the beneficial effect associated with ameliorated endothelial dysfunction. Thus, despite having shown to improve endothelial dysfunction and progression of carotid artery wall thickening (CLAS and ARIC study), there is still no hard evidence that antioxidative vitamins such as vitamin E improve outcome (ATVB, CHAOS, GISSI, HOPE) [64]. One reason for the failure of vitamin E might be that in the presence of oxidative stress the radical -tocopherol (vitamin E) produces further radicals, which might result in enhanced, rather than reduced, oxidative stress within the vessel wall [65]. Similarly, oestrogens certainly improve endothelium-dependent vasodilation in several vascular beds, but, in contrast to such functional findings, long-term oestrogen administration to post-menopausal women appears to have deleterious effects on rates of cardiovascular events such as myocardial infarction, strokes or venous thromboembolism (HERS [66], WHI [67]). Open label extension of the HERS trial (HERS II) demonstrated that this risk is not limited to the first years of therapy but rather continues throughout the treatment [68]. Thus, presumption of benefit of hormone therapybased on pathophysiological and epidemiological studiesclearly changed into demonstration of harm. Probably, the adverse effects of hormone replacement on cardiovascular events may be related to a different noxious effect of oestrogens on the vasculature, namely the induction of a pro-inflammatory response, as shown by an increase of high sensitivity CRP levels [69].
Perspectives
The growing knowledge of the pathophysiology of atherogenesis including the important role of endothelial activation may have a profound impact on the management of patients at risk of atherosclerotic disease.
Risk stratification
Assessment of endothelial dysfunction or inflammatory parameters might serve as a surrogate marker of disease activity to more precisely predict the risk of cardiovascular events. However, before their routine use in clinical practice a variety of questions have to be answered by pending prospective studies [70].
Which is the optimal test for prognostic evaluation? Non- or minimal-invasive tests for endothelial vasodilator function such as forearm plethysmography will have to be compared with other activity markers, such as inflammatory parameters (e.g. CRP, phospholipase or heat shock protein), to see whether they are better than traditional risk assessment by the Framingham risk factors. It will further have to be clarified which marker provides additive information and which marker is redundant. In addition, the role of methods to detect morphological manifestations of the disease, such as coronary calcium by computerized tomography, intima-media thickness by ultrasound or evolving magnetic resonance imaging techniques, will have to be determined within this scenario.
What are the predictive values for individual patients? Whatever method will be chosen in future to predict cardiovascular risk, it will have to be standardized with respect to methodology and cut-off values. Positive and negative predictive value for the individual patient and the clinical condition will have to be identified.
Guidance of therapy
Since retrospective studies suggest enhanced effectiveness of statins in patients with elevated inflammatory parameters [53], it has been suggested to use CRP for the decision when to initiate treatment in order to improve cost-effectiveness. However, prospective studies are still lacking. They are needed before such a concept can be applied to clinical routine. Likewise, inflammatory markers or endothelial function may serve as an index to guide the effectiveness and dose of protective cardiovascular therapy, such as ACE-inhibitors. However, again, prospective studies to support this concept are lacking.
New therapeutic concepts
Probably the most exciting aspects derived from better knowledge of the fundamental role of endothelial activation and inflammation in atherogenesis are new therapeutic concepts of how to interfere with disease progression.
Anti-inflammatory therapy
Selective blockade of cytokines such as TNF improves endothelial function [71]. However, clinical studies with TNF
blockers in heart failure have been stopped because of adverse side effects. Other targets such as CRP receptors might be interesting to study in the future. However, as the example with TNF
blockers demonstrates, a proof of the concept using pathophysiological surrogate markers, e.g. by demonstrating improved endothelial vasodilator dysfunction, must be complemented by clinical end point studies.
Gene therapy
Currently, gene therapy of atherosclerosis passes the threshold from experimental to clinical therapy [72]. Experimentally, transfection of endothelial NO synthase improved endothelium-dependent vasodilator function [73]. However, to achieve a benefit by gene therapy might be more difficult in a multifactorial disease such as endothelial dysfunction and atherosclerosis where numerous counteracting and balancing systems exist together compared with unifactorial diseases such a certain haemostatic disorders [9].
Stem cell therapy
Bone marrow derived endothelial progenitor cells, involved in post-natal vascularization, are mobilized during ischaemia [74]. Experimentally, transplantation of endothelial progenitor cells can rescue ischaemic tissue and improve heart function after myocardial infarction [75,76]. Thus the concept is attractive to use adult stem cells in order to regenerate the endothelium (altered by the atherosclerotic process) or induce therapeutic neovascularization in the setting of an acute coronary syndrome. First clinical feasibility studies are ongoing.
Acknowledgments
The authors are supported by a grant from the Deutsche Forschungsgemeinschaft (TP C5, SFB 553).
Notes
Correspondence and offprint requests to: Volker Schächinger, Department of Medicine IV, Division of Cardiology, J. W. Goethe University, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany. Email: schaechinger{at}em.uni-frankfurt.de
References