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Endothelial dysfunction in acute and chronic coronary syndromes: evidence for a pathogenetic role of oxidative stress
ABB Archives of Biochemistry and Biophysics 420 (2003) 255–261 www.elsevier.com/locate/yabbi
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Endothelial dysfunction in acute and chronic coronary syndromes: evidence for a pathogenetic role of oxidative stress Marco Valgimigli,* Elisa Merli, Patrizia Malagutti, Olga Soukhomovskaia, Giordano Cicchitelli, Gaetano Macrı, and Roberto Ferrari Department of Cardiology, University of Ferrara c/o Arcispedale S.Anna, C.rso Giovecca 203, 44100 Ferrara, Italy Received 12 February 2003, and in revised form 16 July 2003 Communicated by Luke I. Szweda
Abstract The past two decades have highlighted the pivotal role of the endothelium in preserving vascular homeostasis. Among others, nitric oxide (NO) is currently believed to be the main component responsible for endothelium dependent vasorelaxation and therefore for endothelial function integrity. Reduced NO bioavailability causes the so-called ‘‘endothelial dysfunction,’’ which seems to be the common molecular disorder comprising stable atherosclerotic narrowing lesions or acute plaque rupture causing unstable angina or myocardial infarction. Compelling evidence is accumulating, stressing the role of oxidative stress in causing reduced NO bioavailability and subsequently endothelial dysfunction (ED). More recently, the role of endothelial cell (EC) apoptosis as a possible final stage of ED and plaque activation has been suggested. In vitro and in vivo evidence suggests a role of oxidative stress also as a putative mechanism finally leading to plaque denudation and activation through increased EC apoptosis. Thus, oxidative stress, irrespective of atherosclerotic disease stages, seems to represent a key phenomenon in vascular disease progression and possible prevention. Ó 2003 Elsevier Inc. All rights reserved.
Crucial advances in the understanding of the pathogenesis of atherosclerosis have been achieved during the last two decades. Several factors are likely to be involved in the initiation and evolution of this disease [1–4]. Among the most critical are endothelial dysfunction and inflammation that have a role in the initiation and progression of atherosclerotic disease. Since the atherosclerotic and thrombotic processes appear somewhat interdependent, they are usually incorporated in a single term: ‘‘atherothrombosis.’’ Atherothrombosis is a systemic disease involving the intima of large- and medium-sized arteries, including the aorta, carotid, coronary, and peripheral arteries. In more advanced stages of the disease, secondary changes may occur in the underlying media and adventitia [5]. Evidence indicates that both plaque composition and its propensity to rupture are major determinants of fu* Corresponding author. Fax: +39-0532-241885. E-mail address: [email protected] (M. Valgimigli).
0003-9861/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2003.07.006
ture ischemic events. Disruption-prone plaques in the coronary arteries (the so-called ‘‘high-risk’’ or ‘‘vulnerable’’ plaques) tend to have a thin fibrous cap, a large lipid core, and a high macrophage content [6]. Acute coronary syndromes (ACS)1 often result from disruption of such modestly stenotic, lipid-rich, vulnerable plaques (which are not detectable by X-ray angiography), leading to thrombotic complications [7]. By contrast with vulnerable plaques in the coronary artery, the disruption-prone, high-risk plaques in the carotid arteries are severely stenotic. Therefore, viewed from the more global perspective of systemic atherothrombotic 1 Abbreviations used: ACS, acute coronary syndromes; SMC, smooth muscle cells; ET-1, endothelin-1; ACh, acetylcholine; cGMP, cyclic guanosine monophosphate; NOS, NO synthase; TNF-a, tumor necrosis factor-a; ecNOS, constitutive form of NOS; ED, endothelial dysfunction; EC, endothelial cell; iNOS, inducible form of NOS; CAD, coronary artery disease; AVD, atherosclerotic vascular disease; LDL, low-density lipoprotein; UA, unstable angina; MI, myocardial infarction; HUVEC, human umbilical vein endothelial cell.
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disease, the term ‘‘high-risk’’ plaque, rather than the classic term ‘‘vulnerable’’ plaque (that implies only the presence of a lipid-rich core), is more appropriate for defining a disruption-prone plaque.
Initiation of atherothrombotic disease: the role of the endothelium The past two decades have highlighted the pivotal role of the endothelium in preserving vascular homeostasis (by controlling vasomotion) and hemostasis (by balancing pro- and anti-thrombotic properties). The endothelium, the inner layer of blood vessels, is a dynamic autocrine and paracrine organ. It regulates contractile, secretory, and mitogenic activities in the vessel wall, as well as blood thrombogenicity, by producing several locally active substances. ‘‘Vascular homeostasis,’’ defined as the ability of the vascular system to maintain normal hemorheological conditions, is guaranteed by adequate control of vasomotion through the balanced production of potent vasodilators such as nitric oxide (NO) and/or adenosine as well as vasoconstrictors such as endothelin-1 (ET-1) and angiotensin II.
Endothelial dysfunction Pioneering experiments by Furchgott and Zawadzki [8] showed that the presence of intact endothelium is essential for acetylcholine (ACh) to induce dilation of isolated arteries. In contrast, if the endothelium is removed, the arteries constrict in response to ACh. Subsequent studies revealed that ACh stimulated the release of a potent vasodilating substance by the endothelium, identified as NO [9,10]. When NO is lost—as after mechanical denudation of the endothelium or pathologic conditions affecting the endothelium—the normal vasodilator response to ACh is replaced by paradoxical constriction resulting from the direct effect of ACh on vascular smooth muscle. NO exerts its relaxing effect on vascular smooth muscle by activatimg guanylate cyclase which, in turn, leads to increased production of cyclic guanosine monophosphate (cGMP) and reduction in intracellular calcium. The role of the endothelium is not confined to the regulation of vascular tone and vasomotor function but extends to the regulation of inflammation, platelet activation, and thrombosis. NO actively mediates many of these functions exerted by intact endothelium. In addition to its potent vasodilating effect, NO exerts a potent inhibitory effect on vascular smooth muscle proliferation [11] and platelet aggregation [12]. Furthermore, NO also promotes platelet disaggregation. These biologic actions of NO make it an important component in the endogenous
defense against vascular injury, inflammation, and thrombosis, all key events involved throughout the course of atherosclerosis. Therefore, it is not surprising that, in much of the literature, this term has been used to refer to an impairment of endothelium-dependent vasorelaxation caused by a deficit of NO bioactivity in the vessel wall. Experimentally, endothelial function and dysfunction can be measured by directly monitoring endothelial NO production from the amino-acid precursor L -arginine, or indirectly by measuring citrulline, a terminal product of NO synthase (NOS), the enzyme that catalyzes the conversion of L -arginine to NO and L -citrulline. Another way to determine endothelial dysfunction is by directly measuring either NOS mRNA or the expression and activity of the protein. Two isoenzymes of NOS have been identified in the endothelium [13]. Despite very close homology in gene sequences, their physiological actions differ markedly. The predominant isoenzyme is the constitutive form of NOS (ecNOS) that requires the presence of calcium, calmodulin, NADPH or other electron donors as cofactors [14]. ecNOS produces relatively small amounts of NO; it is a highly regulated system, closely linked to the control local vasomotor tone. Under particular circumstances, the endothelium can also express a second isoenzyme: the inducible form of NOS (iNOS). This form does not require calcium or calmodulin for its activity and, once expressed, produces large amount of NO, albeit for a limited time [15]. iNOS is found predominantly in activated macrophages in response to cytokine activation that induces cytotoxic amounts of NO. This form of NOS seems particularly important in chronic heart failure and ACS, two linked conditions in which cytokines in general and tumor necrosis factor-a (TNF-a) in particular are highly activated [16,17]. Endothelial synthesis of NO is induced by hormonal stimuli including acetylcholine, bradykinin, and serotonin [18]. These agents act through a receptormediated activation of ecNOS and can be utilized clinically to measure the degree of endothelial function. Usually, this is achieved by using various techniques (e.g., plathysmography or ultrasound) to monitor the vasodilatatory responses to topical administration of an endothelium-dependent vasodilator such as acetylcholine. A decreased response to acetylcholine, but not to endothelium-independent vasodilator nitroprusside, is considered evidence for endothelial dysfunction. Putative role of endothelial dysfunction in the genesis and development of chronic and acute coronary syndromes Many studies demonstrate that in the absence of angiographic evidence of atherosclerosis, endothelial
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dysfunction and activation are one of the earliest markers of disease in patients with atherogenic risk factors. Such markers include male gender [19], aging [20,21], hypertension [22], diabetes mellitus [23], smoking [24], and family history [25]. There is a negative correlation between the number of risk factors and the severity of endothelial dysfunction [19,26]. Vasoconstriction of large epicardial vessels and small resistance coronary arteries is a resultant of endothelial dysfunction in response to physiologic stimuli [27–30], therefore endothelial dysfunction of coronary arteries is presumed to contribute to effort-related angina pectoris. In addition, impaired endothelial function of the coronary microcirculation leads to reduced blood flow supply to the myocardium during increased metabolic demand [28]. Therefore, endothelial dysfunction of the coronary microcirculation alone can cause or promote myocardial ischemia, even in the absence of flow-limiting stenosis of the large epicardial arteries. Indeed, patients with microvascular angina pectoris have been shown to have endothelial dysfunction of the coronary microcirculation [31,32]. On the other hand, there is a debate over the role of endothelial dysfunction in the pathogenesis of epicardial coronary artery spasms in patients with vasospastic angina pectoris [33,34]. Besides contributing to myocardial ischemia, endothelial dysfunction may have a crucial role in ACS, such as unstable angina (UA) or myocardial infarction (MI) [4,35], that are characterized by plaque rupture or erosion followed by platelet aggregation and local thrombus formation [4]. The endothelium has a crucial role in modulating these events because in the presence of endothelial dysfunction, increased constriction in response to platelet products can trigger plaque rupture. Thus, endothelial dysfunction is likely to contribute to ACS worsening while its improvement is actually a therapeutic goal that contributes, at least in part, to reduce clinical events in the long-term. Given this apparent link between loss of NO activity and atherosclerosis, several groups have been interested
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in the concept that endothelium-dependent vasodilatation, a surrogate for NO bioavailability, may predict cardiovascular events (Table 1). Indeed, Suawaidi et al. [36] followed 157 patients with mildly diseased coronary arteries for an average of 28 months and observed cardiac events only in the patients with the lowest tertile of coronary vasodilatation to acetylcholine. Similarly, Schaechinger et al. [37] used three different stimuli for endothelial release of NO: acetylcholine, cold pressor testing, and increased blood flow. The authors showed that responses to each of these stimuli were independent predictors of cardiovascular events during a follow-up period of 8 years. Perticone et al. [38] also demonstrated that endothelial dysfunction in the forearm circulation predicts cardiovascular events in hypertensive patients. More recently, Heitzer et al. [39] have defined further the relationship between endothelial dysfunction and cardiovascular prognosis and have also interestingly linked both phenomena to oxidative stress. The authors examined forearm blood flow in response to acetylcholine in 276 patients with coronary artery disease (CAD). In addition, in a subset of these patients, the authors repeated the testing of acetylcholine responses during intra-arterial infusion of vitamin C. These subjects were then followed for 6 80 months. The authors found that the amount of increase in forearm blood flow in response to acetylcholine was an excellent prognostic indicator, i.e., the subsequent event rate was high in those with blunted responses to acetylcholine. The truly novel aspect of this study is that the abnormal responses to acetylcholine could be dramatically improved by the intra-arterial administration of vitamin C only in the group with cardiovascular events. In contrast, vitamin C infusion had minimal effect on forearm blood flow responses in the subjects that subsequently had no cardiovascular events. The authorsÕ interpretation is that vitamin C infusion corrects oxidative stress, and when the response to vitamin C is large, a large amount of oxidative stress must be present. If this assumption were correct, a high level of oxidant
Table 1 Endothelial dysfunction as a predictor of cardiovascular prognosis Endothelial function as a prognostic marker for cardiovascular disease
Al Suwaidi et al. Circulation 101 (9) (2000) 948–954 Schachinger et al. Circulation 101 (10) (2000) 1899–1906 Neunteufl et al. Am. J. Cardiol. 86 (2) (2000) 207–210 Heitzer et al. Circulation 2001 Gokce et al. Circulation 105 (13) (2002) 1567–1572 Halcox et al. Circulation 106 (6) (2002) 653–658
No. of pts
Disease
Follow-up (months)
Vascular district
157 145 73 281 187 308
CAD CAD CAD CAD AVD CAD
28 (11–52) 80 (13–126) 60 (52–66) 53 (30–87) 1 46 (6–96)
Coronary flow, eicardial arteries Epicardial arteries Brachial artery Forearm microcirculation Brachial artery Coronary flow,eicardial arteries
Endothelial dysfunction measured in different vascular districts in different populations in these publications resulted to be a strong predictor for cardiovascular prognosis independently from baseline cardiovascular risk factors. CAD, coronary artery disease; AVD, atherosclerotic vascular disease. * Clinical events after cardiovascular surgery.
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stress portends a poor prognosis from the standpoint of cardiovascular disease. There is a large amount of data in the literature to support this concept. Central role of oxidative stress in the development of endothelial dysfunction and atherosclerotic disease Numerous studies have demonstrated that oxidative stress plays a pivotal role in the pathogenesis of cardiovascular diseases [40,41]. Superoxide anion is formed by univalent reduction of molecular oxygen. Although several enzymes are involved in the generation of superoxide anion, including xanthine oxidase, NADH/ NADPH oxidase, lipoxygenase, and nitric oxide synthase, one of the largest factories producing superoxide anion in vivo are the mitochondria [42,43]. In patients with mitochondrial disease, vascular complications—not only stroke but also CAD—are commonly observed in young subject without common risk factors for atherosclerosis [44–47]. Abnormal mitochondria, which have defect in substrate utilization in the respiratory chain or in oxidative-phosphorylation coupling, leading to leakage of superoxide anions [48] are accumulated in the endothelium and vascular smooth muscle cells in mitochondrial diseases [49,50]. Endothelium-dependent vasodilation was lost at basal level, which was restored by anti-oxidant ascorbic acid in patients with mitochondrial diseases, suggesting that oxidative stress may be involved in premature cardiovascular diseases and antioxidant may become a therapeutic tool in mitochondrial diseases [51]. Via spontaneous or enzymatically catalyzed dismutation, superoxide anion is reduced to hydrogen peroxide. Transition metal, such as iron or copper-catalyzed interaction with hydrogen peroxide, produces highly toxic hydroxyl radicals [52,53]. A decreased concentration of NO also favors the Haber– Weiss and Fenton reactions, because NO binds to ferrous complexes and inhibits the reaction of iron with hydrogen peroxide [54]. Several epidemiological studies have demonstrated that there is a close association between iron or iron stores and cardiovascular diseases [55,56]. Parenteral iron supplementation impaired endothelial function, which was accompanied by increase in lipid peroxides [57], suggesting that iron overload may play a causal role in the pathogenesis of atherosclerosis through endothelial dysfunction by oxidative stress. Reactive oxygen species have detrimental effect on vascular function through several mechanisms [40,41]. First, as their direct effect, reactive oxygen species, especially hydroxyl radicals, injure cell membrane and nuclei. Second, by interacting with endogenous vasoactive mediators formed in endothelial cells (ECs), reactive oxygen species modulate vasomotion and atherogenic process. For instance, superoxide anions react with endothelium-derived NO rapidly and inactivate its effect [58]. In addition, reactive oxygen species oxidize
an essential cofactor of nitric oxide synthase namely tetrahydrobiopterin, yielding de novo synthesis of superoxide anion, instead of NO, from this enzyme [59,60]. Third, reactive oxygen species peroxidize lipid components, leading to formation of oxidized low-density lipoproteins (LDLs), which are one of the key mediators of atherosclerosis [61,62]. Oxidized LDL is not only incorporated into macrophages yielding to the formation of foam cells, but also stimulates production of cytokines from vascular tissues. These cytokines induce phenotypic change of vascular smooth muscle cell and stimulate the migration of monocyte and platelet activation. Like hydroxyl radical, oxidized LDLs are also directly cytotoxic to vascular cells, thus promoting the release of lipids and lysozomal enzymes aggravating tissue injury. In addition, oxidative stress stimulates the synthesis of homocysteine, a known risk factor for atherosclerosis [63,64], and methylarginine, an endogenous inhibitor of NO synthase [65,66]. Taken together, oxidative stress induced by reactive oxygen species and their reactants may impair the biological activity of endothelium-derived NO leading to the progression of atherosclerosis (Fig. 1).
Fig. 1. ROS impairs biological activity of NO. Oxidative stress induced by reactive oxygen species impairs the biological activities of endothelium-derived NO through several mechanisms. Modified from Matsuoka H. Endothelial dysfunction associated with oxidative stress in human. Diabetes Research and Clinical Practice 2001;54 Suppl 2:S65–S72.
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Endothelial cell apoptosis as the final stage of endothelial dysfunction and a possible link to atherosclerotic plaque activation It has been suggested that increased endothelial apoptosis leads to atheroma denudation and subsequent coronary thrombosis [4,67–71]. Endothelial apoptosis could also increase susceptibility toward coronary thrombosis through the release of apoptotic cell debris into the bloodstream, which is known to directly activate the coagulation cascade [72–74]. Lutgens et al. [75] have reported that the number of apoptotic cells in the plaque is related to the stage of plaque development, being higher in advanced lesions, suggesting a possible cause-effect relation. On the other hand, it is also known that the plaque itself could release, once activated, biochemical substances such as pro-oxidants [76–78], which, in turn, can stimulate the apoptotic process [79–81]. NO induces apoptosis in smooth muscle cells (SMC) and some leukocytes, but confers resistance to apoptosis in EC [82]. Molecular studies have indicated that NO may inhibit EC apoptosis through an attenuation of caspase activity. S-nitrosylation of these proteases inhibits their activity and protects cells from apoptosis [83]. NO nitrosylates caspase-3 at cys-163 in EC. Shear stress protects the arterial wall from atherosclerosis and inhibits ECs apoptosis. Apoptotic ECs are localized to areas of decreased blood flow in atherosclerotic lesion. Shear stress is currently considered to inhibit EC apoptosis through up-regulation of eNOS. Decreased blood flow may therefore sensitize EC to apoptosis, thereby initiating atherosclerosis. Also, loss of EC integrity during atherosclerosis may decrease the blood concentration of NO, leading to an increase in SMC proliferation, leukocyte extravasation, and platelet aggregation. OxLDL can decrease the synthesis and secretion of NO in EC. OxLDL may decrease EC uptake of L -arginine and in this way inhibit synthesis of NO. Therefore, a reduction of endothelial NO by stimuli such as oxLDL may be an additional mechanism for EC sensitization to apoptosis in atherosclerotic lesions. When taken together, these data seem to indicate that increased oxidative stress inside arterial wall, by decreasing NO bio-availability can cause endothelial dysfunction during first stages of atherosclerotic lesion and subsequently enhance EC apoptosis, resulting in atherosclerosis progression and possibly plaque from stable to unstable transition. Some recently published data from our group tend to support this view [84]. We evaluated whether incubation with serum from patients with ACS is pro-apoptotic on human umbilical vein endothelial cells (HUVECs) compared with that from patients with stable angina (SA) and from healthy subjects (Fig. 2). Serum from patients with ACS has also been re-evaluated at 1-year follow-up, in stable clinical conditions. To gain further insights into this mechanism,
Fig. 2. Nuclear fragmentation during apoptotic process. Representative image showing a nucleus of human endothelial cells (colored in red with propidium iodide) at fluorescence microscopy with typical nuclear fragmentation (arrow) that identifies a specific phase of apoptotic process.
the roles of cytokine activation and oxidative stress have been investigated by in vitro addition of anti-human cytokines (TNF-a and interleukin [IL]-6) monoclonal antibodies and of the antioxidant Trolox on HUVECs incubated with serum from patients with ACS. We found that serum from patients with ACS is markedly pro-apoptotic towards HUVECs, while patients with stable atherosclerotic lesions (e.g., patients with SA) did not differ in terms of apoptosis from healthy subjects (Fig. 3). Interestingly enough, when
Fig. 3. Serum-induced HUVEC apoptosis in controls and patients. HUVECs treated either with serum subtraction (positive control) or with serum from either healthy subjects (Group 1) and patients with SA (Group 2) or ACS (Group 3). Mean data SD are reported. (*p < 0:001 vs. Group 1).
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more advanced stages of the disease, and especially during ACS, the integrity of endothelial layer is lost by EC apoptotic process activation and subsequently plaque destabilization and denudation. Oxidative stress is currently considered to play a pathogenetic role both during early and later stages of the atherosclerotic disease. In the future, the control and modulation of oxidative imbalance inside the arterial wall could provide a novel mechanism for atherosclerosis prevention and protection.
References
Fig. 4. Effect of addition of anti-cytokine antibodies and Trolox on HUVEC apoptosis. Co-incubation of HUVECs with serum from patients of Group 3 resulted in an amount of apoptotic nuclei (white column) that was not modified by in vitro addition of monoclonal antibodies against human TNF-a or IL-6, either alone or together. The addition of Trolox (black column) induced a significant reduction of apoptotic nuclei (*p < 0:001 vs. baseline). Mean data SD are reported.
patients with ACS have been reconsidered at 12-month follow-up, they did not differ anymore from patients with stable lesions, suggesting that increased apoptotic activity of serum is temporally linked to ACS. The pro-apoptotic effect was not related to the degree of necrosis (i.e., UA vs. MI) or to the level of cytokine activation and was not significantly affected by coincubation with anti-TNF-a or anti-IL6 human antibodies. On the contrary, coincubation with the antioxidant Trolox showed a reduction of apoptotic nuclei by >50% (Fig. 4). When taken together, these data indicate that the acute setting of coronary events is not necessarily a localized vascular insult because the systemic proapoptotic activity of the serum could exert deleterious effects at a distance. This could contribute to the explanation of the pan-coronary syndromes [85,86].
Conclusions Atherosclerosis appears to be a life-long disease, starting inside the maternal uterus [87] but finally leading patients to medical attention only during advanced ages. The endothelium is currently considered to play a central role in atherosclerotic lesion generation and progression. We have accumulated a large amount of evidence suggesting that during the first phases of the disease, endothelium is morphologically normal but functionally impaired through the decreased bioavailability of NO (i.e., endothelial dysfunction). During
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Endothelial dysfunction in acute and chronic coronary syndromes: evidence for a pathogenetic role of oxidative stress Marco Valgimigli,* Elisa Merli, Patrizia Malagutti, Olga Soukhomovskaia, Giordano Cicchitelli, Gaetano Macrı, and Roberto Ferrari Department of Cardiology, University of Ferrara c/o Arcispedale S.Anna, C.rso Giovecca 203, 44100 Ferrara, Italy Received 12 February 2003, and in revised form 16 July 2003 Communicated by Luke I. Szweda
Abstract The past two decades have highlighted the pivotal role of the endothelium in preserving vascular homeostasis. Among others, nitric oxide (NO) is currently believed to be the main component responsible for endothelium dependent vasorelaxation and therefore for endothelial function integrity. Reduced NO bioavailability causes the so-called ‘‘endothelial dysfunction,’’ which seems to be the common molecular disorder comprising stable atherosclerotic narrowing lesions or acute plaque rupture causing unstable angina or myocardial infarction. Compelling evidence is accumulating, stressing the role of oxidative stress in causing reduced NO bioavailability and subsequently endothelial dysfunction (ED). More recently, the role of endothelial cell (EC) apoptosis as a possible final stage of ED and plaque activation has been suggested. In vitro and in vivo evidence suggests a role of oxidative stress also as a putative mechanism finally leading to plaque denudation and activation through increased EC apoptosis. Thus, oxidative stress, irrespective of atherosclerotic disease stages, seems to represent a key phenomenon in vascular disease progression and possible prevention. Ó 2003 Elsevier Inc. All rights reserved.
Crucial advances in the understanding of the pathogenesis of atherosclerosis have been achieved during the last two decades. Several factors are likely to be involved in the initiation and evolution of this disease [1–4]. Among the most critical are endothelial dysfunction and inflammation that have a role in the initiation and progression of atherosclerotic disease. Since the atherosclerotic and thrombotic processes appear somewhat interdependent, they are usually incorporated in a single term: ‘‘atherothrombosis.’’ Atherothrombosis is a systemic disease involving the intima of large- and medium-sized arteries, including the aorta, carotid, coronary, and peripheral arteries. In more advanced stages of the disease, secondary changes may occur in the underlying media and adventitia [5]. Evidence indicates that both plaque composition and its propensity to rupture are major determinants of fu* Corresponding author. Fax: +39-0532-241885. E-mail address: [email protected] (M. Valgimigli).
0003-9861/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2003.07.006
ture ischemic events. Disruption-prone plaques in the coronary arteries (the so-called ‘‘high-risk’’ or ‘‘vulnerable’’ plaques) tend to have a thin fibrous cap, a large lipid core, and a high macrophage content [6]. Acute coronary syndromes (ACS)1 often result from disruption of such modestly stenotic, lipid-rich, vulnerable plaques (which are not detectable by X-ray angiography), leading to thrombotic complications [7]. By contrast with vulnerable plaques in the coronary artery, the disruption-prone, high-risk plaques in the carotid arteries are severely stenotic. Therefore, viewed from the more global perspective of systemic atherothrombotic 1 Abbreviations used: ACS, acute coronary syndromes; SMC, smooth muscle cells; ET-1, endothelin-1; ACh, acetylcholine; cGMP, cyclic guanosine monophosphate; NOS, NO synthase; TNF-a, tumor necrosis factor-a; ecNOS, constitutive form of NOS; ED, endothelial dysfunction; EC, endothelial cell; iNOS, inducible form of NOS; CAD, coronary artery disease; AVD, atherosclerotic vascular disease; LDL, low-density lipoprotein; UA, unstable angina; MI, myocardial infarction; HUVEC, human umbilical vein endothelial cell.
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disease, the term ‘‘high-risk’’ plaque, rather than the classic term ‘‘vulnerable’’ plaque (that implies only the presence of a lipid-rich core), is more appropriate for defining a disruption-prone plaque.
Initiation of atherothrombotic disease: the role of the endothelium The past two decades have highlighted the pivotal role of the endothelium in preserving vascular homeostasis (by controlling vasomotion) and hemostasis (by balancing pro- and anti-thrombotic properties). The endothelium, the inner layer of blood vessels, is a dynamic autocrine and paracrine organ. It regulates contractile, secretory, and mitogenic activities in the vessel wall, as well as blood thrombogenicity, by producing several locally active substances. ‘‘Vascular homeostasis,’’ defined as the ability of the vascular system to maintain normal hemorheological conditions, is guaranteed by adequate control of vasomotion through the balanced production of potent vasodilators such as nitric oxide (NO) and/or adenosine as well as vasoconstrictors such as endothelin-1 (ET-1) and angiotensin II.
Endothelial dysfunction Pioneering experiments by Furchgott and Zawadzki [8] showed that the presence of intact endothelium is essential for acetylcholine (ACh) to induce dilation of isolated arteries. In contrast, if the endothelium is removed, the arteries constrict in response to ACh. Subsequent studies revealed that ACh stimulated the release of a potent vasodilating substance by the endothelium, identified as NO [9,10]. When NO is lost—as after mechanical denudation of the endothelium or pathologic conditions affecting the endothelium—the normal vasodilator response to ACh is replaced by paradoxical constriction resulting from the direct effect of ACh on vascular smooth muscle. NO exerts its relaxing effect on vascular smooth muscle by activatimg guanylate cyclase which, in turn, leads to increased production of cyclic guanosine monophosphate (cGMP) and reduction in intracellular calcium. The role of the endothelium is not confined to the regulation of vascular tone and vasomotor function but extends to the regulation of inflammation, platelet activation, and thrombosis. NO actively mediates many of these functions exerted by intact endothelium. In addition to its potent vasodilating effect, NO exerts a potent inhibitory effect on vascular smooth muscle proliferation [11] and platelet aggregation [12]. Furthermore, NO also promotes platelet disaggregation. These biologic actions of NO make it an important component in the endogenous
defense against vascular injury, inflammation, and thrombosis, all key events involved throughout the course of atherosclerosis. Therefore, it is not surprising that, in much of the literature, this term has been used to refer to an impairment of endothelium-dependent vasorelaxation caused by a deficit of NO bioactivity in the vessel wall. Experimentally, endothelial function and dysfunction can be measured by directly monitoring endothelial NO production from the amino-acid precursor L -arginine, or indirectly by measuring citrulline, a terminal product of NO synthase (NOS), the enzyme that catalyzes the conversion of L -arginine to NO and L -citrulline. Another way to determine endothelial dysfunction is by directly measuring either NOS mRNA or the expression and activity of the protein. Two isoenzymes of NOS have been identified in the endothelium [13]. Despite very close homology in gene sequences, their physiological actions differ markedly. The predominant isoenzyme is the constitutive form of NOS (ecNOS) that requires the presence of calcium, calmodulin, NADPH or other electron donors as cofactors [14]. ecNOS produces relatively small amounts of NO; it is a highly regulated system, closely linked to the control local vasomotor tone. Under particular circumstances, the endothelium can also express a second isoenzyme: the inducible form of NOS (iNOS). This form does not require calcium or calmodulin for its activity and, once expressed, produces large amount of NO, albeit for a limited time [15]. iNOS is found predominantly in activated macrophages in response to cytokine activation that induces cytotoxic amounts of NO. This form of NOS seems particularly important in chronic heart failure and ACS, two linked conditions in which cytokines in general and tumor necrosis factor-a (TNF-a) in particular are highly activated [16,17]. Endothelial synthesis of NO is induced by hormonal stimuli including acetylcholine, bradykinin, and serotonin [18]. These agents act through a receptormediated activation of ecNOS and can be utilized clinically to measure the degree of endothelial function. Usually, this is achieved by using various techniques (e.g., plathysmography or ultrasound) to monitor the vasodilatatory responses to topical administration of an endothelium-dependent vasodilator such as acetylcholine. A decreased response to acetylcholine, but not to endothelium-independent vasodilator nitroprusside, is considered evidence for endothelial dysfunction. Putative role of endothelial dysfunction in the genesis and development of chronic and acute coronary syndromes Many studies demonstrate that in the absence of angiographic evidence of atherosclerosis, endothelial
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dysfunction and activation are one of the earliest markers of disease in patients with atherogenic risk factors. Such markers include male gender [19], aging [20,21], hypertension [22], diabetes mellitus [23], smoking [24], and family history [25]. There is a negative correlation between the number of risk factors and the severity of endothelial dysfunction [19,26]. Vasoconstriction of large epicardial vessels and small resistance coronary arteries is a resultant of endothelial dysfunction in response to physiologic stimuli [27–30], therefore endothelial dysfunction of coronary arteries is presumed to contribute to effort-related angina pectoris. In addition, impaired endothelial function of the coronary microcirculation leads to reduced blood flow supply to the myocardium during increased metabolic demand [28]. Therefore, endothelial dysfunction of the coronary microcirculation alone can cause or promote myocardial ischemia, even in the absence of flow-limiting stenosis of the large epicardial arteries. Indeed, patients with microvascular angina pectoris have been shown to have endothelial dysfunction of the coronary microcirculation [31,32]. On the other hand, there is a debate over the role of endothelial dysfunction in the pathogenesis of epicardial coronary artery spasms in patients with vasospastic angina pectoris [33,34]. Besides contributing to myocardial ischemia, endothelial dysfunction may have a crucial role in ACS, such as unstable angina (UA) or myocardial infarction (MI) [4,35], that are characterized by plaque rupture or erosion followed by platelet aggregation and local thrombus formation [4]. The endothelium has a crucial role in modulating these events because in the presence of endothelial dysfunction, increased constriction in response to platelet products can trigger plaque rupture. Thus, endothelial dysfunction is likely to contribute to ACS worsening while its improvement is actually a therapeutic goal that contributes, at least in part, to reduce clinical events in the long-term. Given this apparent link between loss of NO activity and atherosclerosis, several groups have been interested
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in the concept that endothelium-dependent vasodilatation, a surrogate for NO bioavailability, may predict cardiovascular events (Table 1). Indeed, Suawaidi et al. [36] followed 157 patients with mildly diseased coronary arteries for an average of 28 months and observed cardiac events only in the patients with the lowest tertile of coronary vasodilatation to acetylcholine. Similarly, Schaechinger et al. [37] used three different stimuli for endothelial release of NO: acetylcholine, cold pressor testing, and increased blood flow. The authors showed that responses to each of these stimuli were independent predictors of cardiovascular events during a follow-up period of 8 years. Perticone et al. [38] also demonstrated that endothelial dysfunction in the forearm circulation predicts cardiovascular events in hypertensive patients. More recently, Heitzer et al. [39] have defined further the relationship between endothelial dysfunction and cardiovascular prognosis and have also interestingly linked both phenomena to oxidative stress. The authors examined forearm blood flow in response to acetylcholine in 276 patients with coronary artery disease (CAD). In addition, in a subset of these patients, the authors repeated the testing of acetylcholine responses during intra-arterial infusion of vitamin C. These subjects were then followed for 6 80 months. The authors found that the amount of increase in forearm blood flow in response to acetylcholine was an excellent prognostic indicator, i.e., the subsequent event rate was high in those with blunted responses to acetylcholine. The truly novel aspect of this study is that the abnormal responses to acetylcholine could be dramatically improved by the intra-arterial administration of vitamin C only in the group with cardiovascular events. In contrast, vitamin C infusion had minimal effect on forearm blood flow responses in the subjects that subsequently had no cardiovascular events. The authorsÕ interpretation is that vitamin C infusion corrects oxidative stress, and when the response to vitamin C is large, a large amount of oxidative stress must be present. If this assumption were correct, a high level of oxidant
Table 1 Endothelial dysfunction as a predictor of cardiovascular prognosis Endothelial function as a prognostic marker for cardiovascular disease
Al Suwaidi et al. Circulation 101 (9) (2000) 948–954 Schachinger et al. Circulation 101 (10) (2000) 1899–1906 Neunteufl et al. Am. J. Cardiol. 86 (2) (2000) 207–210 Heitzer et al. Circulation 2001 Gokce et al. Circulation 105 (13) (2002) 1567–1572 Halcox et al. Circulation 106 (6) (2002) 653–658
No. of pts
Disease
Follow-up (months)
Vascular district
157 145 73 281 187 308
CAD CAD CAD CAD AVD CAD
28 (11–52) 80 (13–126) 60 (52–66) 53 (30–87) 1 46 (6–96)
Coronary flow, eicardial arteries Epicardial arteries Brachial artery Forearm microcirculation Brachial artery Coronary flow,eicardial arteries
Endothelial dysfunction measured in different vascular districts in different populations in these publications resulted to be a strong predictor for cardiovascular prognosis independently from baseline cardiovascular risk factors. CAD, coronary artery disease; AVD, atherosclerotic vascular disease. * Clinical events after cardiovascular surgery.
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stress portends a poor prognosis from the standpoint of cardiovascular disease. There is a large amount of data in the literature to support this concept. Central role of oxidative stress in the development of endothelial dysfunction and atherosclerotic disease Numerous studies have demonstrated that oxidative stress plays a pivotal role in the pathogenesis of cardiovascular diseases [40,41]. Superoxide anion is formed by univalent reduction of molecular oxygen. Although several enzymes are involved in the generation of superoxide anion, including xanthine oxidase, NADH/ NADPH oxidase, lipoxygenase, and nitric oxide synthase, one of the largest factories producing superoxide anion in vivo are the mitochondria [42,43]. In patients with mitochondrial disease, vascular complications—not only stroke but also CAD—are commonly observed in young subject without common risk factors for atherosclerosis [44–47]. Abnormal mitochondria, which have defect in substrate utilization in the respiratory chain or in oxidative-phosphorylation coupling, leading to leakage of superoxide anions [48] are accumulated in the endothelium and vascular smooth muscle cells in mitochondrial diseases [49,50]. Endothelium-dependent vasodilation was lost at basal level, which was restored by anti-oxidant ascorbic acid in patients with mitochondrial diseases, suggesting that oxidative stress may be involved in premature cardiovascular diseases and antioxidant may become a therapeutic tool in mitochondrial diseases [51]. Via spontaneous or enzymatically catalyzed dismutation, superoxide anion is reduced to hydrogen peroxide. Transition metal, such as iron or copper-catalyzed interaction with hydrogen peroxide, produces highly toxic hydroxyl radicals [52,53]. A decreased concentration of NO also favors the Haber– Weiss and Fenton reactions, because NO binds to ferrous complexes and inhibits the reaction of iron with hydrogen peroxide [54]. Several epidemiological studies have demonstrated that there is a close association between iron or iron stores and cardiovascular diseases [55,56]. Parenteral iron supplementation impaired endothelial function, which was accompanied by increase in lipid peroxides [57], suggesting that iron overload may play a causal role in the pathogenesis of atherosclerosis through endothelial dysfunction by oxidative stress. Reactive oxygen species have detrimental effect on vascular function through several mechanisms [40,41]. First, as their direct effect, reactive oxygen species, especially hydroxyl radicals, injure cell membrane and nuclei. Second, by interacting with endogenous vasoactive mediators formed in endothelial cells (ECs), reactive oxygen species modulate vasomotion and atherogenic process. For instance, superoxide anions react with endothelium-derived NO rapidly and inactivate its effect [58]. In addition, reactive oxygen species oxidize
an essential cofactor of nitric oxide synthase namely tetrahydrobiopterin, yielding de novo synthesis of superoxide anion, instead of NO, from this enzyme [59,60]. Third, reactive oxygen species peroxidize lipid components, leading to formation of oxidized low-density lipoproteins (LDLs), which are one of the key mediators of atherosclerosis [61,62]. Oxidized LDL is not only incorporated into macrophages yielding to the formation of foam cells, but also stimulates production of cytokines from vascular tissues. These cytokines induce phenotypic change of vascular smooth muscle cell and stimulate the migration of monocyte and platelet activation. Like hydroxyl radical, oxidized LDLs are also directly cytotoxic to vascular cells, thus promoting the release of lipids and lysozomal enzymes aggravating tissue injury. In addition, oxidative stress stimulates the synthesis of homocysteine, a known risk factor for atherosclerosis [63,64], and methylarginine, an endogenous inhibitor of NO synthase [65,66]. Taken together, oxidative stress induced by reactive oxygen species and their reactants may impair the biological activity of endothelium-derived NO leading to the progression of atherosclerosis (Fig. 1).
Fig. 1. ROS impairs biological activity of NO. Oxidative stress induced by reactive oxygen species impairs the biological activities of endothelium-derived NO through several mechanisms. Modified from Matsuoka H. Endothelial dysfunction associated with oxidative stress in human. Diabetes Research and Clinical Practice 2001;54 Suppl 2:S65–S72.
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Endothelial cell apoptosis as the final stage of endothelial dysfunction and a possible link to atherosclerotic plaque activation It has been suggested that increased endothelial apoptosis leads to atheroma denudation and subsequent coronary thrombosis [4,67–71]. Endothelial apoptosis could also increase susceptibility toward coronary thrombosis through the release of apoptotic cell debris into the bloodstream, which is known to directly activate the coagulation cascade [72–74]. Lutgens et al. [75] have reported that the number of apoptotic cells in the plaque is related to the stage of plaque development, being higher in advanced lesions, suggesting a possible cause-effect relation. On the other hand, it is also known that the plaque itself could release, once activated, biochemical substances such as pro-oxidants [76–78], which, in turn, can stimulate the apoptotic process [79–81]. NO induces apoptosis in smooth muscle cells (SMC) and some leukocytes, but confers resistance to apoptosis in EC [82]. Molecular studies have indicated that NO may inhibit EC apoptosis through an attenuation of caspase activity. S-nitrosylation of these proteases inhibits their activity and protects cells from apoptosis [83]. NO nitrosylates caspase-3 at cys-163 in EC. Shear stress protects the arterial wall from atherosclerosis and inhibits ECs apoptosis. Apoptotic ECs are localized to areas of decreased blood flow in atherosclerotic lesion. Shear stress is currently considered to inhibit EC apoptosis through up-regulation of eNOS. Decreased blood flow may therefore sensitize EC to apoptosis, thereby initiating atherosclerosis. Also, loss of EC integrity during atherosclerosis may decrease the blood concentration of NO, leading to an increase in SMC proliferation, leukocyte extravasation, and platelet aggregation. OxLDL can decrease the synthesis and secretion of NO in EC. OxLDL may decrease EC uptake of L -arginine and in this way inhibit synthesis of NO. Therefore, a reduction of endothelial NO by stimuli such as oxLDL may be an additional mechanism for EC sensitization to apoptosis in atherosclerotic lesions. When taken together, these data seem to indicate that increased oxidative stress inside arterial wall, by decreasing NO bio-availability can cause endothelial dysfunction during first stages of atherosclerotic lesion and subsequently enhance EC apoptosis, resulting in atherosclerosis progression and possibly plaque from stable to unstable transition. Some recently published data from our group tend to support this view [84]. We evaluated whether incubation with serum from patients with ACS is pro-apoptotic on human umbilical vein endothelial cells (HUVECs) compared with that from patients with stable angina (SA) and from healthy subjects (Fig. 2). Serum from patients with ACS has also been re-evaluated at 1-year follow-up, in stable clinical conditions. To gain further insights into this mechanism,
Fig. 2. Nuclear fragmentation during apoptotic process. Representative image showing a nucleus of human endothelial cells (colored in red with propidium iodide) at fluorescence microscopy with typical nuclear fragmentation (arrow) that identifies a specific phase of apoptotic process.
the roles of cytokine activation and oxidative stress have been investigated by in vitro addition of anti-human cytokines (TNF-a and interleukin [IL]-6) monoclonal antibodies and of the antioxidant Trolox on HUVECs incubated with serum from patients with ACS. We found that serum from patients with ACS is markedly pro-apoptotic towards HUVECs, while patients with stable atherosclerotic lesions (e.g., patients with SA) did not differ in terms of apoptosis from healthy subjects (Fig. 3). Interestingly enough, when
Fig. 3. Serum-induced HUVEC apoptosis in controls and patients. HUVECs treated either with serum subtraction (positive control) or with serum from either healthy subjects (Group 1) and patients with SA (Group 2) or ACS (Group 3). Mean data SD are reported. (*p < 0:001 vs. Group 1).
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more advanced stages of the disease, and especially during ACS, the integrity of endothelial layer is lost by EC apoptotic process activation and subsequently plaque destabilization and denudation. Oxidative stress is currently considered to play a pathogenetic role both during early and later stages of the atherosclerotic disease. In the future, the control and modulation of oxidative imbalance inside the arterial wall could provide a novel mechanism for atherosclerosis prevention and protection.
References
Fig. 4. Effect of addition of anti-cytokine antibodies and Trolox on HUVEC apoptosis. Co-incubation of HUVECs with serum from patients of Group 3 resulted in an amount of apoptotic nuclei (white column) that was not modified by in vitro addition of monoclonal antibodies against human TNF-a or IL-6, either alone or together. The addition of Trolox (black column) induced a significant reduction of apoptotic nuclei (*p < 0:001 vs. baseline). Mean data SD are reported.
patients with ACS have been reconsidered at 12-month follow-up, they did not differ anymore from patients with stable lesions, suggesting that increased apoptotic activity of serum is temporally linked to ACS. The pro-apoptotic effect was not related to the degree of necrosis (i.e., UA vs. MI) or to the level of cytokine activation and was not significantly affected by coincubation with anti-TNF-a or anti-IL6 human antibodies. On the contrary, coincubation with the antioxidant Trolox showed a reduction of apoptotic nuclei by >50% (Fig. 4). When taken together, these data indicate that the acute setting of coronary events is not necessarily a localized vascular insult because the systemic proapoptotic activity of the serum could exert deleterious effects at a distance. This could contribute to the explanation of the pan-coronary syndromes [85,86].
Conclusions Atherosclerosis appears to be a life-long disease, starting inside the maternal uterus [87] but finally leading patients to medical attention only during advanced ages. The endothelium is currently considered to play a central role in atherosclerotic lesion generation and progression. We have accumulated a large amount of evidence suggesting that during the first phases of the disease, endothelium is morphologically normal but functionally impaired through the decreased bioavailability of NO (i.e., endothelial dysfunction). During
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