26 Cell Death in the Cardiovascular System

1. INTRODUCTION

Cardiovascular disease is the most common cause of death in the world. Regulated forms of cell death play critical roles in cardiovascular disease. In particular, apoptosis and necrosis, and perhaps autophagic cell death, are causal components in the pathogenesis of the most common and lethal cardiovascular syndromes: myocardial infarction and heart failure. This chapter summarizes the mechanisms and physiologic impact of regulated cell death in the cardiovascular system.

2. CELL DEATH IN THE VASCULATURE

2.1. Apoptosis in the developing blood vessels

Vascular development requires not only the formation, but also the regression, of blood vessels. For example, components of the first, second, and fifth aortic arches involute during embryonic development. Similarly, the ductus arteriosus, which shunts blood past the lungs in fetal life, becomes a fibrotic vestigial structure after the initiation of postnatal pulmonary function. Changes of this sort are accompanied by apoptosis of endothelial and smooth muscle cells,1,2,3,4 suggesting that cell death is involved in vascular regression and remodeling.

A causal connection between cell death and vascular remodeling has been demonstrated by genetic manipulations in the mouse. For example, loss of survival pathways can cause marked reductions in blood vessel abundance. This is illustrated by endothelial cellspecific deletion of IKKβ (IκB [inhibitor of κB] kinase β), which results in caspase activation, marked reductions in liver blood vessels, and lethality at embryonic days 13.5 through 15.5.5 Similarly, knockout of Bcl-2 (B- cell leukemia/lymphoma-2) results in apoptosis, reduc-

tion in the abundance of endothelial cells and pericytes, and decreased retinal artery density in postnatal mice.6 Conversely, loss of apoptosis signaling can lead to extra vessels. This is illustrated by combined knockouts of Bax (Bcl-2–associated X protein) and Bak (Bcl-2 homologous antagonist/killer), which display loss of normally occurring endothelial cell apoptosis with persistence of fetal retinal vessels.7

A variety of physiologic stimuli, including shear stress, interactions with extracellular matrix, and soluble factors, such as vascular endothelial growth factor, promote endothelial cell survival.8 Conversely, endothelial cells in regressing capillary beds can be killed by Wnts secreted from macrophages.9 Moreover, reductions in capillary flow resulting from the killed endothelial cells can then reduce shear stress and delivery of nutrients, thereby leading to further endothelial cell death.10,11

In contrast, the role of apoptosis in the remodeling of larger vessels is not known. For example, reduction of carotid blood flow in adult rabbits and mice stimulates endothelial cell and/or smooth muscle cell apoptosis. The vascular lumen becomes smaller, but this may be due to reactive smooth muscle cell proliferation, matrix deposition, and overall vessel shrinkage. Moreover, apoptosis of vascular cells in arteries may cause only variable and, in some cases, transient vascular changes.12,13 For these reasons, the significance of flow-mediated cell death in the remodeling of large vessels remains unclear.

2.2. Apoptosis in atherosclerosis

The advanced human atherosclerotic plaque is formed through a complex series of events that involve all arterial cell types (Figure 26-1), as follows: (1) Endothelial cell dysfunction/damage is an initiating event. (2) This

Tunica adventitia connective tissue

Tunica intima

endothelium

internal elastic lamina

external elastic lamina

smooth muscle cells

Tunica media

Figure 26-1. Normal human artery consists of three layers. The tunica intima (intima), the layer closest to the lumen of the vessel through which blood flows, is composed of a single layer of endothelial cells resting on a basement membrane. The tunica media (media) is comprised of multiple layers of vascular smooth muscle cells. The tunica adventitia (adventitia), the outermost layer, is composed of fibroblasts and collagen-rich matrix containing nerves, lymphatics, and small blood vessels. Internal and external elastic laminae separate intima from media and media from adventitia, respectively. Reprinted with permission from School of Anatomy and Human Biology – The University of Western Australia. See Color Plate 28.

Levels of apoptosis are low to undetectable in the normal vessel wall18 but increase progressively during plaque development.18,19,20,21 This cell death occurs in both the necrotic core and the fibrous cap. Most apoptosis in plaques involves vascular smooth muscle cells and macrophages. We focus first on smooth muscle cell death.

2.2.1. Vascular smooth muscle cells

Transgenic mice that express diphtheria toxin receptor exclusively in arterial smooth muscle cells have been used to investigate a causal connection between smooth muscle cell apoptosis and atherogenesis (Figure 26-3a). These studies show that modestly elevated levels of vascular smooth muscle cell apoptosis (0.8%– 1.1%) – comparable to those seen in human plaques – are sufficient to accelerate plaque progression in an atherogenic milieu (apolipoprotein E−/– [apoe−/–] mice on a high-fat diet).22 The underlying mechanisms are incompletely understood but involve proinflammatory effects of apoptotic cells.23

Studies have also linked vascular smooth muscle cell apoptosis with plaque rupture (Figure 26-3b). Increased levels of vascular smooth muscle cell apoptosis are associated with rupture-prone coronary artery plaques24,25 in patients with unstable angina as compared with those with stable angina.26 The most direct evidence, however,

leads to recruitment of monocytes/macrophages into the intima. (3) Uptake of lipids into the macrophages results in their transformation to foam cells. (4) Foam and endothelial cells signal the migration of vascular smooth muscle cells from media to intima, where their replication and collagen/matrix production form a fibrous cap. This fibrous cap separates the thrombogenic, lipid-rich “necrotic core” of the plaque from the flowing blood.14,15 Myocardial infarction (“heart attack,” discussed later in this chapter), is the acute death of heart muscle cells resulting from the sudden cessation of blood flow in a coronary artery. Rather than being precipitated by progressive arterial narrowing, most myocardial infarctions are triggered by acute rupture of the fibrous cap of the plaque (Figure 26-2).16,17 Contact between thrombogenic factors in the plaque and the flowing blood then activates platelets, leading to subsequent thrombosis and coronary artery occlusion.

Atherosclerosis

Plaque Instability

Myocardial Infarction

Heart Failure

Figure 26-2. Relationship between atherosclerosis, myocardial infarction, and heart failure. Rupture of an atherosclerotic plaque acutely precipitates myocardial infarction. Myocardial infarction can lead to heart failure. See text for details.

Figure 26-3. Vascular smooth muscle cell apoptosis accelerates atherosclerotic plaque progression22 and induces plaque vulnerability.27 Transgenic mice were created in which expression of the human diphtheria toxin receptor was targeted to arterial smooth muscle cells. Animals were crossed onto an apoe−/– background and fed a highfat diet to induce atherosclerosis. Apoptosis of arterial smooth muscle cells was induced by administration of diphtheria toxin. This model was used to study the e ects of arterial smooth muscle cell apoptosis on plaque progression and instability. Apoptosis accelerated plaque formation in the brachiocephalic artery as shown by increased plaque area by hematoxylin and eosin staining (a). Plaque vulnerability was increased by apoptosis (not shown) in the carotid artery, as illustrated by thinning of the fibrous cap, loss of collagen and matrix, increased necrotic core size, cellular debris, and inflammation (Masson trichrome staining in b and not shown). Space bars 100 μM (a) and 50 μM (b). (a) Reproduced with permission from Clarke MC, Littlewood TD, Figg N, Maguire JJ, Davenport AP, Goddard M, Bennett MR. Chronic apoptosis of vascular smooth muscle cells accelerates atherosclerosis and promotes calcification and medial degeneration. Circ Res. 2008;102:1529– 38. (b) Reprinted by permission from Macmillan Publishers Ltd: Clark MC, Figg N, Maguire JJ, Davenport AP, Goddard M, Littlewood TD, Bennett MR. Apoptosis of vascular smooth muscle cells induces features of plaque vulnerability in atherosclerosis. Nat Med. 2006;12:1075–80. See Color Plate 29.

is provided by the diphtheria toxin receptor transgenic mouse, described in the previous paragraph. Induction of vascular smooth muscle cell apoptosis results in thinning of the fibrous cap, loss of collagen and matrix, accumulation of lipids and cellular debris within an increased necrotic core, and formation of inflammatory foci within the atherosclerotic lesion.27 Although thinning of the fibrous cap does not progress to overt rupture in this model, these data suggest that vascular smooth muscle cell apoptosis can precipitate features of plaque instability in an atherosclerotic context. Although these studies demonstrate the sufficiency of vascular smooth muscle cell apoptosis for plaque instability, the necessity of cell death in this process remains to be evaluated.

2.2.2. Macrophages

Macrophages are the most frequent apoptotic cell type in advanced lesions.18 Apoptosis of these cells may have varying effects on atherosclerosis at different times in the disease process. During atherogenesis, macrophage apoptosis appears to reduce lesion formation. Consistent with this, diphtheria toxin-mediated killing of macrophages in apoe−/– mice fed a high-fat diet results in reduced plaque and necrotic core size.28 Conversely, adoptive transfer of bax−/– bone marrow into mice lacking the low-density-lipoprotein receptor and on a highfat diet showed increased plaque area compared with mice reconstituted with wild-type cells.29 These studies suggest that macrophage apoptosis limits plaque development. In contrast, macrophage apoptosis in established plaques may increase the necrotic core size without affecting plaque size.30

A related theory is that changes in clearance of apoptotic bodies promote progression of established plaque. Inhibition of phagocytosis, through loss-of- function mutations in Mertk (MER tyrosine kinase) or lactadherin, accelerates atherosclerosis in established plaques and is accompanied by increases in necrotic core size.31,32,33 In fact, the efficiency of phagocytosis appears decreased in the milieu of atherosclerotic plaques.34 The precise relationship between apoptosis and phagocytosis with regard to atherogenesis remains to be delineated.

Endoplasmic reticulum (ER) stress has also been implicated in the role of macrophages in atherosclerosis. ER stress may be stimulated by lipid-mediated oxidative damage and/or increased accumulation of free cholesterol within macrophages.35, 36 Markers of the unfolded protein response (UPR) are activated during all phases of plaque development.37 Deletion of CHOP [C/EBP (CCAAT/enhancer binding protein)-homologous protein], which transcriptionally activates genes that mediate both UPR and ER stress-induced apoptosis, lowers rates of macrophage apoptosis and reduces necrotic core size in atherosclerosis-prone mice.38 Furthermore, CHOP and GRP78 (glucose-regulated protein 78, another UPR marker), are increased in ruptured, but not stable, human plaques. These observations suggest a role for ER stress in necrotic core formation and human plaque rupture.39

2.2.3. Regulation of apoptosis in atherosclerosis

Resident vessel wall cells are resistant to apoptosis induced by death ligands, in part because of increased levels of FLIP [FLICE (FADD-Like IL-1β-converting