Vascular endothelial function in health and diseases

Transcription

1 Pathophysiology 15 (2008) Review Vascular endothelial function in health and diseases M. Khazaei a, F. Moien-afshari b, I. Laher b, a Department of Physiology, Isfahan University of Medical Sciences, Isfahan, Iran b Department of Pharmacology and Therapeutics, University of British Columbia, Vancouver, Canada Received 16 December 2007; received in revised form 7 February 2008; accepted 8 February 2008 Abstract The vascular endothelium constitutes approximately 1% of body mass (1 kg) and has a surface area of approximately 5000 m 2. The endothelium is a multifunctional endocrine organ strategically placed between the vessel wall and the circulating blood, and has a key role in vascular homeostasis. The endothelium is both a target for and mediator of cardiovascular disease. The endothelium releases several relaxing and constricting factors, which can affect vascular homeostasis. Endothelial dysfunction, whether caused by physical injury or cellular damage, leads to compensatory responses that alter the normal homeostatic properties of the endothelium. In this review, we summarized some physiological aspects of endothelial function and then we discussed endothelial dysfunction during some pathological conditions Elsevier Ireland Ltd. All rights reserved. Keywords: Endothelium; Health; Homeostasis; Cardiovascular diseases; Membrane receptors; Nitric oxide Contents 1. Introduction Endothelium-derived relaxing factors Nitric oxide Prostacyclin Endothelium-derived hyperpolarizing factor EETs Gap junctions Reactive oxygen species and hydrogen peroxide Potassium ions Contribution and nature of endothelium-derived contracting factors Endothelin Constricting prostaglandins HETE Hemostasis Angiogenesis Endothelial cells, inflammation and immune response Endothelial permeability Endothelium in diseases Endothelial cells in hypertension Endothelium in Atherosclerosis and hyperlipidemia Endothelial cells and hypertrophic cardiomyopathy Corresponding author at: Department of Pharmacology and Therapeutics, Faculty of Medicine, University of British Columbia, 2176 Health Sciences Mall, Vancouver, BC, Canada V6T 1Z3. Tel.: ; fax: address: ilaher@interchange.ubc.ca (I. Laher) /$ see front matter 2008 Elsevier Ireland Ltd. All rights reserved. doi: /j.pathophys

2 50 M. Khazaei et al. / Pathophysiology 15 (2008) Endothelial cells and cigarette smoke Endothelial cells and postmenopausal estrogens Endothelial cells and diabetes Endothelium and viral myocarditis Cardiac allograft vasculopathy (CAV) and endothelium References Introduction A single layer of endothelial cells (ECs) lines inner surface of the entire vascular system. This continuous layer separates blood from the vessel wall. The endothelial layer surface consists of cells, covers an area of approximately 1 7 m 2, and weighs approximately 1 kg in an adult human [1]. Irie and Tavassoli named this layer as tissue-blood barrier [2]. As a barrier, the endothelium regulates the transfer of molecules and acts as a semipermeable layer. In addition to serving as a physical barrier, endothelial cells also control many important functions in vascular homeostasis. Therefore, the endothelial layer is a dynamic and active tissue that has both metabolic and synthetic functions. ECs have significant autocrine, paracrine and endocrine actions. The endothelium is also able to sense changes in hemodynamic forces by membrane receptor mechanisms and respond by synthesis or release of various vasoactive substances, subjects recently reviewed by [3]. For example, the arterial endothelium responds to shear stress and flow via production of nitric oxide (NO) that induces vasodilation [4]. ECs contribute to the regulation of blood flow and blood pressure by releasing vasodilator and vasoconstrictor substances and can be important in control of vascular tone [5,3]. NO is the predominant vasodilator released from ECs and has several effects on the cardiovascular system such as inhibition of platelet aggregation, smooth muscle cell proliferation and anti-atherosclerotic effects [6]. The other endothelium-derived vasodilators are prostacyclin (PGI 2 ) and the putative endothelium-derived hyperpolarizing factor (EDHF) [7 10]. Vasoconstrictors released by ECs include endothelin (ET)-1 and prostaglandins H2. Furthermore, the endothelium is involved in haemostatic processes, platelet activation and aggregation, inflammation and immune modulation, vascular permeability, vascular smooth muscle cell proliferation and angiogenesis [3]. Endothelial dysfunction is a marker of vascular disease and plays an important role in the initiation and progression of disease. Much interest in the functioning of ECs has originated from the concept that EC injury is involved in many disease processes, including atherosclerosis, hypertension, diabetes mellitus, hypertrophic cardiomyopathy (HCM) or viral myocarditis. We reviewed normal endothelial cell function and present evidence that functional endothelial impairment that occurs in many diseases. Where possible, we focus on data obtained from resistance arteries. Endothelial cells are important regulators of vascular tone [5,3]. These cells release NO, PGI 2, EDHF and ET-1 in response to both humoral and mechanical stimuli, and can affect many aspects of smooth muscle function [1]. 2. Endothelium-derived relaxing factors 2.1. Nitric oxide The existence of an endothelium-derived relaxing factor was first described by Furchgott and Zawadzki in 1980 [4]. They showed that in the presence of an intact endothelium, rabbit aortic rings relaxed in response to acetylcholine (ACh). In contrast, when the endothelium was removed, the arteries constricted in response to ACh. Several years later, it was established that EDRF was in fact NO [11]. In pathological states affecting the endothelium or after mechanical denudation of endothelium, the normal vasodilator response of ACh due to release of NO is replaced by constriction resulting from the direct effect of ACh on vascular smooth muscle cells [12]. NO is generated through the conversion of the amino acid l-arginine to l-citrulline by the enzyme, NO synthase (NOS) [13] (Fig. 1). Three isoforms of NOS have been cloned: NOS-I, NOS-II and NOS-III. NOS-I or neuronaltype NOS is found predominantly in nervous system. NOS-II or inos is found in macrophages, neutrophils and other inflammatory cells. NOS-III or enos is found in endothelial cells and also in cardiac myocytes, osteoclasts, osteoblasts and renal mesangial cells to a smaller degree [14]. The activation of enos depends on the intracellular concentration of calcium ions and therefore is calcium-calmodulin dependent [15]. The activation of this enzyme also requires nicotinamide adenine dinucleotide phosphate and tetrahydrobiopterin as cofactors [15]. NO can be synthesized by endothelial cells in response to physiological stimuli such as shear stress or chemical stimuli such as acetylcholine, arachidonic acid and 5-hydroxytryptamine [15,16]. These substances can release NO through activation of specific EC membrane receptors [17]. Shear stress-induced enos activation is most likely mediated by phosphorylation by serine/threonine protein kinase Akt/PKB [18]. NO diffuses from ECs and can affect vascular smooth muscle cell by activation of soluble guanylyl cyclase after binding to the haem group of the enzyme [19]. Activation of this enzyme will lead to increased production of cyclic guanosine monophosphate (cgmp), activation of cgmp-dependent protein kinase and phosphorylation of K + channels leading to hyperpolarization and reduction of intra-

3 M. Khazaei et al. / Pathophysiology 15 (2008) Fig. 1. Nitric oxide regulation of the cardiovascular system. cellular Ca 2+ ions [20]. These processes combine to result in the relaxation of smooth muscle cells. There are two cgmpdependent protein kinases (I and II) of which protein kinase type I is the major kinase mediating vasodilation and inhibition of platelet aggregation [21,22]. The activity of cgmp is terminated by its conversion to GMP by phosphodiesterase. NO is involved in regulation of vasomotion and blood pressure [11]. In large arteries such as aorta, coronary and brain arteries, NO is the major contributor to endothelium-dependent relaxation [15]. Inhibition of NOS causes vasoconstriction in most vascular beds and an increase in systemic blood pressure in animals and human [11]. During the last decade, it became apparent that NO is not only important in the control of vasomotor tone but also in vascular and immunological homeostasis. NO is released toward both the smooth muscle cells and into the lumen of blood vessels. In many pathological processes such as diabetes, shock, infarction and chronic inflammation, impairment of NO synthesis occurs [23]. NO inhibits platelet adhesion and aggregation and promotes platelet disaggregation, in part through a cgmp-dependent mechanism [24]. NO also inhibits leukocyte adhesion to the endothelium [25] and smooth muscle cell migration and proliferation [26,27]. These biological effects make NO an important component of the endogenous prevention of vascular injury, inflammation and thrombosis-events involved in the atherosclerotic process [12] Prostacyclin Another major endothelium-derived vasodilator is prostacyclin. PGI 2 was first described in 1979 [28] and is formed primarily in ECs from arachidonic acid through the enzyme cyclooxygenase [29]. Cyclooxygenase- 1 (COX1) is expressed constitutively in ECs and is the main isoform involved in the production of PGI 2 [30]. Phospholipase A 2, which converts membrane phospholipids to arachidonic acid, is the rate-limiting step in PGI 2 synthesis. Synthesis of PGI 2 by ECs, like NO, is a Ca 2+ -dependent process, because phospholipase A 2 is Ca 2+ sensitive [31]. PGI 2 activates an IP receptor [32] and causes vascular smooth muscle relaxation through stimulation of adenylate cyclase and increases intracellular levels of cyclic AMP [17]. Activation of protein kinase A decreases myosin light chain kinase activity, leading to vasodilation. PGI 2 also can diffuse into the blood and inhibit platelet aggregation [33]. The effect of PGI 2 is tightly related to NO effects since PGI 2 potentiates NO release and in turn NO potentiates the effect of PGI 2 on vascular smooth muscle cells [28]. NO inhibits phosphodiesterase, an enzyme that degrades cyclic AMP [28]. Therefore, increases in cyclic AMP prolong the effects of PGI 2 in smooth muscle cells.

4 52 M. Khazaei et al. / Pathophysiology 15 (2008) Endothelium-derived hyperpolarizing factor In 1988, Taylor and Weston [34] described an endothelium released vasorelaxant that was distinct from NO and PGI 2. They reported that this factor induced vasorelaxation by hyperpolarization of vascular smooth muscle cells, and is not affected by L-NMMA, a blocker of NOS or indomethacin, an inhibitor of cyclooxygenase [34]. In contrast, ouabain, a Na + K + ATPase inhibitor, or depolarization of smooth muscle cells with high extracellular potassium, prevented the vascular relaxation. Later, this factor was named endothelium-derived hyperpolarizing factor [35,36]. It is proposed that EDHF exerts its effect by increasing intracellular calcium levels and subsequent activation of Ca 2+ -sensitive potassium channels that cause vascular smooth muscle cell relaxation [37]. However, the exact nature of EDHF remains unclear. There are several candidates for EDHF such as epoxyeicosatrienoic acids (EETs), potassium ions, reactive oxygen species (ROS) and hydrogen peroxide. The literature concerning EDHF has been extensively reviewed [35,36,38 40] EETs Some investigators propose that EETs, the metabolites of arachidonic acid produced by cytochrome P-450 epoxygenase, can diffuse from endothelial cells to smooth muscle cells and act as EDHFs [38,41]. The following evidence supports the role of EETs as candidate molecules for EDHF: 1. ACh causes non-no, non-prostaglandin-mediated relaxations that are sensitive to inhibition of cytochrome P-450 or phospholipase A 2 [42]. 2. Bradykinin or acetylcholine stimulate EETs synthesis from ECs [43,44]. 3. EETs are synthesized by vascular endothelium, open Ca 2+ -activated K + channels and hyperpolarize and relax smooth muscle cells [44]. EDHF-mediated relaxation is generally inhibited by the combination of apamin and chaybdotoxin [10]. 4. Ouabain, an inhibitor of Na + K + ATPase, inhibits EDHFmediated vasodilation also inhibits Ca 2+ -activated K + channels channel activation, hyperpolarization and relaxation to EETs [10]. Although there is some evidence that EETs can act as EDHF, inhibition of Ca 2+ -activated K + channels with apamin and charybdotoxin inhibits the EDHF-mediated response in most vascular beds, whereas the relaxation and hyperpolarization to EETs is mediated by iberiotoxin-sensitive BKCa channels [45] Gap junctions Some studies propose that myoendothelial gap junctions may mediate endothelium-dependent vasorelaxation associated with hyperpolarization of smooth muscle. Gap junctions can facilitate electrical coupling between endothelial cells and smooth muscle cells and cause a bi-directional coupling between these cells [46]. Thus induced hyperpolarization of endothelial cells causes relaxation of smooth muscle cells and denudation of endothelium impaires this response [47]. This response is attenuated in arteries of mice deficient in connexin, the protein component of endothelial gap junctions [48]. These studies support a role for myoendothelial cell gap junction in electrical coupling between these cells. Connexins are the most important protein which make up gap junctions. There are 13 rodent connexins have been indicated [49]. The principal gap junction protein in endothelial cells and vascular smooth muscle cells is connexin 43 [50]. Its reported that myoendothelial gap junctions in resistance arteries have more important role than conduit arteries [51] Reactive oxygen species and hydrogen peroxide Reactive oxygen species are oxygen-derived molecules. The most important ROS are superoxide (O 2 ), hydrogen peroxide (H 2 O 2 ) and hydroxyl radical (OH ). Generation of ROS occurs under normal physiological conditions and is not restricted to pathological conditions. The enzyme superoxide dismutase (SOD) catalayzes superoxide into H 2 O 2. SOD exists in several forms: copper zinc superoxide dismutase (Cu/Zn-SOD), mitochondrial or manganese superoxide dismutase (Mn-SOD) and iron-containing superoxide dismutase (Fe-SOD) which are localized both within or on the surface of endothelial cells [52]. In addition, catalase or glutathione peroxidase also metabolizes H 2 O 2 into water. The balance between oxidant and antioxidant processes regulates the amount of ROS generated by endothelial cells, and an imbalance between these two processes will impair endothelial function. The superoxide anion reacts with NO to form peroxynitrite (ONOO ) [53] which impairs endothelium-dependent function. However, there is also some evidence suggesting that the ROS H 2 O 2 has a direct relaxant effect [54,55]. The suggestion that EDHF could be a ROS is quite valid given that the endothelial cells produce and release significant amounts of free radical species (see recent review [56]). Oxygen radicals are formed in ECs in response to increases in blood pressure and activation by endothelial agonists [28]. ROS secreted by endothelial cells by NADPH oxidase, xanthine oxidereductase, uncoupled endothelial nitric oxide synthase and cytochrom P-450. There are numerous reports demonstrating that ROS have relaxant effects on vascular smooth muscle cells [56]. Hydrogen peroxide is formed from superoxide by superoxide dismutase. Intracellular expression and localization of superoxide dismutase in endothelial cells has been shown [56]. Some studies report that hydrogen peroxide acts as EDHF in mesenteric arteries from enos knockout mice or L-NAME treated mice as both the relaxation and hyperpolarization to acetylcholine

5 M. Khazaei et al. / Pathophysiology 15 (2008) are inhibited by catalase, an enzyme that inactivates H 2 O 2 [39]. However, others have not been able to reproduce these findings [56]. EDHF-mediated relaxation of ROS is assumed to be directly or indirectly linked to the activation of potassium channels. H 2 O 2 can alter many of the transport mechanisms related to cellular calcium handling, resulting in elevation of intracellular concentrations of calcium in ECs [57]. This increase in cytosolic calcium may be sufficient to open calcium-activated potassium channels, the channel subtype most likely responsible for EDHF-mediated vasodilator response [58]. Hydrogen peroxide-mediated vasodilation is abolished by tetraethylammonium (TEA) or high extracellular potassium [56]. Additional support comes from studies showing that H 2 O 2 increases the open probability of large-conductance calcium-activated potassium channels in isolated porcine coronary myocytes. However, there is evidence that EDHF-mediated relaxations are not always inhibited by catalase, an enzyme that degrades H 2 O 2 [40] Potassium ions A novel finding related to the possible nature of EDHFmediated vasodilatation was initially reported by Edwards et al. [59] who put forward the hypothesis that K + is an EDHF in the rat hepatic artery; they observed that ACh-mediated an increase in extracellular K + (from 4.6 to 11.6 mm) as measured with K + -sensitive microelectrodes. A role for Na + /K + -ATPase in ACh-induced relaxation of arteries was previously reported by De Mey and Vanhoutte [60]. Activation of Na + /K + -ATPase leads to hyperpolarization of smooth muscle [61]. A small increase in extracellular K + (up to 5 mm) dilates blood vessels via the activation of the electrogenic Na + /K + -ATPase. An increase in extracellular K + from 6 to 16 mm induces relaxation of pressurized rat coronary and cerebral arteries in a barium-sensitive manner [62]. A low concentrations (<50 mm) of barium selectively inhibits inwardly rectifying K + channels. Activation of inward rectifying potassium channels by K + hyperpolarizes endothelial cells, an effect that can be transmitted to smooth muscle cells through myoendothelial junctions, thus supporting the speculation that potassium can act as EDHF [63,64]. In human subcutaneous arteries, K + causes relaxation and removal of the endothelium reduces this relaxation [42,65]. 4. Contribution and nature of endothelium-derived contracting factors 4.1. Endothelin-1 ET-1 is synthesized in several cell types, including the endothelial cells. There are three types of ET, but vascular ECs produce only ET-1 [66]. The precursor of ET-1 is preproendothelin-1. Cleavage of preproendothelin by endopeptidases generates big endothelin-1 which is converted to ET-1 by endothelin-converting enzyme [67].ET-1 is a potent vasoconstrictor [3] and because of its short half-life [68], this peptide acts mainly as a locally active vasoregulator. ET-1 is released in response to a variety of stimuli such as hypoxia, adrenaline [69], shear stress and ischemia [70]. ET-1 binds two two G-protein coupled ET-1 receptors: vasoconstrictor ETA receptors are located on vascular smooth muscle cells, and ETB receptor mediating vasodilation andlocated on endothelial cells [3,69]. Vasoconstriction due to ET-1 activation is associated with increases in intracellular Ca 2+ concentrations. When ET-1 binds to G-protein coupled ET A receptor, it causes activation of phospholipase C enzyme and cleavage of phosphatidyl inositol biphosphate to inositol triphosphate (IP 3 ) and diacylglycerol (DAG). IP 3 releases Ca 2+ from intracellular stores and DAG increases the influx of Ca 2+ [71,72]. The vasodilatory effects of ET-1 are mediated by ET B receptor located on the surface of endothelial cells. Activation of ET B receptors activates cytosolic phospholipase A 2 and the synthesis of other endothelium-derived factors such as PGI 2 [73]. In addition, ET B receptors can also modulate the formation of NO and so be protective against excessive vasoconstriction [74] Constricting prostaglandins Endothelial cells release PGH 2 and TXA 2 as endotheliumderived contracting factors. PGH 2 which is a precursor of all prostanoids including TXA 2 [28], is derived from arachidonic acid. Arachidonic acid is the cleavage product of the breakdown of membrane phospholipids by the action of phospholipase A 2. Cyclooxygenase-1 and -2 convert the arachidonic acid to PGH 2, which is then convertd to other biologically active eicosanoids such as TXA 2 through the action of TX synthase. Both PGH 2 and TXA 2 cause vasoconstriction reducing vascular smooth muscle intracellular camp concentrations [75] and/or increases in cytosolic calcium levels [76] HETE One of the major metabolites of arachidonic acid is 20- hydroxyeicosatetraenoic acid (20-HETE). Some evidence suggest that 20-HETE has a central role in regulation of pulmonary and renal vascular function [77,78]. 20-HETE is a potent constrictor of renal and cerebral arteries [79,80]. The elevation of cytoplasmic Ca 2+ activates phospholipase A 2, which converts arachidonic acid to 20-HETE. Subsequent inhibition of large conductance K ca channels in vascular smooth muscle cells promotes the membrane depolarization and Ca 2+ entry and vasoconstriction [80]. Blockade of 20- HETE impairs the autoregulation of blood flow [80,81] as well as eliminating pressure-induced constriction of isolated cerebral arteries [80].

6 54 M. Khazaei et al. / Pathophysiology 15 (2008) Table 1 Prothrombotic and antithrombotic factors and their mechanisms Pro/antithrombotic Mechanism Reference Proteoglycans Antithrombotic Negatively charged proteoglycans prevent platelet adhesion, some [83,84] proteoglycans binds to antithrombin III which inhibit thrombin activity, dermatan sulfate in subendothelium promotes the antithrombotic activity of heparin cofator II Protein C Antithrombotic Inactivates factor Va and VIIIa Thrombomodulin Antithrombotic Catalyses the activation of protein C by thrombin [85] Protein S Antithrombotic Cofactor for action of activated protein C Tissue factor pathway inhibitor (TFPI) Antithrombotic Bind to VIIa and Xa and inhibit activity of tissue factor and extrinsic [86] coagulation pathway Annexin V Antithrombotic Binds to negatively charged phospholipids and inhibits the anchoring [87] coagualtion factor to the endothelium Ectonucleotidase Antithrombotic Degrades platelet-stimulated ADP into AMP [88] Tissue plasminogen activator Antithrombotic Increase fibrinolytic activity [89] vwf Prothrombotic Promote thrombosis NO, PGI 2 Antithrombotic Suppressing platelet adhesion and activation [24,33] Annexin II Prothrombotic Binds to t-pa and plasminogen and enhances plasmin generation [90] 5. Hemostasis Hemostasis is mediated by a balance of precoagulant and anticoagulant factors. Under normal condition, there is a balance between thrombotic and antithrombotic properties of endothelium. Table 1 illustrates some of the important prothrombotic and antithrombotic factors. Perturbation of endothelial function by various stimuli can contribute to loss of the antithrombotic state of these cells, leading to various pathologies. For example, thrombomodulin, which is present on endothelial surfaces, binds to thrombin and prevents platelet activation and fibrin production [82]. In contrast, von Willebrand factor (vwf), which is also expressed on the surface of endothelial cells, interacts with the platelet glycoprotein (GP) receptor complex, initiating platelet adhesion and aggregation. In the presence of a normal endothelium, platelets circulate without adhesion to the vascular wall. Endothelial production of prostacyclin and NO are important for the prevention of platelet adhesion and aggregation [24] through increases in camp and cgmp in platelet cytoplasm, respectively. 6. Angiogenesis Angiogenesis, the growth of new blood vessels from pre-existing ones, is an important process involved in physiological processes such as growth, reproduction and wound healing and in some pathological processes such as diabetic retinopathy, rheumatoid arthritis, cancer development and inflammation [91]. Growth factors are the most potent mediators of angiogenesis. Basophilic fibroblast growth factor (bfgf), acidic FGF (afgf), vascular endothelial growth factor (VEGF), ECGF and hepatocyte growth factor (HGF) are bound to heparin and heparan sulfate in the extracellular matrix. During neovascularization, these mediators are mobilized by endothelial cell-derived heparanase and plasmin [92,93]. Angiogenic mediators augment EC proliferation and migration or through indirect stimulation of the production of other angiogenic factors [92 94] such as VEGF, a potent inducer of EC migration and proliferation [91]. VEGF and other angiogenic factors activate ECs to secrete proteases which disrupt EC basement membranes and EC matrix interactions. This allows ECs to proliferate, migrate and synthesize a new basement membrane leading to formation of a sprout. In addition, there are several chemokine receptors on the surface of ECs, possibly playing a role in chemokinederived angiogenesis. As was briefly mentioned, angiogenesis requires degradation of the EC matrix by proteolytic enzymes, such as matrix metalloproteases; including collagenase, gelatinase and stromelysin, as well as urokinase-plasminogen activators and tissue-type plasminogen activators [92,94]. It is hypothesized that when ECs are stimulated by angiogenic factors, plasminogen and urokinase bind to their receptors to activate ECs. This leads to the formation of plasmin that activates prometalloproteases and degrades the EC matrix. This is a path for endothelial cell migration and induction of angiogenesis [95]. In addition, there are several ECs adhesion molecules such as 1 integrins, E-selectin, L-selectin, vascular cell adhesion molecule-1 (VCAM-1), platelet endothelial cells adhesion molecule-1 (PECAM-1), endoglin and vascular endothelial cadherin (or cadherin-5) which are important for angiogenesis [92,93,96,97]. For example, vascular endothelial cadherin from one EC can bind to vascular endothelium cadherin expressed on an adjacent cell and form a weak junction. Endothelial cells also secrete cyclooxygenase enzymes that convert arachidonic acid to prostaglandins. The cyclooxygenase/prostaglandin system also is involved in angiogenesis. For example, prostaglandin E2 is angiogenic [92,93]. Of the two types of cyclooxygenase, cyclooxygenase II has been implicated in VEGF-dependent neovascularization [98].

7 M. Khazaei et al. / Pathophysiology 15 (2008) Fig. 2. Various means of leukocyte migration to the interstitial tissue in inflamed endothelium. (A and B) Endothelial cell junctional defect due to injury (induced by antibodies against endothelium or reactive oxygen species) or endothelial cell contraction (by histamine-type mediators). (C) Transmigration has four main steps. (1 and 2) Rolling and adhesion is selectin-mediated and terminates at junction of endothelial cells by chemokines acting as arrest signalling. (3) Activation by chemokines increases the expression of adhesion molecules (VCAM-1, ICAM-1 and ICAM-2) and activates integrins ( 4(1, L(2, M(2). (4) The interaction between adhesion molecules and integrins further tightens the link between leukocytes and endothelial cells leading to leukocyte transmigration to interstitial space via the junction between endothelial cells (diapedes). PSGL-1: P-selectin glycoprotein ligand-1; VCAM-1: vascular endothelial cell adhesion molecule-1; ICAM-1 and ICAM-2: intercellular adhesion molecule 1 and 2; Abs: antibodies. 7. Endothelial cells, inflammation and immune response Endothelial cells produce several inflammatory mediators and express cellular adhesion molecules and also undergo some morphological changes during inflammation (Fig. 2). Therefore, endothelial cells can be targets of inflammatory mediators and leukocytes during inflammation. Adhesion of leukocytes to the endothelium is a key event in inflammation that leads to leukocyte transendothelial migration into inflammatory sites [99]. Adhesion of leukocyte to endothelial cells occurs in at least four distinct steps. An early rolling adhesion of leukocytes occurs within the first 1 2 h. The adhesion of leukocytes to endothelial cells is through endothelial cellular adhesion molecules such as ICAM-1, ICAM-2 and VCAM-1 [99,100]. Leukocyte activation and triggering occurs next as a result of the interactions between chemokine receptors on leukocytes and proteoglycans on endothelial cells. In this step upregulation of selectins, synthesis of chemokines and platelet activating factor occur [70]. Transendothelial migration or diapedesis that involves integrins, ICAM-1 and VCAM-1, occurs when secreted chemokines bind to endothelial heparan sulfate. Endothelial cell adhesion to leukocytes and to the extracellular matrix is mediated by cell adhesion molecules such as integrins, selectins and immunoglobulins [99 101]. Integrins are mainly involved in endothelial cell adhesion to extracellular matrix macromolecules, whereas immunoglobulins and selectins play a role in endothelial cell adhesion to other cells [99 102]. The endothelium also undergoes morphologic changes during inflammation, leading to increased vascular permeability and leakage. Vascular leakage results from endothelial cell contraction and retraction. In addition, endothelial cells release NO and PGI2 which are vasodilators. Vasodilatation indirectly enhances vascular permeability. Leukocytes that interact with the vascular wall and anti-endothelial cells antibody causes endothelial cell injury and increased vascular permeability. The mediators in this process are reactive oxygen intermediates and some matrix metalloproteinases [103]. 8. Endothelial permeability The endothelium forms a macromolecular barrier between the blood vessels and underlying tissues and has been termed a tissue-blood barrier [104]. Although the microvascular endothelium acts as a barrier to the movement of macromolecules, it is not an absolute barrier and macromolecules

8 56 M. Khazaei et al. / Pathophysiology 15 (2008) do cross the microvascular endothelium. Several elements regulate the integrity of the endothelium and thus endothelial permeability. These include: (a) intercellular junctions, (b) cell surface binding proteins, (c) the electrostatic charge of endothelial membranes and (d) the composition of the basement membrane. The most important of these are intercellular junctions, which create a physical attachment between two contiguous cell membranes. Three major types of intercellular junctions have been identified in endothelial cells: tight junctions, gap junctions and adherence junctions [105]. Endothelial cell dysfunction characterized by apoptosis and abnormal immune activation is, at least in part, induced by anti-endothelial cell antibodies in some cases of autoimmune disease. However, the molecular mechanisms of anti-endothelial cell antibodies-mediated damage to the vascular system remains unclear. Antibodies that bind to the plasma membrane of endothelial cells can activate endothelial cells and induce the expression of cell adhesion molecules [106]. Furthermore, some anti-endothelial antibodies have been shown to induce apoptosis of endothelial cells [107]. Several biological factors also can modulate endothelial junctions and thus influence permeability across the endothelium [108]. Inflammatory mediators such as thrombin and histamine increase endothelial permeability within minutes. The possible mechanisms of permeability modulation include phosphorylation of proteins involved in the organization of endothelial junctions with subsequent actin myosin contraction and an increased interendothelial gap formation. Thus, it appears changes in transportation of macromolecules through endothelial junctions is a major mechanism for increased endothelial permeability, at least in response to selected inflammatory mediators [109,110]. There are several endothelium-derived factors that can modulate endothelial permeability. For example, inhibition of the basal production of endothelial NO decreases permeability across vascular endothelium. On the other hand, enhanced levels of NO can mediate increased permeability induced by vascular endothelial growth factor. Overproduction of endothelin-1, angiotensin II as well as inflammatory cytokines, such as TNF and interleukin-1 also contribute to increased permeability across the vascular endothelium [105]. Endothelial dysfunction increases the permeability of the endothelium to lipoproteins, monocytes and macrophages, so increasing smooth muscle cell migration and proliferation [17] and aiding the formation of an intermediate lesion and progression to an atherosclerotic plaque [111]. 9. Endothelium in diseases Alterations in endothelial function are important in a variety of pathological conditions. Endothelial dysfunction whether caused by physical injury or cellular damage, leads to compensatory responses that alter the normal homeostatic properties of the endothelium [111]. Most of the common forms of cardiovascular disease are caused by functional and structural changes in the blood-vessel wall. These vascular abnormalities have an important role in the pathogenesis of angina pectoris, myocardial infarction, stroke and vascular forms of renal failure. The following section will investigate endothelial dysfunction in some pathologic states Endothelial cells in hypertension Endothelial cell damage occurs in many vascular bed during hypertension [ ]. However, it is not clear whether hypertension is the cause or the result of this damage. In 1978, Moncada and Vane [116] suggested that endothelial dysfunction follows the course of chronic blood pressure increases and was therefore a consequence of hypertension. On the other hand, Panza et al. [117] found that treatment of hypertension did not improve endothelial function, arguing against endothelial dysfunction as being a consequence of hypertension. An important consideration in this context is the mode of treatment of hypertension. For example, lowering blood pressure with beta-blockers does not improve endothelial function while treatment with angiotensin converting enzyme inhibitors (ACEIs) or angiotensin receptor blockers (ARBs) caused significantly improved endothelial function [112,118]. Supporting the notion that endothelial dysfunction is one of the causes of hypertension is the finding of impaired endothelial function in the normotensive offspring of patients with essential hypertension [119]. A common abnormal finding in hypertensive states is a lower production of endothelial vasodilatory factors or their ineffectiveness and/or over production of or sensitivity to vasoconstrictor agents. One suggestion is that increased oxidative stress in hypertensive states leads to decreased availability of NO [120]. In agreement with this is the finding that antioxidants improve endothelium-dependent relaxation [121]. Higher oxidative stress in some hypertensive states could be due to increased levels of angiotensin-ii, which stimulates NADPH oxidase to generate ROS [122] causing vascular inflammation [123]. By preventing these effects, ACEIs and ARBs improve vasorelaxation in hypertensive patients. Other than angiotensin-ii, endothelial vasoconstrictor prostanoids are also implicated in production of increased vascular tone in patients with essential hypertension [124].It is also likely that over production of endothelin-1 may play a role in hypertension. Measurement of plasma levels of ET-1 in hypertensive rats has been promising but is confounded by opposite results in human patients. In pulmonary hypertension, however, higher plasma level of ET-1 occurs both in human and animal disease [125]. There is some success in the clinical use of the ET-1 receptor A and B antagonist, bosentan in patients with pulmonary hypertension [126] Endothelium in Atherosclerosis and hyperlipidemia Predisposing risk factors for atherosclerosis, which include hypertension, diabetes, smoking and hypercholes-

9 M. Khazaei et al. / Pathophysiology 15 (2008) terolemia are all associated with endothelial dysfunction. In these conditions, the endothelial phenotype changes to a proinflammatory and prothrombotic state [127] by increased expression of leukocyte adhesion molecules (such as VCAM-1) and cytokines such as monocyte chemoattractant protein-1. These changes augment monocyte adhesion to and penetration through the vascular wall. A reduction in endothelium-derived NO is suggested as one of the causes of such endothelial phenotypical changes. The antiatherogenic role of NO is supported by several studies in apo-e knockout mice and other animal models of atherosclerosis. In these models, the inhibition of endothelial NO production accelerates lesion formation in the aorta and coronary arteries, and l-arginine treatment preserves vessel morphology. One mechanism for lower NO bioavailability in arteries predisposed to atherosclerosis could be the increased production of superoxide [128,129]. The ROS will either degrade NO or tetrahydrobiopterin (BH4), a cofactor in its synthetic pathway [ ]. The source for ROS in the arterial bed is through augmented production by NADPH oxidase [134], xanthine oxidase [135,136] or reduced degradation by superoxide desmutase [137,138]. Endothelial dysfunction also occurs in inflammatory conditions that promote atherosclerosis due to increased levels of C-reactive protein (CRP) [139]. CRP decreases enosmediated NO production by decreasing the stability of enos mrna [139]. Moreover, in vitro experiments suggest that oxidized lipoproteins and lysophosphatidylcholine, two important mediators of atherogenesis, inhibit both NO and EDHF release [140]. Oxidized LDL also decreases the expression of enos [141] or its function in vitro [142], effects that are reversed by antioxidants. Lipids and oxidants also decrease the bioavailability of NO by impairing the structure of caveolae and activation of proinflammatory pathways such as NF B. Further, in response to inflammatory mediators (IL-1, TNF- ) that are released from activated monocytes, endothelial cells as well as SMCs will secrete growth factors that will enhance atherogenesis [1]. Excessive endothelial secretions such as ET-1 can have a significant role in atherogenesis since both plasma and coronary vascular endothelin immunoreactivity is increased in hyperlipidemia as well as in early and advanced atherosclerosis. Endothelin enhances atherogenesis through several mechanisms. First, it is a strong chemoattractant by stimulating ET B receptors on circulating monocytes. Second, ET-1 activates macrophages leading to over secretion of inflammatory mediators such as IL-6 and IL-8, TNF, PG E2 and superoxide anion [143,144]. Third, ET-1 stimulates smooth muscle cell migration and hypertrophy and the production of firoblast growth factor-2, making them hyper responsive to angiotensin-2 [145]. Forth, ET-1 increases fibroblasts proliferation, chemotaxis and matrix biosynthesis [145]. Fifth, ET-1 causes PKC activation and increases platelet adherence through increased P-selectin, however, little is known about the effect of ET-1 on platelets in vivo [146]. Finally, endothelial dysfunction also causes atheromatous plaque instability. It will worsen the consequences of plaque rupture due to ineffective antiaggregation, antithrombotic and fibrinolytic functions. In fact, a dysfunctional endothelium may be prothrombotic by the over production of plasminogen activator inhibitor-1, an inhibitor of fibrinolysis [12] Endothelial cells and hypertrophic cardiomyopathy An abnormality in the physiology of the vascular ET-1 system has been reported in the transgenic mice expressing a hypertrophic cardiomyopathy phenotype. At 11 months of age, the pressure-induced response of isolated coronary small arteries was reduced in the transgenic mice compared to their wild type (WT) counterparts. This difference disappeared after treatment with the endothelin-1 dual-receptor antagonist, bosentan. In this model, the constrictor response to exogenously applied ET-1 was also attenuated. The latter finding together with the fact that potassium-induced constriction was the same in both transgeninc and WT mice serves as a strong evidence that a reduction of responsiveness to ET-1 is involved in the attenuated pressure-induced response in the transgenic mice. Since the responses to acetylcholine and sodium nitroprusside were identical in the two groups, the NO-cGMP axis was not affected [147]. However, in 3 month-old hypertrophic cardiomyopathy (HCM) mice the pressure-induced response was not impaired indicating that the vascular expression of the disease may not completely manifest until later in adulthood when growth and development have occurred. Krams et al. [148] also reported that coronary resistance reserve and lumen in HCM patients were lower under resting conditions but similar to control patients during reactive hyperemia. Although this finding may have many explanations, it is also consistent with reduced pressure-induced tone of the coronary arteries in these patients. The decreased pressure-induced response would also tend to increase capillary pressure for a given change in blood pressure, potentially leading to edema formation in the heart. An aberrant vasoregulation may thus lead to ischemia and in part be responsible for the observation that many patients with HCM have small vessel coronary disease [ ]. Ultrastructural changes of small arteries and arterioles such as swelling of the endothelial cells accompanied by luminal stenosis has may be related to impaired microcirculation in HCM patients [152]. In conclusion, swelling of endothelial cells as well as luminal stenosis of small arteries and arterioles may be related to a disturbed coronary microcirculation in HCM patients Endothelial cells and cigarette smoke Cigarette smoking is one of the three major independent risks for coronary heart disease (CHD). Cigarette smoking is more prevalent in the population than is either of the other two major risk factors (hypertension and hypercholesterolemia), and thus represents the largest modifiable cause of CHD.

10 58 M. Khazaei et al. / Pathophysiology 15 (2008) Cigarette smoking also acts synergistically with the other major risk factors to greatly increase the risk for CHD [153]. One of the primary sites of damage by cigarette smoke is the vascular endothelium. There are several studies showing that cigarette smoking impairs endothelium-dependent vasodilatation in humans (carotid [154], brachial [ ], coronary [ ] arteries and saphenous vein [159]) and animals (rat carotid [160], rabbit aorta [161], hamster cheek artery [162,163]). It is important to note that loss of coronary vasodilator function due to cigarette smoking occurs regardless of the presence or absence of coronary atherosclerotic lesions [164]. However, a significant positive correlation exists between cigarette smoking and atherosclerotic lesions in the coronary circulation, at least for those most highly at risk [165]. Cigarette smoking is also associated with other pathologies, such as thrombosis, hemorrhage or vasoconstriction, which lead to occlusion and ischemia that can also be explained by defective endothelial cell function in smokers. The relative risk for coronary artery disease versus cerebral artery disease in active smokers is nearly doubled, however, the reason for this difference is not known [166]. In fact there are few mechanistic studies on the effect of cigarette smoking on the function of resistance arteries from either the coronary or cerebral circulation. In the only study on cerebral (pial) artery function [167], acute cigarette smoking under acute conditions reduced endothelial function in rats. It is also shown that following acute exposure of rats to cigarette smoke, mean arterial pressure increased [167]; on a chronic basis, this would have profound effects on cerebral blood flow autoregulation. Since endothelin-1 does mediate acute cigarette smoke-induced cell proliferation of rat airways and pulmonary arterial vessels [168] it may also play a significant role in cerebral and coronary vessels in smokers. Endothelial dysfunction in smokers can also result from circulating factors such as cigarette toxins and free radicals in the blood. In fact one puff of cigarette smoke contains 1015 oxidant radicals [169]. The circulation of these toxins may lead to initiation and progression of atherosclerosis. It is also indicated that smokers have increased levels of advanced glycation end products, which react with the endothelium and lead to vasculopathy [170]. A key dysfunctional manifestation in smokers may be excessive endothelial apoptosis, which can promote platelet aggregation, monocyte adhesion and increased coagulability of blood leading to atherosclerosis and coronary heart diseases. There is a recent report that in isolated human umbilical artery endothelial cells in vitro, cigarette smoke causes increased apoptosis [171]. Cigarette smoke-induced cell death causes clustering (foci) of apoptotic endothelial cells; this results in exposure of a naked extracellular matrix, leukocyte and platelet adhesions, thrombogenesis and atherogenesis. There are numerous reports of cigarette-induced apoptosis in non-vascular rat tissues such as alveolar macropages [172], gastric mucosa [173], testis [174]. However, it is not known if cigarette smoke-induced endothelial apoptosis occurs in vivo, and what the functional vascular alterations are in terms of augmented myogenic vasoconstriction in resistance arteries Endothelial cells and postmenopausal estrogens The most widely studied vascular effect of estrogen is its regulation of endothelial function. In postmenopausal women, intracoronary infusion of estrogen selectively potentiates endothelium-dependent dilation by ACh in both conductance and resistance arteries [175]. In women with early atherosclerosis, ACh acts as a coronary vasoconstrictor; in these subjects, estrogen replacement therapy (ERT) transforms constriction into vasodilatation [176]. Likewise, the extent of exertional angina is significantly reduced by sublingual estrogen therapy [177]. Acute sublingual administration of estrogen in postmenopausal women increases blood flow and decreases resistance to flow in the forearm without altering mean arterial pressure [178]. It is likely that estrogen exerts its beneficial effects on the cardiovascular system through augmenting the release of NO from the endothelium [179]. A positive correlation exists between plasma estrogen levels and stable metabolites of NO (nitrate/nitrite) in postmenopausal women [180]. Estrogen regulates the activity of enos, the synthetic enzyme for NO in human [ ] and rat endothelial cells [184]. Such potential benefits are at menopause by virtue of a number of factors beyond the activity and expression of NOS regulation by estrogen [ ], including changes in VEGF [ ]. As well, NO has anti-apoptotic effects in human endothelial cells in culture, where it enhances Bcl-2 protein stabilization, Akt activation and inhibits caspase activity [189]. In a recent study in aged ovarectomized rats, we [190] reported that female rats had a greater endothelium-dependent vasodilation compared to age-matched male rats. In these rats ovariectomy caused an immediate loss in plasma estradiol, a delayed increase in apoptosis of endothelial cells, and attendant loss of endothelium-dependent vasodilation. In this study, replacement of estradiol in ovariectomized rats protected endothelial cells against apoptosis and improved their function. One mechanism by which estrogen increases plasma markers of NO is by decreasing age-related oxidation of NO [191]. Estrogen also augments vasodilatation by reducing plasma endothelin-1 levels in postmenopausal women [192]. Higher levels of nitric oxide confer cardiovascular protection by ensuring greater patency and reducing formation of emboli [193]. Despite the beneficial effects of estrogen on improving endothelial cell function, randomized control trials have failed to demonstrate a significant effect of hormone replacement therapy on primary [194] or secondary [195] prevention of coronary vascular disease. Therefore, HRT is no longer indicated for CVD risk factor reduction [5] Endothelial cells and diabetes Endothelial dysfunction occurs in resistance and conduit arteries from diabetic animal models [ ]. There are

11 M. Khazaei et al. / Pathophysiology 15 (2008) multiple mechanisms by which hyperglycemia associated with diabetes mellitus (DM) can modify the endothelium, including glycation of proteins and lipids [199], oxidative stress [200] and activation of protein kinase C [201]. The evidence summarizing endothelial dysfunction in human and animal diabetes has been extensively reviewed [ ]. Specifically in coronary vessels, studies in animal models of type II DM clearly demonstrate markedly reduced endothelium-dependent relaxation [197,198, ] as well as exaggerated responses to vasoconstrictors, both of which compromise the ability of coronary vessels to dilate effectively, resulting in reduced blood flow to the heart. Furthermore, peripheral (mesenteric, skeletal muscle and adipose tissue) arterioles of type II diabetic subjects have increased myogenic tone, which may adversely impact blood flow (e.g. susceptibility to ischemic events) [197,216]. Lower NO production or availability in diabetic vessels, which causes attenuated endothelium-dependent relaxation, is not due to decreased enos protein expression [196]. In fact, diabetes might even upregulate enos protein expression in endothelial cells [217]. Although enos is present, it is possible that the activity [218] and regulation of this enzyme may negatively impacted in diabetes [219]. Moreover, decreased availability of enos substrate, l-arginine, or cofactors such as NADPH, Ca 2+ (ET influx), and tetrahydrobiopterin occurs in diabetes [220,221]. Lower NO bioavailability can also be the result of post-translational changes in diabetes. Moreover, hyperglycemia attenuates guanylate cyclase activity in endothelial cells without directly affecting enos [222].Itis also possible that in diabetes there is a higher degradation of NO due to increased oxidative destruction [212, ]. Finally, diabetes alters prostaglandin synthesis, which will then affect vascular tone. For example, attenuation of prostacyclin synthesis [223] changes NO production or vascular reactivity to this agent, and over production of constrictor prostaglandins may enhance the contractile response to alpha adrenergic stimulation [227,228] Endothelium and viral myocarditis In mouse models of this disease, vasospasms are observed in histological sections of perfusion-fixed hearts early after viral infection [229]. These vascular abnormalities are associated with the development of late cardiomyopathy, and both the apparent vascular dysfunction and development of cardiomyopathy can be ameliorated by treatment with agents that prevent vasospasms, such as calcium channel blockers, ACE inhibitors, or 1- and -adrenergic blockers [ ]. More recently, Ono et al. [232] suggested that increased ET-1 in virally infected hearts contributes to vascular dysfunction by inducing vasoconstriction. Indeed, high levels of ET-1 occur in the myocardium after infection with ECMV, and inhibition of ET-1A and B receptors in vivo by treatment of animals with bosentan significantly reduces the pathological markers of myocarditis, such as myocardial necrosis and heart weight/body weight, indicating that ET-1 contributes to cardiac damage and resultant cardiomyopathy following viral infection [232]. Although this apparent vascular dysfunction occurs early in the disease process in several mouse models, the prevention of vasospasms by treatment with verapamil also attenuates the development of cardiomyopathy during the chronic stages of viral myocarditis, suggesting that this event contributes to cardiac remodeling. To directly assess the nature of vascular dysfunction in coronary arteries during viral myocarditis, we have performed pressure myography on isolated septal arteries from coxsackievirus-infected mice [233]. Prior to these studies, the assessment of vascular dysfunction in virus-infected mice had relied on histological examination of perfusion-fixed tissues. In a CD-1 mouse model of coxsackievirus-induced myocarditis, there is no detectable vasomotor dysfunction of isolated coronary resistance arteries during the early course of this disease despite an appreciable virus titer and inflammatory response in the myocardium. However, subsequent to virus clearance, there is significant vasomotor dysfunction of coronary arteries that is characterized by an increase in pressure-induced constriction as well as a reduction in acetylcholine (ACh)-induced vasodilation. In normal arteries, ACh exerts its vasodilatory effects by stimulating the release of NO from enos of the endothelium. However, in the absence of enos, ACh directly stimulates SMC to constrict. As such, a reduction in ACh-induced vasodilation, or the constriction of vessels to this agonist, is an indication of reduced enos activity in the endothelium of arteries. In addition, inhibition of ET-1 (a vasoconstrictor shown to be increased in other models of viral myocarditis) with the ET-1 receptors A and B inhibitor bosentan did not change the responsiveness of isolated arteries to Ach-induced vasodilation. Taken together, these data suggest that vascular dysfunction that occurs during the late stages of viral myocarditis does not involve ET-1. Indeed, there is no increase in ET-1 levels during this stage of myocarditis in CD-1 mice. Finally, because Ach-induced vasodilation in CD-1 mice is regulated exclusively by ET-1 and enos, the lack of contribution of ET-1 to this virus-induced vascular dysfunction indicates that the observed vasomotor abnormalities are most likely a result of reduced enos expression and/or activity [234]. In humans, there is also severe endothelial dysfunction during viral myocarditis. Specifically, in patients afflicted with this disease the epicardial coronary arteries constrict rather than dilate in response to ACh, indicating that the vasodilating properties of ACh (which are mediated by nitric oxide) are defective [235]. Similarly, coronary blood flow is not increased in response to ACh in patients with diabetic cardiomyopathy while this treatment significantly increases blood flow in control patients [236]. These findings are consistent with our observation of reduced endothelium-derived NO, characterized by a deficiency in ACh-induced vasodilation, in our mouse model of this disease. Interestingly, the less dramatic dysfunction observed in human diabetic cardiomyopathy patients strikingly resembles the late EC dysfunction in our animal studies.