Oxidation-sensitive mechanisms, vascular apoptosis and atherosclerosis

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Oxidation-sensitive mechanisms, vascular apoptosis and atherosclerosis Filomena de Nigris1, Amir Lerman2, Louis J. Ignarro3, Sharon Williams-Ignarro4, Vincenzo Sica5, Andrew H. Baker6, Lilach O. Lerman7, Yong J. Geng8 and Claudio Napoli5,9 1

Department of Pharmacological Sciences, University of Salerno, 84084 Salerno, Italy Division of Cardiovascular Diseases, Mayo Clinic and Foundation, Rochester, MN 55905, USA 3 Department of Molecular and Medical Pharmacology, Center for the Health Sciences, University of California Los Angeles, CA 90095, USA 4 Department of Anesthesiology, University of California Los Angeles, CA 90095, USA 5 Departments of Medicine and Clinical Pathology, University of Naples, 80131 Naples, Italy 6 Division of Cardiovascular and Medical Sciences, University of Glasgow, Glasgow, UK G116NT 7 Division of Hypertension, Mayo Clinic and Foundation, Rochester, MN 55905, USA 8 Department of Internal Medicine, Center for Cardiovascular Biology and Atherosclerosis, University of Texas Houston Medical School, Houston, TX 77030, USA 9 Department of Medicine, University of California San Diego, CA 92093, USA 2

Increased generation of oxidants, resulting from disruption of aerobic metabolism and from respiratory burst, is an essential defense mechanism against pathogens and aberrant cells. However, oxidative stress can also trigger and enhance deregulated apoptosis or programmed cell death, characteristic of atherosclerotic lesions. Oxidation-sensitive mechanisms also modulate cellular signaling pathways that regulate vascular expression of cytokines and growth factors, and influence atherogenesis, in particular when increased levels of plasma lipoproteins provide ample substrate for lipid peroxidation and lead to increased formation of adducts with lipoprotein amino acids. In some cases, increased oxidation and apoptosis in a group of cells might be beneficial for survival and function of other groups of arterial cells. However, overall, oxidation and apoptosis appear to promote the progression of diseased arteries towards a lesion that is vulnerable to rupture, and to give rise to myocardial infarction and ischemic stroke. Recent rapid advances in our understanding of the interactions between oxidative stress, apoptosis and arterial gene regulation suggest that selective interventions targeting these biological functions have great therapeutic potential. Oxidation and nitration of macromolecules, such as proteins, DNA and lipids, are prominent in atherosclerotic arteries. In many cases, nitration occurs at the aromatic amino acids incorporated in proteins, and this phenomenon can be modulated by oxidation-sensitive mechanisms. Extensive evidence has accumulated demonstrating that rather than being epiphenomenon, chemical modification of biologically active macromolecules takes place in vivo and is an integral part of the pathogenic or Corresponding author: Claudio Napoli ([email protected]).

defensive mechanisms that dictate the progression of atherosclerosis. The generation of oxidants results from an active process crucial for innate, nonspecific immune defense, and not simply from an accidental disruption of aerobic metabolism. For example, superoxide released by macrophages during ‘respiratory burst’ contributes to the killing of bacterial pathogens. Although essential for survival, inappropriate or excessive activation of these processes can cause tissue degeneration. Arterial cells are highly susceptible to oxidative stress, which can induce both necrosis and apoptosis (programmed cell death) [1,2]. Among the mechanisms that influence the progression of atherosclerosis, apoptosis is one of the most intriguing. By counterbalancing cell proliferation in the arterial intima, apoptosis might reduce atherogenesis, whereas decreased apoptosis could increase the tissue cellularity and the mass of vascular smooth muscle cells (VSMCs). However, excessive apoptosis of VSMCs might weaken an atherosclerotic arterial wall. By synthesizing various extracellular matrix proteins, VSMCs are known to play a central role in maintaining the integrity of arterial structures. If no new cells are generated, arterial plaques with extensive VSMC death can become unstable and ultimately rupture. By contrast, the death of lipid-laden macrophages and foam cells might lead to a more favorable outcome. By reducing the number of these cells, apoptosis might limit local accumulation of the most abundant inflammatory lipid-rich cells, thereby stabilizing atherosclerotic plaques. Hence, caution must be exercised when designing nonspecific anti-apoptotic strategies to prevent plaque rupture, because these could lead to greater resistance of foam cells. Plaque rupture is the major complication of atherosclerotic lesions, and can cause acute coronary syndromes (ACS) and thrombosis. Selective induction of apoptosis in lipid-laden foam cells might be preferable,

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because these cells are key elements in the atherogenic process and secrete many pro-inflammatory and proatherogenic cytokines. Part of the difficulty in establishing whether apoptosis contributes to or inhibits atherosclerosis stems from the fact that advanced atherosclerotic lesions always progress to more complex, less cell-dense and more matrix-rich stages. These compositional changes determine the mechanical stability of the plaque. Arteries were long regarded merely as blood conduits that become encrusted with lipid detritus as part of the aging process. Thus, mechanical interventions that restore the lumen, such as bypass surgery or balloon catheterization with or without insertion of a stent, seemed the most logical therapeutic approach. These interventions rapidly improve downstream blood supply and alleviate symptoms caused by the stenosis (such as angina), with the relationship between the intervention and mortality depending partly on the branch and location of the stenosis. However, this form of intervention does not affect mortality resulting from ACS. It is now recognized that ACS is caused by rupture of less-advanced plaques that do not necessarily cause much stenosis. These plaques, commonly referred to as ‘vulnerable’, usually contain a lipid-rich necrotic core underneath a weak fibrous cap. Rupture of the cap, as a result of enzymatic degradation and physical stress, produces thrombosis, which in turn triggers ACS, including acute myocardial infarction (AMI). Angiographic studies have confirmed that the ‘culprit’ lesions of many infarctions do not cause hemodynamically relevant stenoses of the coronary arteries [3,4]. Only a few decades ago, some pathologists regarded intracoronary thrombi as a post hoc phenomenon rather than the cause of AMI [5,6]. In addition to the shift of attention away from the lumen (i.e. the degree of stenosis) towards the thickness and composition of the arterial wall, modern attitudes towards atherosclerosis are increasingly influenced by the recognition that pathogenesis of the disease starts much earlier than previously assumed, and that therapeutic interventions beginning in adulthood might come at a time when many lesions are already well established [7 – 10]. In fact, several large studies have established that lesions are prevalent in children [11 – 13], and fatty streaks (the earliest atherosclerotic lesions) are already present in human fetal arteries, in particular when the mother is hypercholesterolemic during pregnancy [14]. In this review, we will discuss the complex interactions between oxidation-sensitive mechanisms [15] in the context of vascular apoptosis and in the development of atherosclerosis. Extensive evidence also indicates that humoral and cellular immune responses against lipid peroxidation products occur in vivo and modulate the progression of atherosclerosis (reviewed in [16,17]). Evaluation of the role in atherosclerosis of immune mechanisms, and their interaction with vascular apoptosis, scavenger receptors and oxidized low-density lipoprotein (oxLDL), is further complicated by the fact that other chronic inflammatory conditions induce similar humoral immune responses to oxidative neo-epitopes [18]. LDL is a particle made up of a large array of molecules, with a core of nonpolar cholesteryl ester and triglycerides, surrounded http://tmm.trends.com

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by an emulsifying agent, a monolayer of phospholipids and unesterified cholesterol, as well as a single molecule of apolipoprotein B100. All biological events induced by LDL and oxLDL are closely related to the structure of these compounds and their modifications. Such pathophysiological issues have been addressed elsewhere [2,15,16,19,20]. Pathogenic events in vascular apoptosis The first evidence that endothelial cell apoptosis might contribute to the initiation of atherogenesis came from the observation that all classical risk factors known to promote endothelial dysfunction and atherogenesis can induce vascular cell apoptosis. One such risk factor is oxLDL, which exert pro-apoptotic effects on a variety of cell lineages, including endothelial cells and VSMCs, both in cultured cells and in arteries. [19– 26]. Increased formation of oxygen radicals (and other radical species) facilitates LDL oxidation and influences oxidation-sensitive mechanisms [15]. It is likely that the apoptotic effects of oxLDL are caused by interference of such radicals with oxidation-sensitive signaling pathways. Indeed, various antioxidant enzymes and free-radical scavengers inhibit caspase activation and other apoptotic effects of oxLDL [27,28]. Not surprisingly, high concentrations of exogenous reactive oxygen species (ROS), generated by enzymatic reactions or by radiation, also stimulate the apoptosis of endothelial cells in vitro [29– 31]. Consistent with these findings, vascular apoptosis and atherogenesis decline in a mouse model of reduced oxidative stress [32]. These processes can occur in the atherosclerotic arterial wall even in the presence of only moderate degrees of LDL oxidation [27]. The peptide hormone angiotensin II and pro-inflammatory cytokines also trigger the apoptotic program in endothelial cells [33,34]. Aging, one of the major predictors for atherosclerotic lesion formation, increases the sensitivity of endothelial cells to apoptosis induced by in vitro and in vivo stimuli [35– 37]. A plethora of signal transduction and nuclear events are involved in vascular apoptosis, including tumornecrosis-factor-receptor- and Fas-receptor-mediated activation, the regulatory interactions between anti- and proapoptotic members of the Bcl-2 family, the activation of class I and II death-effector caspases, mitochondrial dysfunction and lipid-intermediate release from the sphingomyelinase system. The regulatory action of nuclear factor kB (NF-kB) and other transcription factors (and their co-regulators) might also contribute to oxLDLinduced apoptosis. These death signaling pathways have been reviewed in detail elsewhere [19,21,38,39]. Exposure of arterial cells to physiological shear stress might attenuate the apoptosis of endothelial cells induced by a variety of stimuli, including oxidized lipids, ROS, inflammatory cytokines and growth-factor depletion [40]. Most importantly, the occurrence of apoptotic endothelial cells overlying human atherosclerotic plaques spatially relates to hemodynamic forces, with high incidence of apoptosis in areas with low or turbulent flow [41,42]. Thus, the progressive thickening of arterial intima, affecting blood flow and hemodynamics, might initiate anti-apoptotic mechanisms. Indeed, mechanical stimulation of

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endothelial cells by shear stress activates a variety of intracellular signaling pathways, thereby dramatically altering gene expression profiles [43]. Shear stress enhances expression of the gene encoding the endothelial nitric oxide synthase (eNOS) and further stimulates its enzymatic activity, leading to physiologic low concentrations of nitric oxide (NO) within endothelial cells [44 – 46]. This continuous generation of NO prevents the apoptosis of endothelial cells, thereby protecting the endothelial monolayer from injury [47,48]. The important anti-atherosclerotic function of NO is evidenced by clinical and experimental studies [49], although in some murine models, manipulation of NO (or eNOS) production did not significantly affect atherosclerosis [47– 49]. More importantly, intervention with L -arginine has induced beneficial effects on atherosclerosis in experimental and human studies [46,49]. Despite the wealth of results delineating the mechanisms of apoptosis and its consequences in vitro, in vivo assessment of the consequences of vascular apoptosis for the progression of the disease remains difficult. Both oxLDL and apoptotic and necrotic cells are present in atherosclerotic lesions, but it is difficult to establish a direct causal pathogenic link between oxLDL and apoptosis in vivo, because interventions with antioxidants affect atherosclerosis not only via apoptosis but also by (a)

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reducing many other atherogenic effects of oxLDL. Similarly, it is difficult to enhance or reduce vascular apoptosis without also influencing other mechanisms, such as cell proliferation. Furthermore, oxLDL can induce peroxide-mediated macrophage death via a caspase-3independent pathway, which results in cellular necrosis rather than apoptosis, and thereby contributes to the development of advanced atherosclerotic lesions [50]. Nevertheless, there is some in vivo evidence for a proatherogenic effect of apoptosis. A study in monkeys revealed that endothelial cell apoptosis correlates with impairment of endothelial vasodilator function [35]. Apoptotic vascular cells are also found in hypercholesterolemic pigs and mice (Fig. 1). In addition, there is evidence that microparticles (the residual bodies of apoptotic cells) can directly induce endothelial dysfunction [51]. These data suggest that circulating endothelial microparticles, which are elevated in patients with ACS [52], can activate endothelial cells and thereby induce endothelial dysfunction. Another mechanism that potentially promotes endothelial apoptosis relates to the dependence of endothelial function on interactions among healthy neighboring endothelial cells. It is well established that regenerated endothelial cells are functionally impaired [53], and hence one might speculate that, as a consequence of initial

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Fig. 1. Apoptosis of arterial cells is a prominent feature of atherosclerotic lesions. Atherosclerotic lesions are rich in oxidized low-density lipoprotein (oxLDL), which promotes apoptosis in vitro. Techniques such as terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick-end labeling (TUNEL) indicate a higher percentage of apoptosis-positive arterial cells than is reconcilable with the slow evolution of lesions. However, the number of cells actually dying as a result of apoptosis and the role of apoptosis in promoting or inhibiting atherogenesis remains to be determined. (a) TUNEL performed in a coronary artery obtained from a hypercholesterolemic pig. Staining is positive in all layers of the vessel. In the adventitia, cells with positive staining (black arrows) are particularly present in the region corresponding to the external elastic membrane. Several endothelial cells are also positively stained. (b –d) Transmission electron micrographs of sections of coronary arteries from normal (b) and highcholesterol-treated (c,d) pigs. A smooth muscle cell from a normal animal displays normal ultrastructure, whereas cells from hypercholesterolemic animals show varying degrees of nuclear ultrastructural changes associated with apoptosis. One cell shows lobulation of the nucleus (c), whereas another cell displays a more advanced apoptotic state with marked chromatin condensation and peripheral margination (d). (e) An apoptotic residual body. (f) Microphotograph of TUNEL-positive coronary cells (indicated by arrows) obtained from a hypercholesterolemic mouse. Magnification is 50 £ in (a), 12 000 £ in (b,c), 9000 £ in (d), 8000 £ in (e) and 840 £ in (f). Panels (a– e) are modified, with permission, from Ref. [96]. http://tmm.trends.com

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Table 1. Stimulatory and inhibitory factors for apoptosis in the development of atherosclerosisa,b Stimulatory factors

Inhibitory factors

Pro-inflammatory cytokines (e.g. IFN-g, TNF-a, IL-1 and FasL)

Growth factors and anti-inflammatory cytokines (e.g. TGF-b, PDGF, IGF and HGF) Antioxidants and radical scavengers (e.g. vitamins C and E, SOD, catalase and Hb) Scavenging proteins (e.g. scavenger receptors and HDL) Caspase inhibitors Mitochondria protectors DNA repair enzymes

Reactive oxygen and nitrogen species (e.g. O22, H2O2, ONOO2 and NO2) Chemically modified lipids (e.g. oxysterols, lysoPC and ceramides) Caspase activators Mitochondria membrane destabilizers DNA damaging agents

a Abbreviations: FasL, Fas ligand; Hb, hemoglobin; HDL, high-density lipoprotein; HGF, hepatocyte growth factor; IFN-g, interferon-g; IGF, insulin-like growth factor; IL-1, interleukin-1; lysoPC, lysophosphatidylcholine; PDGF, platelet-derived growth factor; SOD, superoxide dismutase; TGF-b, transforming growth factor b; TNF-a, tumor necrosis factor a b The complex interplay between these environmental and endogenous apoptosis-regulating factors is crucial to the balance between cell death and proliferation in atherosclerosis. A variety of environmental factors and drugs might trigger apoptosis, through a range of signaling pathways. These include the caspase cascade, the Bcl-2 protein family and the oncogene–anti-oncogene system. Weakening of anti-apoptotic mechanisms might also contribute to deregulated apoptosis found in advanced atherosclerosis. Clarification of the molecular mechanisms responsible for vascular apoptosis might aid in the development of novel therapeutic strategies to treat atherosclerosis-related diseases and their complications.

endothelial cell apoptosis, the endothelial monolayer is partially replaced by dysfunctional cells that cannot provide the appropriate atheroprotective activity, such as NO synthesis. Alternatively, endothelial progenitor cells derived from the bone marrow might contribute to endothelial regeneration, at least after balloon injury [54]. Interestingly, patients with risk factors for coronary heart disease show lower numbers and functional impairment of endothelial progenitor cells [55]. Therefore, regeneration of injured endothelial cells by functionally impaired endothelial progenitor cells might also lead to a dysfunctional endothelium, further accelerating vascular inflammation and atherogenesis. In addition, the endothelial progenitor cells themselves might be prone to apoptosis [56], which could reduce the endogenous regenerative capacity. Redox mechanisms, lipid peroxidation products and apoptosis are intricately linked ROS are involved in various biological processes, such as cell activation, proliferation, survival and apoptosis, mediated by several signaling pathways, including mitogen-activated protein kinases (extracellular-signal-regulated kinase 1/2, stress-activated protein kinase and JUN N-terminal kinase), NF-kB, Akt, caspases and calcium [57]. A role for ROS in oxLDL-mediated cytotoxicity has been reported recently, mediated largely through the activation of class I and II caspases and apoptosis. OxLDL induces the generation of ROS in different vascular cell types [15,58], and might also promote a rise in ROS levels through a decrease in ROS degradation, because oxLDL induces a reduction in the antioxidant enzymes Cu/Zn superoxide dismutase [59] and glutathione peroxidase [60]. Although many studies implicate oxidized lipids and ROS in apoptosis induction, the identity of other cellular oxidants and the mechanisms of their cytotoxic actions are only partly known. Because oxidative stress can injure vascular cells and promote apoptosis, antioxidants might mitigate this process. Inhibitors of apoptosis relevant to atherosclerosis include oxygen-radical scavenger enzymes (e.g. superoxide dismutase) [61,62] and pharmacological doses of some antioxidant vitamins [28,63]. For instance, during 7-ketocholesterol-induced apoptosis of U937 cells, http://tmm.trends.com

the cellular antioxidant content falls rapidly [64], but administration of two potent antioxidants, the aminothiols glutathione and N-acetylcysteine, can protect the monocytic cells from 7-ketocholesterol-induced apoptotic cell death [64]. In addition, another oxygen-radical scavenger enzyme, catalase, can prevent VSMC apoptosis triggered by hydrogen peroxide [27,65]. Table 1 lists factors that promote or inhibit vascular apoptosis. Different ROS exert different effects on different cell types, making it hard to establish their effects on atherosclerotic progression in vivo. Recent data indicate a potentially important role of oxidation-sensitive regulation of c-myc in experimental atherosclerosis [66,67]. The proto-oncogene c-myc can promote cell death (probably mediated through p53) or cell proliferation, depending on its expression level. It functions as a nuclear phosphoprotein with particular properties of a transcription factor [68,69]. In serumdeprived cultures, cells overexpressing c-myc readily undergo apoptosis. In addition, deregulation of c-myc causes apoptosis of VSMCs deprived of growth factors or treated with cytokines such as interferon-g [70]. Although increased oxidative stress (or lipid peroxidation) modulates c-myc, it remains to be determined whether this is the main regulator of c-myc in the arterial wall and whether it plays a major role in promoting apoptosis in vivo. Moreover, further studies are needed to establish the potential protective role against vascular apoptosis of antioxidants and newly developed vitamin E analogs [71]. Apoptosis and its possible role in thrombogenicity of atherosclerotic plaques Vascular apoptosis could be relevant not only because it influences the progression of atherosclerosis, but also for the ensuing ACS, plaque rupture and formation of (occluding) thrombi or emboli. The contribution of apoptosis to these events might include weakening of the fibrous cap, enlargement of the amorphous necrotic core and, most importantly, enhanced thrombogenicity of atherosclerotic plaque components. Tissue factor (TF) is a 47 kDa transmembrane glycoprotein that initiates blood coagulation by binding coagulation factor VII and its activated form (factor VIIa), to produce a high-affinity complex [72] that proteolytically activates factors IX and X, leading to

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thrombin generation. Cellular and extracellular TF are major determinants of the thrombogenicity of the lipid core in humans: specific inhibition of vascular TF using a recombinant TF-pathway inhibitor is associated with a significant reduction in acute thrombus formation in lipidrich plaques [73]. TF works at the surface of cell membranes, and its activity is highly dependent on the presence of phosphatidylserine (PS), an anionic phospholipid that is redistributed on the cell surface during apoptotic death and confers a potent pro-coagulant activity to the apoptotic cell [74]. During apoptosis, there is significant exposure of PS on the cell surface, leading to the shedding of PS-containing membrane microparticles. It is likely that if the cells undergoing apoptosis are not engulfed immediately, the apoptotic bodies or PS-containing membrane microparticles could cause TF activation within the plaque. Furthermore, TF expression (cellular and extracellular) and apoptotic death can take place in the same lesions, particularly in the lipid core, indicating that TF might be released in apoptotic microparticles during cell death [75]. Most of the microparticles seem to originate from macrophages and lymphocytes, which are known to be abundant at sites of plaque rupture [76]. These data suggest that shed membrane apoptotic microparticles play a major role in initiation of the coagulation cascade after plaque rupture and exposure of the lipid core to the circulating blood. Consistent with this notion, macrophage apoptotic death is significantly increased at sites of plaque rupture and thrombosis in patients with sudden coronary death [77]. Apoptosis is also significantly increased in unstable versus stable human plaques [78]. Overall, vascular inflammation that might induce apoptosis is associated with advanced atheroma (Box 1). Rupture of a thin fibrous cap overlying a lipid core is not the only event that can lead to the formation of coronary thrombi: plaque erosion without rupture is also an important predisposing factor for ACS and sudden cardiac death [79,80]. Apoptosis of luminal endothelial cells might be one of the mechanisms leading to erosion and thrombosis and, indeed, analysis of longitudinal plaque sections revealed the presence of endothelial apoptosis in Box 1. Advanced atherosclerosis and vascular inflammation Atherosclerosis is a diffuse inflammatory process that starts at an early stage of life, in response to exposure to known and novel risk factors. Initiation and progression of the disease is highly dependent on the integrity of the endothelium. The endothelium is at an important strategic location between the vascular wall and the circulation, and can therefore regulate vascular tone and growth. Moreover, although the main focus of clinical and basic research was previously on the main lumen of the vessel, it has become apparent that the adventitia and, more specifically, the vasa vasorum might play a seminal role in the initiation and progression of atherosclerosis. There is accumulating evidence that inflammation and neovascularization occur at the adventitia in the early stage of the disease. Thus, understanding the interactions between oxidationsensitive mechanisms, vascular apoptosis and neovascularization might help us to reduce the progression of atherosclerotic lesions, plaque instability and their clinical manifestations http://tmm.trends.com

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60% of plaques examined. Endothelial cells are exposed to many other forms of injury – infectious, immunological, chemical, radiation, as well as mechanical (e.g. iatrogenic) – that have an impact on their cellular structure and function. In human carotid atherosclerotic plaques, alteration of the blood flow directly influences endothelial survival [42]. An increase in apoptosis is not necessarily balanced by an increase in cell proliferation, suggesting that there could be relatively large areas of endothelial erosion in the distal part of atherosclerotic plaques, as a consequence of endothelial apoptosis. However, not all endothelial damage is pro-apoptotic. Indeed, pro-apoptotic signaling and effectors might be counteracted by cell survival signals, some of which could be activated via a negative-feedback mechanism. Given the high pro-coagulant and pro-adhesive potential of apoptotic cells, and the propensity of denuded vessel segments to increased vasospasm and platelet aggregation, primary endothelial apoptosis and secondary denudation might lead to lumen thrombosis, favoring plaque progression or ACS. However, extensive endothelial denudation is prevalent over advanced lesions, but the only consequence usually seen is platelet adhesion, not thrombosis. Furthermore, in studies with experimental models in which massive apoptosis was induced within the vessel, thrombosis was not observed [81,82]. In these experimental settings, apoptosis was limited to subendothelial cells, and there was no direct contact between apoptotic cells and the circulating blood. Clarification of the role of apoptosis in plaque rupture and thrombosis will have to rely on experimental models of plaque instability and rupture, which are difficult to establish but might become more available in the near future [83]. Similarly, improved understanding of key mechanisms of the basal machinery of the cell increases the possibility of selective pharmacological modulation of apoptosis [84]. Future directions and concluding remarks Apoptotic mechanisms are intricately linked with many pathological alterations in atherosclerosis, including oxidation-sensitive events, immune responses, expression of pro-inflammatory cytokines, plaque growth and mechanical stability, thrombogenic factors, and ultimately plaque rupture and thrombosis (Fig. 2). Many proteins have been shown to be co-regulators (e.g. co-repressors and co-activators) that can be recruited by DNA-binding nuclear receptors to influence transcriptional regulation (reviewed in [85,86]). Levels of co-regulators and coregulatory events are crucial for nuclear-receptormediated transcription and might further modulate the pathogenic framework of vascular apoptosis. This suggests that there might be multiple targets for pharmacological interventions using drugs that influence one or more of these processes, such as statins, fibrates, antioxidants, vitamin E analogs, anti-apoptotic drugs and newly developed tissue-specific anti-apoptotic compounds. However, before we can examine their effects on apoptosis, let alone atherosclerosis and its consequences, several questions need to be resolved. These include the fundamentally unresolved issue of whether, and to what degree, apoptosis is involved in atherosclerosis and its

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Box 2. Modulation of vascular apoptosis and gene transfer Gene therapy represents a unique opportunity to modulate vascular cell apoptosis in vivo, through delivery of either pro- or anti-apoptotic genes, depending on the target pathology (Fig. I ). Gene transfer using adenoviral vectors is particularly suited in this context, because efficient and high-level transgene expression can be achieved locally at sites of disease. Candidate therapeutic genes – the anti-apoptotic genes Bcl-2 and CrmA or the pro-apoptotic genes encoding Fas ligand (FasL) and tissue inhibitor of metalloproteinases 3 (TIMP-3) – provide an adequate means through which to achieve the required phenotypic response in the vasculature. Expression of Bcl-2 or CrmA requires a high proportion of cells to be transduced, because their protein products are intracellular and act by blocking caspase activation (CrmA) or mitochondrial apoptotic events (Bcl-2), with the net effect of inhibiting apoptosis. Potential applications of this technology include promoting cell survival in regions of ischemia in the brain [91] and reducing apoptotic events in advanced atherosclerosis.

Induction of cell death in the vessel wall, particularly to treat postangioplasty re-stenosis [92] or late vein-graft disease [93] by promotion of smooth-muscle cell death, is also an attractive gene therapy strategy. Here, adenoviral-mediated overexpression of pro-apoptotic proteins TIMP-3 or FasL leads to high-level secretion into the extracellular milieu. These transgenes possess potent bystander effects, because neighboring non-infected cells can also be phenotypically modulated through death-receptor-mediated mechanisms. The resulting cleavage of caspase-8 and activation of classical apoptotic pathways leads to caspase-3 cleavage and apoptotic cell death. TIMP-3 can be anti-angiogenic by direct binding to vascular endothelial growth factor (VEGF) receptor 2 in a matrix-metalloproteinase-independent manner [94]. This is intriguing because TIMP-3 is highly expressed in macrophages in plaques [95]. Hence, in this context, gene therapy might be better than pharmacological intervention, because the therapy can be targeted locally and could be therapeutic with a single administration.

Anti-apoptosis (Bcl-2 or CrmA)

Pro-apoptosis (FasL or TIMP-3)

FasL Adenovirus binding and internalization

TIMP-3

Secretion

Pathology-induced death-receptor activation

Endosome disruption

CrmA

FADD-mediated caspase-8 activation TIMP-3

CrmA

Autocrine and paracrine death-receptor activation

Mitochondrial cytochrome c release

FFasL

Bcl-2 Caspase-9 activation Caspase-3-mediated substrate cleavage

No caspase-3 activation

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Fig. I. Modulation of apoptosis by gene transfer. Adenoviral vectors (blue) can be used to deliver anti-apoptotic genes (e.g. Bcl-2 or CrmA) or genes encoding pro-apoptotic proteins [e.g. Fas ligand (FasL) or tissue inhibitor of metalloproteinases (TIMP-3)]. Bcl-2 and CrmA inhibit apoptosis (left) by preventing the activation of caspase3. FasL and TIMP-3 induce apoptosis (right) by activating death-receptor-mediated mechanisms. Abbreviation: FADD, Fas-associated death domain.

progression. Recent insights into the prevention of restenosis through Fas-induced apoptosis [87,88] and the reduction of myocardial infarction using caspase inhibitors [89,90] might be helpful for designing future clinical http://tmm.trends.com

trials targeting apoptosis. Gene therapy approaches are also feasible (Box 2) [91– 95]. Further in vivo studies are needed to establish the possible causal relationship between apoptosis and plaque instability.

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Fig. 2. A proposed pathophysiological scenario, in which cellular and nuclear oxidation-sensitive signaling and transcription pathways are depicted in relation to vascular apoptosis. On the cellular membrane, different receptors are shown (right inset), through which oxidized lipids can activate cytoplasmic events involving the kinase cascade and activation of several transcription factors. The target genes are divided into genes that regulate the cell cycle and those that regulate extracellular signaling. The degree to which oxidized low-density lipoprotein (oxLDL) causes vascular apoptosis in vivo remains unknown. The postulated effect of oxidation on apoptosis in vivo is supported by the observation that treatment with antioxidants protects against apoptosis induced by oxLDL. These molecular interactions can subsequently affect the fate of vascular lesions and plaque instability. Abbreviations: AII, angiotensin II; AP, activator protein; ATF-2, activating transcription factor 2; CREB, cAMP-responsive-element-binding protein; eoxLDL, extensively oxidized LDL; IkB, inhibitor of NF-kB; MAP kinase, mitogen-activated protein kinase; moxLDL, mildly oxidized LDL; NF-kB, nuclear factor kB; nLDL, native LDL; NO, nitric oxide; PLT, platelet; ROS, reactive oxygen species; TF, tissue factor; TNFR, tumor-necrosis-factor receptor.

Acknowledgements All the co-authors contributed to the design of the review. We thank Dr Wulf Palinski (San Diego) for critical comments on the manuscript. Our studies were supported by NIH grants HL-56989, HL-63282, HL-58433 and HL-66999, and by the Mayo Foundation.

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