Endothelial cell dysfunction

Antonio Ceriello Department of Pathology and Medicine, Experimental and Clinical, Chair of Internal Medicine, University of Udine, Udine, Italy (ceriello@uniud.it)

Endothelium

Endothelium covers the arterial and venous walls, arranged like cobblestone paving. At the capillary level the endothelium consists of a single cell that folds outwards to cover the entire wall. The endothelial layer is built on an amorphous basal membrane, formed from endothelial cell-derived material. The basal membrane is divided into three layers: (1) rare internal, (2) dense and (3) rare external, formed by type IV collagen, glycoproteins, laminin and fibronectin. In the large vessels the basal membrane is covered by a tunica media and tunica adventitia. At the capillary level the basal membrane layer includes pericytes, which are cells with contractile function involved in vessel diameter regulation and intimal cell turnover. A large number of pericytes are located in the retina and central nervous system. Endothelial morphology changes according to functional need: sinusoid, uninterrupted and uninterrupted with tight junction. Usually, large vessel endothelium is homogenous.
Recent findings have demonstrated that the vascular endothelium is an important regulatory organ for maintaining cardiovascular homeostasis. In physiological conditions it contributes to vascular homeostasis, continuously monitoring blood stimulus and local stimulus and modifying itself in response to environmental changes. The primary endothelial functions are [1, 2]:
— Selective gate: as the primary endothelial function, this permits the transport of glucose, nutrients, metabolites and hormones from blood to tissues and vice versa. The non-insulin-dependent glucose transporter GLUT1 transports glucose through the endothelial cells.
— Basal membrane and growth factor synthesis and catabolism: endothelium synthesizes collagen I, fibronectin, laminin, basic fibroblast growth factor, insulin-like growth factor-I, transforming growth factor-b1, platelet-derived growth factor and vascular endothelial growth factor.
— Leukocyte recruitment and activation: endothelium produces intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 and E-selectin, which promote leukocyte uptake. It also produces monocyte chemotactic factors: monocyte chemoattractant protein and some cytokines (interleukin-1, -6 and -8).
— Lipoprotein metabolism: lipoprotein lipase, mainly produced by adipocytes and macrophages, is also present in the endothelial layer, where it hydrolyses triglycerides of very-low-density lipoprotein and chylomicrons.
— Hemostasis: endothelium produces and releases coagulant and anticoagulant factors. Usually, anticoagulant activity predominates, but during conditions of mechanical stress or altered biohumoral stimulus, procoagulant activity dominates:
• procoagulant factors: thromboxane A2, platelet activating factor, von Willebrand factor;
• anticoagulant factors: heparin-like molecules that activate antithrombin III, production of thrombomodulin (a co-factor of C protein), competitive inhibition of Factor V activation, blockade of extrinsic pathways;
• profibrinolytic factors: tissue plasminogen activator and urokinase;
• antifibrinolytic factors: inhibitor of plasminogen activator-1;
• factors inhibiting platelet activation: prostaglandin I2, nitric oxide and glycosaminoglycans; — Vascular tone modulations:
• vasoconstrictor factors: angiotensin- converting enzyme, angiotensin I and II, endothelin-1, thromboxane A2 and prostaglandin H2;
• vasodilator factors: nitric oxide, prostaglandin I2 and endothelium-derived hyperpolarizing factor.
One of the most important regulatory and vasoactive substances produced by the endothelial cells is nitric oxide.
Nitric oxide modulates vascular tone and inhibits platelet adherence and aggregation, smooth muscle proliferation and endothelial cell-leukocyte interaction.

Endothelial dysfunction

Endothelial dysfunction indicates a generalized alteration in endothelial cell function characterized by an abnormal vasodilatory response, decreased prostacyclin release, increased production of vasoconstrictor substances, impaired endothelial control of fibrinolysis and inflammation, and altered expression of adhesion molecules. Loss of nitric oxide bioactivity is a central feature of this abnormal condition and is an independent predictor of future cardiovascular events in patients with atherosclerosis. Endothelial dysfunction is thought to precede the development of atherosclerosis. Indeed, in the presence of cardiovascular risk factors endothelial dysfunction can be detected before there is any angiographic evidence of disease or increased intima-media ratio on ultrasound examination [3, 4]. Many of the cardiovascular risk factors, including hyperlipidemia, hypertension, diabetes and smoking, are associated with overproduction of reactive oxygen species or increased oxidative stress, both of which reduce vascular nitric oxide bioavailability and promote cellular damage [5]. Hence, increased oxidative stress is considered to be a major mechanism involved in the pathogenesis of endothelial cell dysfunction and may serve as a common pathogenic mechanism of the effect of risk factors on the endothelium [5, 6].

Diabetes, oxidative stress and endothelial dysfunction

Hyperglycemia is a hallmark of both non-insulin-dependent (type 2) and insulin-dependent (type 1) diabetes mellitus. In vivo and in vitro studies have demonstrated that, in both diabetic and normal subjects, hyperglycemia directly induces endothelial dysfunction and attenuates endothelium-dependent relaxation [7–9]. There is considerable evidence that elevated glucose levels are associated with increased production of reactive oxygen species via several different mechanisms [10–14]. The hyperglycemia-dependent increase in the production of reactive oxygen species contributes to the development of impaired endothelial- dependent relaxation [7]. Several experimental and prospective studies have suggested that, in diabetes, oxidative stress plays a key role in the pathogenesis of both micro- and macrovascular complications [15], and an early stage of such damage is thought to be the development of endothelial dysfunction [6, 15].
Recently, Brownlee pointed out the key role in the pathogenesis of diabetic complications of superoxide production in the endothelial cells during hyperglycemia [16]. This is consistent with the four pathways involved in the development of diabetic complications: (1) increased polyol pathway flux, (2) increased advanced glycosylation endproduct formation, (3) activation of protein kinase C, and (4) increased hexosamine pathway flux. However, superoxide generation during hyperglycemia represents only a first step in the process of endothelial dysfunction in diabetes. Nitric oxide production plays a central role in modulating endothelial function [17]. It is generated from the metabolism of L-arginine by the enzyme nitric oxide synthase (NOS), of which there are three isoforms: the constitutive types bNOS and eNOS and the inducible type iNOS [18]; the latter is induced de novo by various stimuli, including hyperglycemia, and leads to the production of large amounts of nitric oxide [19]. The superoxide anion may quench nitric oxide, thereby reducing the efficacy of a potent endothelium-derived vasodilatory system that participates in the general homeostasis of the vasculature [20], and evidence suggests that during hyperglycemia there is reduced nitric oxide availability [8]. In hyperglycemic conditions an overproduction of both superoxide and nitric oxide has consistently been reported, with a threefold increase in superoxide generation [21]. The simultaneous overgeneration of nitric oxide and superoxide favours the production of a toxic reaction product, the peroxynitrite anion [22]. The peroxy-nitrite anion is cytotoxic because it oxidizes sulfhydryl groups in proteins and initiates lipid peroxidation and nitrate amino acids such as tyrosine, which affects several signal transduction pathways [22].

Lipid peroxidation

LDL in its native state is not atherogenic. LDL chemical modification, as occurs during lipid peroxidation and oxidative stress, leads to the formation of oxidized LDL. This modification renders LDL susceptible to macrophage uptake via a number of scavenger receptor pathways, producing foam cells [23]. Moreover, oxidized LDL induces synthesis of monocyte chemotactic protein-1 [24, 25], resulting in the recruitment of inflammatory cells [26] and stimulation of smooth muscle cell proliferation [27]. The presence of oxidized lipids has been discovered in human atherosclerotic lesions [28–32].

Protein oxidation and nitration

In addition to lipid oxidation, there is also good evidence of protein oxidation in human atherosclerotic lesions [33, 34]. Oxidation and nitration products include several reactive species such as hydroperoxides and protein-bound reductants. Some of these products, like nitrotyrosine, have become markers of oxidative stress. Nitrotyrosine formation, derived by nitration of tyrosine, was detected in the artery wall of monkeys during hyperglycemia [35] and preceded the development of endothelial dysfunction in healthy subjects [36] and in the coronary vessels of perfused hearts [37]. This effect is not surprising, since it has been shown that nitrotyrosine can also directly harm endothelial cells [37]. The toxic action of nitrotyrosine is supported by evidence that increased apoptosis of myocytes, endothelial cells and fibroblasts in heart biopsies from diabetic patients [38] is selectively associated with the levels of nitrotyrosine found in those cells, as well as in the hearts of streptozotocin-induced diabetic rats [39] and in the working hearts of rats during hyperglycemia [37]. However, limited information is available regarding the relationship between the accumulation of oxidized and nitrated protein and the severity of oxidative damage.

DNA damage

Peroxynitrite is a potent initiator of DNA single-strand breakage, which is an obligatory stimulus for the activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) [40]. These reactive species trigger DNA single-strand breakage, which induces a rapid activation of PARP [41]. PARP activation in turn depletes the intracellular concentration of its substrate oxidized nicotinamide adenine dinucleotide, slowing the rate of glycolysis, electron transport and adenosine triphosphate formation, and produces adenosine diphosphate (ADP) ribosylation of glyceraldehyde phosphate dehydrogenase (GAPDH). A recent study showed the pivotal role of ADP ribosylation of GAPDH in the activation of the three major pathways of hyperglycemic damage (formation of advanced glycosylation endproducts, activation of protein kinase C and hexosamine pathway flux) [41]. This process resulted in acute endothelial dysfunction in diabetic blood vessels [41]. In addition to the direct cytotoxic pathway regulated by DNA injury and PARP activation, PARP also appears to modulate the activation of nuclear factor-kB and the expression of genes, including intercellular adhesion molecule-1, iNOS and reduced nicotinamide adenine dinucleotide phosphate oxidase genes [42].

Markers of endothelial dysfunction

Assessment of endothelial cell function refers to the measurement of endothelial cell response to stimulation, for example by vasoactive substances released by, or those that interact with, the vascular endothelium. Abnormalities in endothelium-dependent vasodilatation can be detected in the arteries before the development of overt atherosclerosis. There is ample evidence that measurement of flow-mediated vasodilatation sensitively detects endothelial dysfunction in hyperlipidemia, arterial hypertension and diabetes, all of which are considered major cardiovascular risk factors. In addition, markers of oxidative stress, markers of DNA damage and adhesion molecules are other measures of endothelial health.

Flow-mediated vasodilatation

The capacity of the blood vessels to respond to physical and chemical stimuli in the lumen confers the ability to self-regulate tone and to adjust blood flow and distribution in response to changes in the local environment. Many blood vessels respond to an increase in blood flow or, more precisely, shear stress by dilating. This phenomenon is termed flow-mediated vasodilatation and its principal mediator is endothelium-derived nitric oxide [43]. Brachial artery ultrasound is a widely used, non-invasive measure of endothelial cell function [43, 44]. The forearm blood flow is occluded for 5 min using a blood pressure cuff maintained at a standard pressure. When the pressure is released, reactive hyperemia occurs. This technique has the advantage of being non-invasive and can readily identify patients with attenuated endothelial function. Recent studies identified a prognostic role for brachial ultrasound [45, 46], but large-scale multicentre trials might provide a definitive answer to the real prognostic value of endothelial dysfunction in terms of cardiovascular risk and therapeutic approach.

Coronary circulation

Quantitative coronary angiography has been used to evaluate endothelial function in coronary arteries. Dilatation of coronary resistance vessels in order to induce increases in shear stress in the upstream epicardial arteries can be achieved through metabolic stimuli such as exercise or pacemaker stimulation, or through selective infusion of acetylcholine, bradykinin, substance P or serotonin [47]. Endothelial function of the coronary microvasculature was assessed using intracoronary Doppler techniques to measure coronary blood flow in response to pharmacological or physiological stimuli [48]. Although this method permits a good evaluation of endothelial function, assessment of flow-mediated vasodilatation in the epicardial arteries is a much more invasive approach than in the brachial artery.

Vascular stiffness

Non-invasive measures of arterial compliance and waveform morphology also provide a marker of vascular health that may in part be endothelium-dependent. Pulse wave veloc-ity has been used as an index of vascular stiffness. Evidence suggests that endogenous nitric oxide modulates vascular stiffness probably by vascular tone modulation and vascular remodelling [49]. Endothelial dysfunction and increased arterial stiffness commonly coexist in patients at increased risk of cardiovascular disease and some studies have directly related increased stiffness to impaired endothelial function [50, 51].

Biochemical markers of oxidative stress

Nitric oxide, nitrite, nitrates

Direct measurements of free radicals is difficult, particularly in vivo. Nitric oxide is destroyed by a reaction with superoxide after it is produced. Nitric oxide metabolites, nitrite and nitrates can be measured in plasma, but their levels are influenced by physical exercise [52], diet [53], intestinal bacteria and laboratory contaminants [54]. However, the products of radical damage in the cell, namely DNA, lipids and proteins, are reasonable indirect markers of oxidative stress [55].

8-Hydroxy-2'deoxyguanosine (8-OHdG)

Oxidative changes to DNA can occur through a number of routes including oxidative modification of the nucleotide bases or sugars, or through formation of crosslinks. In vivo, damaged DNA is repaired by endonuclease- and glycosylase-liberating deoxynucleotides and bases, respectively, which are excreted in urine. DNA damage can be evaluated by measuring 8-hydroxy-2'deoxyguanosine (8-OHdG) and its free base 8-hydroxyguanine in blood cells or in urine [55]. Studies have shown that hyperglycemia independently increases the levels of 8-OHdG in the urine and plasma of patients with type 2 diabetes mellitus, and the levels of urinary 8-OHdG in diabetes correlated with the severity of diabetic nephropathy and retinopathy [56–58]. Moreover, in human atherosclerotic plaques, increased amounts of 8-OHdG were found [59].

Oxidized LDL

Lipid peroxidation is probably the most extensively investigated process induced by free radicals. These compounds are abundant at membrane level, where most of the reactive radicals, especially reactive oxygen species, are formed. Given its pathophysiological significance, there has been a great deal of interest in the detection of oxidized LDL or antibodies to oxidized LDL. In clinical trials oxidized LDL levels were found to correlate to the development of myocardial infarction and coronary heart disease [60–62].

Isoprostanes

Lipid peroxidation may result in a chain reaction that autopropagates once started, leading to the formation of many lipid peroxide radicals and amplifying the reactive oxygen species effect. Lipid peroxides, derived from polyunsaturated fatty acids, are unstable and decompose to form a complex series of compounds. The most studied are isoprostanes, prostaglandin-like compounds with constrictor properties, generated in vivo by the free-radical catalysed peroxidation of arachidonic acid, independently of cyclo-oxygenase [55]. In patients with diabetes, smokers and hypercholesterolemic patients, evidence has been reported of increased plasma and urine isoprostane levels [63, 64].

Oxidatively modified tyrosines

Modifications of proteins may result in crosslinking, peptide fragmentation and conversion of one amino acid to another or to a modified residue by oxidation of the amino acid side chain. Such modifications can result in the alteration of secondary and tertiary structures of protein and these conformational changes may expose previously shielded regions to further oxidation or to other types of spontaneous modification such as deamination. Recently, nitrotyrosine has been proposed as a new and interesting marker of oxidative damage to protein [65]. When superoxide and nitric oxide exist in close proximity, they can spontaneously form peroxynitrite, a powerful oxidant [66]. Nitration in the 3-position (ortho) of tyrosine is the major product of peroxynitrite attack on protein [65]. The plasma level of nitrotyrosine correlates with the severity of coronary artery disease [67], the level of glycemia [68] and the presence of peripheral vascular disease [69].

Adhesion molecules

Adhesion molecules regulate the interaction between endothelium and leukocytes [70] and are involved in the process of endothelial dysfunction and atherogenesis. An increase in their expression on the endothelial surface causes increased adhesion of leukocytes, particularly monocytes [71]. It is well known that this is one of the first steps in the process leading to atheroma. Among the various pro-adhesive proteins, special concern has been aroused by ICAM-1. The soluble form of ICAM-1 accumulates in cells and can be rapidly expressed on their surface after various stimuli [70]. The cir- culating form of this molecule was found to be increased in subjects with vascular disease [72] and in diabetes mellitus with or without vascular disease [73]. As a result, it can be considered a marker of the activation of the atherogenic process [74]. Acute hyperglycemia in both normal and diabetic subjects is a sufficient stimulus for the circulating concentration of ICAM-1 to increase, thus activating one of the earliest stages of the atherogenic process [75, 76].

Conclusion

Vascular endothelium is an important regulatory organ in maintaining cardiovascular homeostasis. Endothelial-derived nitric oxide modulates vascular tone and inhibits platelet adherence and aggregation, smooth muscle proliferation and endothelial cell-leukocyte interaction. There is considerable evidence to suggest a role of oxidative stress in the pathogenesis of endothelial dysfunction (Fig. 1).

Fig. 1: Hyperglycemia precipitates an overproduction of superoxide by the mitochondrial electron transport chain and favours increased expression of reduced nicotinamide adenine dinucleotide phosphate (NADPH) and iNOS by activation of nuclear factor-kB (NFkB). Consequently, hyperglycemia results in overproduction of superoxide and nitric oxide. This condition is related to overgeneration of a toxic reaction product, the peroxynitrite anion. Peroxynitrite is cytotoxic because it leads to oxidation of sulfhydryl groups in protein and peroxidation of lipids and nitrate amino acids. Moreover, peroxynitrite is a potent initiator of DNA single-strand breakage, which is a stimulus for activation of PARP and which depletes the intracellular concentration of its substrates oxidized nicotinamide adenine dinucleotide (NAD+), slowing the rate of glycolysis, electron transport, adenosine triphosphate formation and ADP ribosylation of GAPDH. This process results in acute endothelial dysfunction in diabetic blood vessels, which contributes to the development of diabetic complications. Assessment of endothelial cell function is now possible with a consistent number of biomarkers: DNA damage can be detected using 8-OHdG, nitrotyrosine is a marker of protein nitration, and oxidized LDL and isoprostanes are markers of lipid peroxidation. Moreover, direct measurement of endothelial dysfunction is possible using flow-mediated vasodilatation, quantitative angiography and measurement of arterial stiffness. AP-1, Activator protein 1; STAT, signal transducers and activators of transcription.

Endothelial dysfunction is thought to precede the development of atherosclerosis. Assessment of endothelial cell function is now possible with a consistent number of biomarkers and with direct measures of endothelial cell response to stimulation. These markers permit an accurate assessment of endothelial dysfunction and thus the possibility of monitoring the efficacy of therapy.

References