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between these factors determines the functional or dysfunctional state of the endothelium.

Vasorelaxing Factors

The principle vasodilating factor secreted by the endothelial cells is NO. It is a free radical that is produced by the oxidation of L-arginine to L-citrulline, by the enzyme nitric oxide synthase (NOS) (Fig. 23.1) [4]. This enzyme has three isoforms: the neuronal NOS (nNOS), the inducible NOS (iNOS), and the endothelial NOS (eNOS). The endothelial cells constitutively express the eNOS, which constantly releases small amounts of NO in systemic and pulmonary circulations [5]. Once released from the endothelial cells, NO interacts with the heme prosthetic group of guanylate cyclase present on the surface of underlying smooth muscle cells. This causes the activation of guanylate cyclase and increased production of cyclic guanidine monophosphate (cGMP). Increased cGMP leads to a decrease in the intracellular

L-arginine L-citrulline

NOS

Nitric Oxide

Anti-inflammatory

Anti-proliferative

Anti-thrombogenic

Anti-atherosclerotic

FIGURE 23.1. Various pathways involved in mediating the antiatherosclerotic effects of nitric oxide. NOS, nitric oxide synthase; VSMCs, vascular smooth muscle cells. ↑, increase; ↓, decrease.

reported a strong association between reduction in the bioavailability of the vasodilator NO and progression of endothelial dysfunction [15–20]. This may result either due to reduced activity of eNOS or due to increased degradation of NO. Since endothelial NO is largely responsible for endothelium-dependent dilation of blood vessels, this dilation is severely blunted in coronary and peripheral arteries during atherosclerosis [18, 21, 22].

In addition to the loss of vasodilator NO, endothelial injury is also associated with an increase in the production of vasoconstrictive factors such as ET-1 [23–25]. This leads to further exaggeration of loss of NO and the vasculature becomes prone to unregulated constriction. This can further lead to the development of hypertension and future coronary heart disease (CHD).

Regulation of Inflammation and Thrombosis by the Endothelium

In addition to the maintenance of vascular tone, a normal endothelium possesses anti-proliferative and anti-inflammatory properties. Endothelium-derived vasodilator NO inhibits leukocyte adhesion to the endothelium [26, 27], migration and proliferation of VSMC [28, 29], and stimulates the proliferation of endothelial cells [30]. NO is further known to inhibit platelet aggregation and promote platelet deaggregation [31] (Fig. 23.1). Prostacyclins, another endothe- lium-derived vasodilator, also interact synergistically with NO, causing inhibition of platelet adhesion and aggregation [32]. Furthermore, the healthy endothelial cells have a negatively charged surface coated with heparans, which exert contact inhibition [33]. Endothelial cells also express anticoagulant factors such as tissue plasminogen activator (tPA), inactivators of thrombin, and thrombomodulin [32]. As a result, leukocytes do not stick to the vascular surface and cell proliferation is tightly controlled [33, 34]. These are among the multiple and redundant healthy defenses that are impaired during the endothelial dysfunction and atherosclerosis.

Loss of Control of Inflammation and Thrombosis

Loss of endothelial function results in an impairment of normal anticoagulant defenses. Reduced bioavailability of NO, abnormal heparans, local thrombin activation, relative lack of tissue PAF-1, and thrombomodulin are the factors that favor local coagulation [35]. Similarly, increased tissue plasminogen activator inhibitor and lack of tissue PAF-1 impair clot lysis [36, 37]. Thrombin activation, membrane-bound platelet adhesion molecules, exposure of collagen, increased production of tissue factor, and loss of NO also favor increased platelet adhesion and aggregation [38, 39].

The various risk factors and pathological stimuli associated with the development of endothelial dysfunction also lead to the abnormal activation of

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the vessel wall. This leads to the stimulation of certain signaling cascades resulting in the onset of adhesion and inflammation in the vasculature. This results in an abnormal increase in the growth of VSMC, fibroblasts, and matrix within the vessel wall. This process further leads to intimal thickening and plaque formation [35].

Endothelial Dysfunction: Role of Oxidative stress

The development of the endothelial dysfunction is associated with a number of altered physiological markers. Oxidative stress or the presence of reactive oxygen species (ROS) is one such marker that is known to be a key player in its development. The mechanism for the generation of intracellular ROS involves multiple enzyme systems including NAD(P)H oxidase, xanthine oxidase, lipoxygenase (LOX), cytochrome p450, monooxygenase, and COX [40, 41]. Presence of ROS in vasculature leads to quenching of NO, resulting in the formation of peroxynitrite [42]. Peroxynitrite, a cytotoxic oxidant, further enhances the oxidative stress in the system by uncoupling of NOS [43]. It causes a reduction of eNOS cofactor tetrahydrobiopterin BH4, resulting in the formation of BH2. When this occurs, eNOS is uncoupled, thus losing the oxygenase and NO formation activity. This further activates the reductase function of eNOS and more ROS are generated. So, NOS switches from its NO-producing oxygenase activity to ROS producing reductase activity. This leads to a positive feedback loop resulting in enhanced ROS generation, thereby causing endothelial dysfunction [44]. Peroxynitrite is also known to be an important mediator of oxidation of low-density lipoproteins (LDL) [45]. Oxidized LDL (Ox LDL), once formed, can induce endothelial dysfunction and thus significantly contribute to the pathogenesis of atherosclerosis. There is evidence that the degree of endothelial dysfunction relates to the number and density of LDL particles, the level of Ox LDL, the susceptibility of LDL to oxidation in an individual and the titer of autoantibodies to Ox LDL [46]. Ox LDL also affects NO production by several mechanisms like: (a) Ox LDL can inhibit agonist-stimulated arginine transport by endothelial cells, (b) Ox LDL can mediate downregulation of eNOS expression both at mRNA and protein levels, thereby directly inhibiting NO production [47], (c) Ox LDL upregulates asymmetric dimethylarginine (ADMA), which is a competitive antagonist of NOS and may reduce NO production via substrate competition with L-arginine [48, 49], (d) Ox LDL increases synthesis of caveolin-1, which is a membrane protein known to bind and inactivate eNOS, thereby inhibiting production of NO [50]. By reducing NO production, Ox LDL also promotes thrombin generation and platelet aggregation [51]. In addition, Ox LDL alters the balance of other endothelial products, such as prostacyclins and ET-1, further contributing to platelet aggregation [52, 35]. Thus, generation of Ox LDL may mediate endothelial dysfunction by a range of molecular mechanisms (Fig. 23.2).

Peroxynitrite

Ox LDL

Endothelial dysfunction

Atherosclerosis

FIGURE 23.2. Different mechanisms by which Ox LDL causes endothelial dysfunction and atherosclerosis. Ox LDL, Oxidized low-density lipoprotein; eNOS, endothelial nitric oxide synthase; ADMA, asymmetric dimethylarginine. ↑, increase; ↓, decrease.

The importance of Ox LDL in atherogenesis has been well appreciated. Ox LDL activates vascular component cells, such as endothelial cells, macrophages, and VSMC. Also, it increases the chemotactic stimulus for monocytes, attracting them to the site of endothelial dysfunction and transforming them into monocyte-derived macrophages. These macrophages take up Ox LDL via the scavenger receptors (CD-36, SR-A, SR-PSOX) and further get transformed to “foam cells”, which are a key feature of atherosclerosis [53].

The ROS and their modified target biomolecules (e.g., Ox LDL) are also defined as true second-messenger molecules that regulate various signal transduction cascades upstream of nuclear transcription factors, including modulation of Ca2+ signaling, protein kinase, and protein phosphatase pathways [54, 55]. Recently, G-protein Ras has been proposed to act as a mediator of ROS signaling, activating a cascade of kinases, including diverse members of the mitogen-activated protein kinase (MAPK) family [56]. In the case of extracellular signal-regulated kinase 5 (ERK5) or big mitogen-activated protein kinase 1 (BMK1), hydrogen peroxide appears to be an exclusive activator [57]. Different agents that induce oxidative stress have been demonstrated to stimulate tyrosine kinase activity, induce tyrosine phosphorylation events, and activate downstream kinases such as protein kinase C (PKC), c-Src, Raf-1, and MAPK [54, 57–59]. MAPK phosphorylates and enhances the transcriptional activity of redox sensitive nuclear transcription factors such as NFκB and activator protein-1 [54, 60]. Activation of these nuclear factors can further upregulate genes encoding proteins that control adhesion and binding of macrophages and leukocytes to the endothelium.

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The increased production of macrophage chemoattractant protein-1 (MCP-1) recruits mononuclear phagocytes and increased expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin leads to an increase in the aggregatory events of these cells to the vessel wall. This upregulation has been proposed to be mediated both directly by Ox LDL and indirectly by changes in the redox state of endothelial cells [61–63]. Decreased NO and increased oxidative stress can also activate matrix metalloproteinases (MMP) namely MMP-2 and MMP-9, which contribute to the progression of atherosclerosis [64, 65]. Thus, endothelial dysfunction with reduced NO bioavailability, increased oxidative stress, and expression of adhesion molecules and cytokines contributes not only to the initiation but also to the progression of the atherosclerotic disease [66].

The vasoconstrictive factor, AngII, also contributes to ROS generation by activating NADPH oxidase leading to the production of superoxide radicals and decreased availability of NO, thus, resulting in impaired vascular function [67]. AngII not only acts as a prooxidant but also stimulates the production of ET-1 [68]. ET-1 and AngII promote proliferation of VSMC and thereby contribute to the development of atherosclerosis [69, 70].

Endothelial Dysfunction: Role of Inflammation

Oxidative stress has been intimately linked to the proinflammatory state of the endothelium. The expression of VCAM-1, ICAM-1, integrins, and E-selectin, in response to oxidative stress, plays an important role in the initiation of the inflammatory process [71]. In fact, the soluble forms of VCAM-1, ICAM-1, and E-selectin in the plasma have been hailed as the markers of endothelial dysfunction [72–75]. Under normal physiological conditions, the endothelium does not allow the circulating immune effector cells to adhere to its surface. However, dysfunction of the endothelium results in the loss of this property of nonadherence. Dysfunctional endothelium expresses a variety of adhesion molecules such as VCAM-1 and ICAM-1 that mediate the recruitment of monocytes, macrophages, T-lymphocytes, and platelets to the endothelium. This results in the production of proinflammatory cytokines such as interferon-γ (IFN-γ), tumor necrosis factor alpha (TNF-α), interleukins (IL-1, 2, 6, 8), and other factors like MCP-1, MCP-4, COX, and MMP by the endothelium, which in turn stimulate the immune cells to secrete these proinflammatory factors [76–77]. A proinflammatory feedback loop is thus established, as these mediators will (a) recruit additional local cytokine releasing immune cells to the endothelium (b) promote LDL receptor expression by the endothelium causing injurious aggregation of Ox LDL particles on its surface, and (c) promote the stimulation of systemic inflammation cascades [78].

The proinflammatory status of endothelium affects its membrane permeability. It allows the passage of adherent Ox LDL into the subendothelial