Since the discovery in 1980 that acetylcholine requires the presence of endothelial cells to elicit vasodilation (1), the importance of the endothelial cell layer for vascular homeostasis has been increasingly appreciated. Dysfunction of the endothelium has been implicated in the pathophysiology of different forms of cardiovascular disease, including hypertension, coronary artery disease, chronic heart failure, peripheral artery disease, diabetes, and chronic renal failure.
The endothelium, the largest organ in the body, is strategically located between the wall of blood vessels and the blood stream. It senses mechanical stimuli, such as pressure and shear stress, and hormonal stimuli, such as vasoactive substances. In response, it releases agents that regulate vasomotor function, trigger inflammatory processes, and affect hemostasis. Among the vasodilatory substances produced by the endothelium are nitric oxide (NO), prostacyclin, different endothelium-derived hyperpolarizing factors, and C-type natriuretic peptide. Vasoconstrictors include endothelin-1 (ET-1), angiotensin II (Ang II), thromboxane A2, and reactive oxygen species (ROS) (2,3). Inflammatory modulators include NO, intercellular adhesion molecule-1 (ICAM-1), vascular adhesion molecule-1 (VCAM-1), E-selectin, and NF-κB. Modulation of hemostasis includes release of plasminogen activator, tissue factor inhibitor, von Willebrand factor, NO, prostacyclin, thromboxane A2, plasminogen-activator inhibitor-1, and fibrinogen. The endothelium also contributes to mitogenesis, angiogenesis, vascular permeability, and fluid balance.
Endothelial dysfunction was initially identified as impaired vasodilation to specific stimuli such as acetylcholine or bradykinin. A broader understanding of the term would include not only reduced vasodilation but also a proinflammatory and prothrombic state associated with dysfunction of the endothelium.
Cardiovascular Disease and Endothelial Dysfunction
Endothelial dysfunction was first described in human hypertension in the forearm vasculature in 1990 (4). Impaired vasodilation in hypertension has been confirmed by many studies in different vascular beds, including small resistance vessels (5,6). In stage I essential hypertension, we have shown that ∼60% of patients exhibit impaired small artery vasodilation when this is studied in vitro on vessels dissected from gluteal subcutaneous biopsies (7). Impairment of vasodilation has also been described in type 1 (8) and type 2 diabetes (9–11), coronary artery disease (12), congestive heart failure (13), and chronic renal failure (14–16). Moreover, this manifestation of endothelial dysfunction not only is associated with cardiovascular disease but may also precede its development, as shown in a study of offspring of hypertensive patients (17). The study subjects displayed endothelial dysfunction despite being normotensive. Another study showed endothelial dysfunction in symptom-free children and young adults at high risk for atherosclerosis (18). Also, in normotensive, normoglycemic, first-degree relatives of patients with type 2 diabetes, endothelial dysfunction was correlated with insulin resistance (19). Endothelial dysfunction has been demonstrated in the metabolic syndrome and in dyslipidemia (20) and may be associated with obesity (21), hyperhomocysteinemia (22), sedentary lifestyle (23), and smoking (24), in the absence of overt cardiovascular disease.
This review concentrates on NO, oxidant excess, inflammation, and the role of endothelial dysfunction in relation to these mechanisms in cardiovascular disease. The pathophysiologic mechanisms related to endothelium-derived hyperpolarizing factors (25) and to endothelin-1 (26) have been reviewed in detail elsewhere and may be consulted by the reader.
Pathophysiology of Endothelial Dysfunction
The pathophysiology of endothelial dysfunction is complex and involves multiple mechanisms. However, some of these seem to be common to most conditions.
One of the most important vasodilating substances released by the endothelium is NO, which acts as a vasodilator, inhibits growth and inflammation, and has anti-aggregant effects on platelets. Reduced NO has often been reported in the presence of impaired endothelial function. It may result from reduced activity of endothelial NO synthase (eNOS; as a result of endogenous or exogenous inhibitors or reduction in the availability of its substrate, L-arginine) and to decreased bioavailability of NO. ROS are known to quench NO with formation of peroxynitrite (27), which is a cytotoxic oxidant, and through nitration of proteins will affect protein function and therefore endothelial function. Peroxynitrite is an important mediator of oxidation of LDL, emphasizing its proatherogenic role (28). Moreover, peroxynitrite leads to degradation of the eNOS cofactor tetrahydrobiopterin (BH4) (29), leading to “uncoupling” of eNOS, as discussed below. Using a novel peroxynitrite decomposition catalyst, FP15, endothelial and cardiac dysfunction could be prevented in diabetic mice (30). Oxidant excess will also result in reduction of BH4 with increase in BH2. When this occurs, formation of the active dimer of eNOS with oxygenase activity and production of NO is curtailed (uncoupling of eNOS). The reductase function of eNOS is activated and more ROS are formed, so NO synthase goes from its oxygenase function producing NO to its reductase function producing ROS, with the consequent exaggeration of oxidant excess and deleterious effect on endothelial and vascular function (31). Oxidative excess is linked to a proinflammatory state of the vessel wall. ROS upregulate adhesion (VCAM-1 and ICAM-1) and chemotactic molecules (macrophage chemoattractant peptide-1 [MCP-1]) (28). Inflammation decreases NO bioavailability. Indeed, C-reactive protein (CRP) has been shown to decrease eNOS activity (32,33). The main source for oxidative excess in the vasculature is NAD(P)H oxidase (34,35). Other sources include xanthine oxidase (13), the mitochondria (36), and uncoupled NOS.
A relatively new and attractive mechanism that leads to reduced NO is asymmetric dimethylarginine (ADMA), an endogenous competitive inhibitor of eNOS that has been linked to endothelial dysfunction. In human endothelial cells, which were stimulated with plasma from patients with chronic renal disease, inhibition of eNOS correlated with plasma ADMA levels (37). ADMA levels were inversely related to endothelium-dependent vasodilation (38) in subjects with hypercholesterolemia, and infusion of L-arginine, the substrate of eNOS and competitor of ADMA, normalized endothelial function. It has been suggested that accumulation of this endogenous eNOS inhibitor leads to reduced effective renal plasma flow and increased renovascular resistance and BP (39). Intravenous low-dose ADMA reduced heart rate and cardiac output and increased mean BP (40). ADMA is a product of protein turnover and is eliminated by excretion through the kidneys or metabolism to citrulline by the enzyme dimethylarginine dimethylaminohydrolase (DDAH). Recently, overexpression of DDAH was shown in transgenic mice to decrease ADMA, increase eNOS activity, and reduce BP (41), underlining the pathophysiologic importance of ADMA. Because ADMA is eliminated through renal excretion and degradation by DDAH, it is not surprising that it is increased in patients not only with chronic renal failure (42–44) but also with other diseases such as in presence of hepatic dysfunction (45). New interest is focusing not only on the elimination but also on the generation of ADMA. Protein-arginine methyltransferases, which produce methylated arginines, namely protein-arginine methyltransferase-1, were shown to be upregulated by shear stress, and this upregulation was associated with enhanced ADMA generation (46). Hypercholesterolemia is a risk factor for atherosclerosis, associated with endothelial dysfunction (47), and there is now also evidence that elevated ADMA levels are associated with hypercholesterolemia (38). Plasma ADMA levels were also increased in elderly hypertensive patients (39) and correlated with age and BP (48). ADMA levels have been associated with increased cardiovascular risk factors in renal failure, such as CRP, carotid intima-media thickness, concentric left ventricular hypertrophy, and left ventricular dysfunction (48–50). Moreover, it was found to be a predictor of acute coronary events (51), overall mortality of patients with chronic renal failure (52), and mortality of critical ill patients (45).
In animal models of hypertension, oxidative excess leads to endothelial dysfunction as evidenced by improvement of the impaired endothelium-dependent relaxation after use of antioxidants (53). Oxidative excess in hypertensive patients leads to diminished NO (54) and correlates with the degree of impairment of endothelium-dependent vasodilation and with cardiovascular events (55). In patients with chronic renal failure, markers of oxidative excess also correlated with endothelial dysfunction (56). Findings in animal models of chronic renal failure suggest that enhanced generation of ROS leads to decreased NO bioavailability and endothelial dysfunction, which may be improved by antioxidant pretreatment (57,58). In humans with chronic renal failure, administration of vitamin C improved endothelial dysfunction of resistance arteries but not of conduit arteries (59). In animal models of diabetes, increased oxidative excess also led to endothelial dysfunction (60,61). ROS seem also to be involved in the mediation of endothelial injury leading to programmed cell death or apoptosis and to a form of apoptosis characterized by detachment of endothelial cells called anoikis (62). Anoikis is induced by the loss of cell-matrix interactions, but its exact mechanisms and pathophysiologic role in cardiovascular disease are not fully understood. Eicosapentaenoic acid, a polyunsaturated fatty acid contained in fish oil, was shown to protect endothelial cells from anoikis (63), which may contribute to the antiatherogenic and cardioprotective effects of fish oil.
Ang II has been implicated in the pathophysiology of hypertension and chronic renal failure. Ang II infusion induces endothelial dysfunction in rats (64–66), increases ROS by stimulating NAD(P)H oxidase (35,67), and promotes vascular inflammation (68). In hypertensive humans, interruption of the renin-angiotensin system with angiotensin-converting enzyme inhibitors or angiotensin receptor blockers restores endothelial function in contrast to a similar degree of BP lowering with a β-blocker, which has no effect on endothelium-dependent vasodilation (6,69,70).
A nontraditional cardiovascular risk factor that leads to endothelial dysfunction is hyperhomocysteinemia. This has been evidenced by animal models of hyperhomocysteinemia (71). Normotensive patients with hyperhomocysteinemia display endothelial dysfunction (22). Folic acid supplementation was able to reduce homocysteine levels and improve endothelial dysfunction in children with chronic renal failure (72). Cellular (73), animal (71), and human studies (22) suggest that homocysteine reduces NO bioavailability by oxidative excess. There is now also evidence that homocysteine may cause ADMA accumulation by inhibition of DDAH (74). Experimental studies in humans have confirmed that hyperhomocysteinemia may lead to endothelial dysfunction via accumulation of ADMA (75,76). However, not all studies support this link (77). These mechanisms may explain the increased cardiovascular risk of patients with hyperhomocysteinemia. This is of special importance for patients with chronic renal failure, who often have increased homocysteine levels, which were shown to predict cardiovascular outcomes in a recent study (78).
In diabetes, additional mechanisms may trigger endothelial dysfunction. In states of insulin resistance, such as in type 2 diabetes, insulin signaling is altered, differently affecting the two major pathways emerging from the insulin receptor. The pathway leading via phosphoinositide 3-kinase, phosphoinositide-dependent kinase-1, and Akt/protein kinase B to phosphorylation and activation of eNOS is dramatically downregulated, whereas the pathway leading via mitogen activated protein kinases to mitogenic effects and growth is unaffected (79–82). Moreover, hyperglycemia leads to advanced glycation end products (AGE), which were shown to quench NO and impair endothelial function, as evidenced by inhibition of advanced glycosylation with aminoguanidine (83). AGE induce ROS and promote vascular inflammation, with enhanced expression of interleukin-6, VCAM-1, and MCP-1 (84). This turns into a vicious circle in diabetic nephropathy, because in renal failure, clearance of AGE is delayed, which further promotes vascular and renal injury (85). Finally, acute hyperglycemia itself can reduce NO (86) and attenuate endothelium-dependent vasodilation in humans in vivo (87).
Pathophysiologic Effects of Endothelial Dysfunction
Endothelial dysfunction has been proposed to be an early event of pathophysiologic importance in the atherosclerotic process (18,88) and provides an important link between diseases such as hypertension, chronic renal failure, or diabetes and the high risk for cardiovascular events that patients with these conditions exhibit. Low NO bioavailability can upregulate VCAM-1 in the endothelial cell layer via induction of NF-κB expression (89). ROS, CRP, CD40 ligand, and lectin-like oxidized LDL receptor-1 (LOX-1) also upregulate endothelial expression of adhesion molecules (90). The expression of VCAM-1, ICAM-1, and E-selectin plays a role in the initiation of the inflammatory process. VCAM-1 binds monocytes and T lymphocytes, the first step of invasion of the vessel wall by inflammatory cells (91). NO inhibits leukocyte adhesion (92). Reduction in NO results in induction of MCP-1 expression, which recruits mononuclear phagocytes (93). Monocytes are transformed to lipid-loaded foam cells. Oxidized LDL, for example, is scavenged through LOX-1 (94), which is highly expressed in blood vessels in hypertension, diabetes, and dyslipidemia (95). Oxidized LDL uptake by LOX-1 triggers a variety of actions: It reduces eNOS expression (96) and further stimulates adhesion molecule expression (97). LOX-1 expression can be stimulated by Ang II and endothelin-1 (98,99). As the atherosclerotic plaque progresses, growth factors secreted by macrophages in the plaque stimulate vascular smooth muscle cell growth and interstitial collagen synthesis (91). The event that initiates a majority of myocardial infarctions is the rupture of the fibrous cap of the plaque, inducing thrombus formation. Decreased NO and oxidative excess may activate matrix metalloproteinases (MMP) (100,101), namely MMP-2 and MMP-9, which weaken the fibrous cap. Because NO inhibits platelet aggregation (102), reduced NO contributes to thrombogenicity and to the severity of the event. Thus, endothelial dysfunction with reduced NO bioavailability, increased oxidant excess, and expression of adhesion molecules contributes not only to initiation but also to progression of atherosclerotic plaque formation and triggering of cardiovascular events.
Local renal endothelial dysfunction has also been implicated in the pathophysiology of acute renal failure, such as acute ischemic renal failure and hemolytic uremic syndrome. This subject is not dealt with here; it has been reviewed extensively, and readers are referred to those publications (103,104).
Prognostic Value of Endothelial Dysfunction
Because endothelial dysfunction is an early event, as pointed out above, it may be of prognostic value. In patients with and without coronary artery disease, endothelial dysfunction in coronary arteries was associated with cardiovascular events (88,105,106). Endothelial dysfunction of the peripheral vasculature also has prognostic value. Noninvasive endothelial function testing predicted cardiovascular events in patients with coronary artery disease (55), peripheral artery disease (107), and hypertension (108) and in patients who underwent vascular surgery (109). Moreover, there is increasing evidence that links markers of endothelial dysfunction and vascular inflammation with cardiovascular events. Soluble VCAM-1 (110) and interleukin-18 (111) predicted cardiovascular death in patients with coronary artery disease independently from other risk factors. In contrast, another study did not find additional predictive value of coronary artery disease to classical risk factors for the soluble adhesion molecules VCAM-1, ICAM-1, and E-selectin (112). Plasminogen activator inhibitor-1 activity has been shown to predict coronary artery disease in hemodialysis patients (113). Soluble VCAM and CRP predicted risk of death in patients with type 2 diabetes (114). Even in otherwise healthy men, soluble ICAM was a predictor of peripheral artery disease (115) and myocardial infarction (116).
Assessment of Endothelial Dysfunction
As pointed out above, the usual parameter assessed when testing endothelial function is endothelium-dependent vasodilation. In coronary arteries, this is performed angiographically by Doppler flow measurements, assessing the effect of endothelium-dependent agonists, mainly acetylcholine (106). Some have proposed the use of cold pressor test with measurement of coronary perfusion by positron emission tomography scanning as a measure of endothelial function (117). Endothelial function may be assessed on forearm resistance arteries by forearm blood flow measurement using strain-gauge plethysmography (108). Acetylcholine or metacholine is administered through an intra-arterial catheter. Because shear stress is a stimulus to the endothelium to release NO, a noninvasive technique consists of inducing increased shear stress during reactive hyperemia to assess flow-mediated vasodilation of the brachial artery by ultrasound (18). We have also assessed endothelial dysfunction of isolated resistance arteries in vitro, by obtaining vessels dissected from biopsies of gluteal subcutaneous tissue, which are then studied on a wire or a pressurized myograph. We found a good correlation between endothelial function of small arteries studied in vitro and flow-mediated dilation of the brachial artery. However, in vitro measurement seemed to be more sensitive (5). Other noninvasive techniques have emerged and include fingertip pulse pressure tonometry and measurement of intima-media thickness, which has been considered a surrogate of endothelial function (118).
Because endothelial dysfunction is paralleled by arterial inflammation, markers of endothelial dysfunction include soluble forms of ICAM-1, VCAM-1, and E-selectin, which can be assessed in plasma. New markers have been proposed, such as LOX-1, CD40 ligand, CRP, and ADMA, to name a few (90). Because ET-1 plays a pathophysiologic role in various forms of cardiovascular disease (119), it has been suggested as a potential marker of endothelial dysfunction. Although plasma ET-1 levels are elevated in patients with symptomatic atherosclerosis (120), heart failure (121), cardiogenic shock (122), primary pulmonary hypertension (123), diabetes (124), and renal failure (125), plasma ET-1 does not reliably represent levels of tissue ET-1 production and has not been found to be elevated in patients with stage 1 hypertension (126). Plasma ET-1 has not proved to be a useful clinical measurement in most conditions.
Microalbuminuria has been considered for some time an expression of endothelial dysfunction. Microalbuminuria in most pathologic conditions seems to be a disorder of the capillary wall in the glomerulus with transcapillary escape of albumin. It therefore is intriguing to assume that endothelial dysfunction parallels or contributes to albuminuria. In diabetes, endothelial dysfunction has been correlated with microalbuminuria (127) and may precede its development (128). Microalbuminuria has also been shown to correlate with markers of endothelial dysfunction (129,130) and risk of death (130) in patients with diabetes. In hypertension, several studies have suggested that microalbuminuria may reflect endothelial dysfunction (131). However, one study did not confirm the association in hypertensive subjects (132). In another study in seemingly healthy subjects, microalbuminuria but not endothelial dysfunction correlated with cardiovascular risk factors, suggesting that microalbuminuria may precede endothelial dysfunction (133). Considering that besides fenestrated endothelial cells, the basement membrane, podocytes, and tubules may contribute to microalbuminuria, it is likely that the time at which microalbuminuria and endothelial dysfunction develop may differ between diseases with different pathophysiology, even if both are somehow related.
Therapy of Endothelial Dysfunction
Endothelial dysfunction is present in various forms of cardiovascular disease. Treatment of the underlying disease may restore endothelial function, albeit only in some conditions. In patients with chronic renal failure, renal transplantation restores renal function and may improve endothelial dysfunction (134). In hypertension, reduction of BP per se does not seem to restore endothelial function. Angiotensin receptor blockers and angiotensin-converting enzyme inhibitors have been shown to be especially beneficial (6,69,70) (Figure 1). Mechanisms whereby the blockade of the renin-angiotensin system may improve endothelial function include reduction of oxidative excess and inflammation (68). In insulin-resistant states and in diabetes, the mechanisms of endothelial dysfunction are complex and the underlying targets are still not clear. It is interesting that we and others have found that peroxisome proliferator-activated receptor-γ activators (insulin sensitizers, e.g., the glitazones pioglitazone and rosiglitazone) and peroxisome proliferator-activated receptor-α activators (fibrates, e.g., fenofibrate) exhibit cardiovascular anti-inflammatory and antioxidant properties and correct endothelial dysfunction induced by Ang II (65,66,135,136) (Figure 2).
Another approach for treatment of endothelial dysfunction is to address the components in the disease process that trigger dysfunction of the endothelium. Thus, decrease of homocysteine levels in hyperhomocysteinemia by supplementation with folic acid can improve endothelial dysfunction (72,73). L-Arginine (137) and tetrahydrobiopterin, as well as tetrahydrobiopterin mimetics (138), may improve endothelial function via increased NO bioavailability. However, some studies have not found L-arginine administration to improve endothelial dysfunction (139). Recently, acetyl salicylic acid has been suggested as an agent that can reduce oxidative stress and improve endothelial function (140). Statins have proved to have beneficial effects on endothelial dysfunction (141), which may be the result in part of lipid lowering but also of their pleiotropic anti-inflammatory effects. For example, statins inhibited upregulation of LOX-1 and increased eNOS expression in human coronary artery endothelial cells (96).
New Research on Endothelium
New insights into the regulation of endothelial function will be obtained through greater understanding of the signaling pathways within endothelial cells. Research on caveolae and caveolin-1 may be of future interest, because endothelial cells exhibit high levels of caveolin-1. Caveolin-1 has been implicated in the regulation of eNOS turnover and consequently in NO release as a result of shear stress (142).
A new fascinating aspect of endothelial function is emerging from research on endothelial progenitor cells. These are primitive bone marrow cells that have the ability to mature into endothelial cells and have a physiologic role in repair of endothelial lesions (143). Levels of circulating endothelial progenitor cells correlate inversely with the degree of endothelial dysfunction in humans at various degrees of cardiovascular risk (144). It is interesting that eNOS expression by bone marrow stroma cells plays an essential role in the recruitment of endothelial progenitor cells (145). Therapy with statins increases the number of circulating endothelial progenitor cells (146). Of special interest for patients with chronic renal failure may be the finding that erythropoietin is a potent physiologic stimulus for endothelial progenitor cell mobilization (147). Moreover, transfer of endothelial progenitor cells may represent a new approach to therapy and has already had some success on peripheral ischemia and on ischemic myocardium, including promoting neovascularization and potentially transdifferentiation into cardiomyocytes (143).
D.H.E. is supported by a grant from the Deutsche Forschungsgemeinschaft. The work of E.L.S. is supported by grants 13570 and 37917 and a group grant to the Multidisciplinary Research Group on Hypertension, all from the Canadian Institutes of Health Research.
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