биохимия атеросклероза

148 Dean Gilham and Richard Lehner

151.Lehner R, Verger R: Purification and characterization of a porcine liver microsomal triacylglycerol hydrolase. Biochemistry 36: 1861–1868, 1997.

152.Lehner R, Cui Z, Vance DE: Subcellullar localization, developmental expression and characterization of a liver triacylglycerol hydrolase. Biochem J 338(Pt 3): 761–768, 1999.

153.Matsushima M, Inoue H, Ichinose M, Tsukada S, Miki K, Kurokawa K,

Takahashi T, Takahashi K: The nucleotide and deduced amino acid sequences of porcine liver proline-β-naphthylamidase. Evidence for the identity with carboxylesterase. FEBS Lett 293: 37–41, 1991.

154.Dolinsky VW, Gilham D, Alam M, Vance DE, Lehner R: Triacylglycerol hydrolase: role in intracellular lipid metabolism. Cell Mol Life Sci 61: 1633–1651, 2004.

155.Lehner R, Vance DE: Cloning and expression of a cDNA encoding a hepatic microsomal lipase that mobilizes stored triacylglycerol. Biochem J 343(Pt 1): 1–10, 1999.

156.Dolinsky VW, Sipione S, Lehner R, Vance DE: The cloning and expression of a murine triacylglycerol hydrolase cDNA and the structure of its corresponding gene. Biochim Biophys Acta 1532: 162–172, 2001.

157.Alam M, Ho S, Vance DE, Lehner R: Heterologous expression, purification, and characterization of human triacylglycerol hydrolase. Protein Expr Purif 24: 33–42, 2002.

158.Douglas DN, Dolinsky VW, Lehner R, Vance DE: A role for Sp1 in the transcriptional regulation of hepatic triacylglycerol hydrolase in the mouse. J Biol Chem 276: 25621–25630, 2001.

159.Gilham D, Lehner R: The physiological role of triacylglycerol hydrolase in lipid metabolism. Rev Endocr Metab Disord 5: 303–309, 2004.

160.Rusinol AE, Cui Z, Chen MH, Vance JE: A unique mitochondria-associated membrane fraction from rat liver has a high capacity for lipid synthesis and contains pre-Golgi secretory proteins including nascent lipoproteins. J Biol Chem 269: 27494–27502, 1994.

161.Gaustad R, Berg T, Fonnum F: Heterogeneity of carboxylesterases in rat liver cells. Biochem Pharmacol 44: 827–829, 1992.

162.Gilham D, Ho S, Rasouli M, Martres P, Vance DE, Lehner R: Inhibitors of hepatic microsomal triacylglycerol hydrolase decrease very low density lipoprotein secretion. FASEB J 17: 1685–1687, 2003.

163.Soni KG, Lehner R, Metalnikov P, O’Donnell P, Semache M, Gao W, Ashman K, Pshezhetsky AV, Mitchell GA: Carboxylesterase 3 (EC 3.1.1.1) is a major adipocyte lipase. J Biol Chem 279: 40683–40689, 2004.

164.Gilham D, Alam M, Gao W, Vance DE, Lehner R: Triacylglycerol hydrolase is localized to the endoplasmic reticulum by an unusual retrieval sequence where it participates in VLDL assembly without utilizing VLDL lipids as substrates. Mol Biol Cell 16: 984–996, 2005.

165.Durrington PN, Newton RS, Weinstein DB, Steinberg D: Effects of insulin and glucose on very low density lipoprotein triglyceride secretion by cultured rat hepatocytes. J Clin Invest 70: 63–73, 1982.

166.Brown AM, Wiggins D, Gibbons GF: Glucose phosphorylation is essential for the turnover of neutral lipid and the second stage assembly of triacylglycerolrich apoB-containing lipoproteins in primary hepatocyte cultures. Arterioscler Thromb Vasc Biol 19: 321–329, 1999.

167.Dolinsky VW, Gilham D, Hatch GM, Agellon LB, Lehner R, Vance DE: Regulation of triacylglycerol hydrolase expression by dietary fatty acids and peroxisomal proliferator-activated receptors. Biochim Biophys Acta 1635: 20–28, 2003.

168.Becker A, Bottcher A, Lackner KJ, Fehringer P, Notka F, Aslanidis C, Schmitz G: Purification, cloning, and expression of a human enzyme with acyl coenzyme A: cholesterol acyltransferase activity, which is identical to liver carboxylesterase. Arterioscler Thromb 14: 1346–1355, 1994.

169.Langmann T, Becker A, Aslanidis C, Notka F, Ullrich H, Schwer H, Schmitz G: Structural organization and characterization of the promoter region of a human carboxylesterase gene. Biochim Biophys Acta 1350: 65–74, 1997.

170.Huang GS, Yang SM, Hong MY, Yang PC, Liu YC: Differential gene expression of livers from apoE deficient mice. Life Sci 68: 19–28, 2000.

molecule-1, HSP: heat shock protein, HMG-CoA: 3-hydroxy-3-methylglutaryl coenzyme A, Statins: HMG-CoA reductase inhibitors, F1+2: prothrombin fragment 1+2, AII: angiotension II, ACE: angiotensin-converting enzyme, AT1: AII receptor type 1

Introduction

Low-density lipoprotein (LDL) is an independent risk factor for ischemic heart disease (IHD) [1]. LDL is removed from blood circulation via the LDL receptor [2]. LDL may be oxidatively modified by a group of biological factors or cells in vitro or in vivo. Particles of oxidized LDL (oxLDL) are recognized by various scavenger receptors (SR) on the surface of vascular smooth muscle cells (SMC), endothelial cells (EC), and macrophages [3, 4]. Oxidative modification substantially increases the atherogenecity of LDL [5]. Growing amounts of evidence suggest that oxLDL also affects the processes of coagulation, fibrinolysis, platelet activation, and thrombosis [6, 7]. This chapter summarizes up-to-date information on the role of oxLDL in thrombogenesis and potential pharmacological management.

Thrombosis and Cardiovascular Events

Thrombosis at the sites of atherosclerotic lesions is a common cause of acute coronary syndrome (ACS). ACS is the leading cause of morbidity and mortality in North America [8]. Thrombus has been frequently detected in coronary arteries of patients with acute myocardial infarction (MI), unstable angina, or sudden death [9]. Vulnerable plaques have been considered as triggers for thrombogenesis and ACS [10]. Exposure of blood components to rough surfaces of plaques may trigger platelet aggregation, coagulation, and the formation of fibrin clots. Under normal condition, fibrin clots are quickly cleared through fibrinolysis. When the activity of fibrinolysis is reduced, fibrin clots may develop to thrombus [11]. Intravascular thrombosis may cause ischemia in tissues or organs where blood supply relies on the involved artery.

LDL and Cardiovascular Diseases

LDL is the major carrier of cholesterol in blood circulation. Apolipoprotein B-100 is the sole apolipoprotein in LDL. The lipid core of LDL is composed of cholesterol, cholesteryl esters, triglycerides, and phospholipids. LDL may be subclassed to small dense and large LDL based on their sizes, density, and lipid components. Small dense LDL contains relatively abundant amounts of triglycerides compared to large LDL. The levels of small dense LDL are closely associated with the incidences of IHD and diabetes [12]. LDL transports cholesterol to peripheral tissues for the synthesis of cellular

coexist with other risk factors (diabetes, hypercholesterolemia, hyperglycemia, hypertriglyceridemia, and low HDL-cholesterol), it is often difficult to separate the effect of oxLDL from the other risk factors. High incidences of thrombosis are characteristic in patients with antiphospholipid syndrome (APS) or systemic lupus erythematosus (SLE). Elevated levels of autoantibody against oxLDL were correlated with venous thrombosis in SLE patients in a cohort study [33]. Significantly increased levels of oxLDL and its antibody were detected in APS patients compared to healthy subjects, or in APS patients with thrombosis compared to those without [34]. Circulating oxLDL may form complexes with β2-glycoprotein I. High levels of oxLDL- β2-glycoprotein I complex were associated with arterial thrombosis in APS patients [35]. These findings suggest that oxLDL may cause thrombosis in patients with immunological disorders. The effects of oxLDL on thrombosisrelated activities are discussed in the following sections.

Effects of oxLDL on Coagulation

The activation of tissue factor (TF) is crucial for triggering the extrinsic coagulation cascade, which leads to the formation of thrombin and fibrin clot. TF may activate factor X and IX. This process is inhibited by tissue factor pathway inhibitor (TFPI). Thrombomodulin is a binding site on EC surface for thrombin. Both oxLDL and minimally modified LDL stimulate the expression of thrombomodulin in EC [36, 37], which may partially compensate the increased activity of coagulation. OxLDL increases the activity of protein C in vascular EC [38]. Protein C is activated by the complex of thrombin and thrombomodulin to generate activated protein C (APC) [39]. The formation of APC is increased when protein C is bound to endothelial cell protein C receptor (EPCR). APC becomes anticoagulant after it dissociates from EPCR and binds to protein S. Protein S–APC complex inactivates the activated forms of factor V and VIII, which inhibits coagulation (Fig. 8.1). OxLDL enhances the expression of TF in monocytes induced by endotoxin

[40].Both LDL and oxLDL induce the expression of TF in vascular SMC

[41].OxLDL increases the binding sites for factor VIII, and the activation of factor VIII and IX on the surface of macrophages and SMC [42]. OxLDL does not affect the expression of TFPI but promotes the degradation of TFPI in EC [43]. These findings suggest that oxLDL activates both extrinsic and intrinsic coagulation pathways.

Role of oxLDL in Platelet Activation

Treatment with oxLDL increases the aggregation and adhesion of platelets [44], and reduces the membrane fluidity of platelets [45]. OxLDL enhances the synthesis of thromboxane A2, one of the strongest biological agonists for platelet aggregation, in platelets [46]. OxLDL increases the production of prostacyclin, a strong inhibitor for platelet aggregation after a short period of incubation with EC, but inhibits the production of prostacyclin following

FIGURE 8.1. Simplified scheme for coagulation and protein C pathway. TF, tissue factor; TFPI, tissue factor pathway inhibitor; VIIa, activated factor VII; X, factor X; Xa, activated X; IX, factor IX; IXa, activated IX; PT, prothrombin; Va, activated factor V; T, thrombin; TM, thrombomodulin; PC, protein C; EPCR, endothelial protein C receptor; V, factor V; VIII, factor VIII; Va, activated V; VIIIa, activated VIII; APC, activated protein C; S, protein S; Vi, inactivated factor V; VIIIi, inactivated factor VIII. (+), stimulation; (−), inhibition.

longer-term incubations [47, 48]. Glycated LDL promotes platelet aggregation that is associated with increased intracellular calcium concentration and nitric oxide (NO) production in platelets [49]. Hypochlorite-modified LDL induces greater platelet aggregation than copper ion-oxLDL [50]. The phosphorylation of p38 kinase mediates the activation of platelet by oxLDL or hypochloride-modified LDL [51]. CD36 mediates oxLDL-induced platelet activation [52]. Thus, oxidatively modified LDL may activate platelets through multiple mechanisms.

Impact of oxLDL on Fibrinolysis

Imbalance between coagulation and fibrinolysis leads to intravascular thrombosis. The biologically active product of fibrinolytic system is plasmin, which functions in dissolving fibrin clot and maintains the fluency of blood flow. The precursor of plasmin is plasminogen, which is synthesized in liver and is abundant in most biological fluids. The generation of plasmin is mainly regulated by tissue and urokinase plasminogen activators (tPA and uPA) (Fig. 8.2). The activity of tPA and uPA is regulated by their major physiological inhibitor, plasminogen activator inhibitor-1 (PAI-1) [53, 54]. Tremoli et al. [55] reported that LDL and oxLDL increased the release of PAI-1 from EC via a LDL receptor-independent pathway. The activity and mRNA levels of PAI-1 in EC are also elevated by oxLDL treatment [56]. The release of tPA from EC is attenuated by incubation with oxLDL [57]. LDL increases the generation of PAI-1 and decreases the release of tPA from EC. Augmentation of the formation of conjugated diene is detected in previously unmodified

Plasminogen Plasmin

(+)

(−)

tPA PAI-1

Blood

oxLDL, LDL

FIGURE 8.2. Simplified scheme for fibrinolytic system and effect of oxLDL and LDL. tPA, tissue plasminogen activator; PAI-1, plasminogen activator inhibitor-1; EC, endothelial cells; LDL, low-density lipoprotein; oxLDL, oxidized LDL. (+), stimulation; (−), inhibition.

LDL following prolonged exposure to EC [58]. The activation of protein kinase C-β is required for oxLDL-induced overproduction of PAI-1 in EC [59]. Glycation enhances the effects of LDL on PAI-1 production and further reduces the release of tPA from EC [58]. LDL isolated from type 2 diabetic patients with suboptimal glucose control stimulates the transcription and generation of PAI-1 from EC [60]. Beside LDL and its modified forms, very low-density lipoprotein (VLDL) and lipoprotein(a) also stimulate the generation of PAI-1 from EC [56, 61, 62]. A VLDL-responsive element has been detected in −672/−657 bp of the PAI-1 promoter [63]. OxLDL and LDL activate a responsive element independent from the VLDL-responsive element in the PAI-1 promoter [64]. The findings from above studies suggest that oxidatively modified LDL may reduce fibrinolytic activity in blood circulation by enhancing the production of PAI-1 and decreasing the generation of tPA in vascular EC.

Endothelial Dysfunction and Thrombosis

Intact endothelium provides an anticoagulant and antiplatelet deposition surface, which is critical for preventing thrombosis in vascular lumen. Endothelial dysfunction may be assessed from abnormal secretion of endothelial products, vascular tone, or the loss of EC [65]. EC synthesize a large numbers of products, including protein C, protein S, tPA, PAI-1,

inflammation, oxidative, shear, and restrain stresses. Heat shock factor is activated during many stresses, which mediates the transcription of heat shock protein (HSP) genes [86]. OxLDL induces the expression of HSP-70 in EC [87]. Hyperthemia increases the expression of PAI-1 in EC [88]. Restrain stress induced the expression of PAI-1 in mice in a tissueand cell-type- specific manner. Increased PAI-1 mRNA and thrombosis was more evident in aged mice than in young mice [89]. The findings suggest that stress may promote thrombosis partially resulting from its inhibition on fibrinolysis. The relationship between oxLDL, stress and the expression of thrombosisrelated factors remains to be exploited.

Thrombosis, Endothelial Functions, and Diabetes

Increased thrombotic vascular diseases were detected in patients with diabetes [90]. Elevated levels of vWF, factor VII, TF, fibrinogen or PAI-1, and decreased levels of antithrombin III, protein C, or tPA were associated with diabetes [91, 92]. Increased platelet activation was found in diabetic patients [93]. Endothelial injury and oxidative stress are potential underlying mechanisms for the increased thrombosis in diabetes. Hyperglycemia, advanced glycation end products, and dyslipidemia may impair endothelial function and increase oxidative stress. LDL from type 2 diabetic patients or glycated LDL increase the production of PAI-1 in EC and decrease the release of tPA from EC compared to unmodified LDL from healthy donors [58, 60]. Endothelial dysfunction is an early finding of diabetic vascular abnormalities. Reduction in flow-mediated dilation has been detected in children with diabetes [94]. Hyperglycemia increases susceptibility of LDL to oxidation. Substantial increases in the generation of hydrogen peroxide are detected in EC treated with glycated LDL or oxLDL for 1–2 h [95]. Insulin resistance is one of the underlying mechanisms for the pathogenesis of type 2 diabetes. Insulin resistance is associated with an upregulation of CD36, a receptor for oxLDL, in macrophages of experimental obesity [96]. Increased uptake of oxLDL in vascular wall may elicit the production of oxidative stress, impairs endothelium, and promote thrombosis-related processes in diabetic patients.

Effects of Statins on Thrombosis

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase is a ratelimiting enzyme for cholesterol synthesis, which regulates the generation of mevalonate from acetyl CoA. Mevalonate is a precursor of a group of active intracellular mediators (farnesyl and geranyl pyrophosphates) in addition to cholesterol. HMG-CoA reductase inhibitors (statins) were designed for lowering cholesterol synthesis [97]. Results from a recent meta-analysis demonstrated that statin treatment reduced coronary event by 27%, stroke by 18%, and all-causes of mortality by 15% in hypercholesterolemic patients in comparison to placebo controls [98]. The beneficial effects of statins have been