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Cardiovascular Research 2000 45(3):756-765; doi:10.1016/S0008-6363(99)00270-9
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Copyright © 2000, European Society of Cardiology

Apoptosis in inflammatory–fibroproliferative disorders of the vessel wall

Kenneth Walsha,b,* and Jeffrey M. Isnera

aDivisions of Cardiovascular Research and Vascular Medicine, St. Elizabeth's Medical Center, 736 Cambridge Street, Boston, MA 02135, USA
bProgram in Cell, Molecular and Developmental Biology, Sackler School of Biomedical Studies, Tufts University School of Medicine, Boston, MA 02135, USA

* Corresponding author. Tel.: +1-617-562-7501; fax: +1-617-562-7506 kwalsh{at}opal.tufts.edu

Received 13 July 1999; accepted 27 August 1999


   Abstract
Top
 Abstract
1 Apoptosis in chronic...
 2 Apoptosis resulting from...
 3 Death receptor/ligand...
 4 Participation of Bcl-2...
 5 Closing remarks
 Acknowledgments
 References
 
Apoptotic cell death is a hallmark of inflammatory–fibroproliferative disorders of the vessel wall. Here, we review what is currently known about cell death within atherosclerotic and restenotic lesions. We also examine evidence suggesting that inflammatory cells contribute to the regulation of cell turnover within these lesions, and discuss the molecules expressed by vascular cells that modulate these processes. In toto, these studies suggest that apoptosis is prevalent in vascular lesions, controlling the viability of both inflammatory and vascular cells, and thus determining the cellular composition of the vessel wall.

KEYWORDS Apoptosis; Arteries; Atherosclerosis; Infection/inflammation; Smooth muscle


   1 Apoptosis in chronic vascular lesions
Top
 Abstract
 1 Apoptosis in chronic...
2 Apoptosis resulting from...
 3 Death receptor/ligand...
 4 Participation of Bcl-2...
 5 Closing remarks
 Acknowledgments
 References
 
Apoptosis appears to be an important feature of the remodeling process that occurs during atherosclerotic and restenotic lesion formation. In the normal vessel wall, vascular cells undergo very low rates of turnover. However, in chronic vessel wall lesions, the frequency of apoptosis is more appreciable and roughly correlates with the increased proliferative capacity of the tissue. Early studies documented vascular smooth muscle cell (VSMC) apoptosis by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) and electron microscopic analysis of human atherectomy and endarterectomy specimens from atherosclerotic and restenotic lesion [1–4]. In these lesions, TUNEL-positive cells display elevated expression of caspases, proteases that are essential for apoptosis [2,4]. Abundant apoptosis is also detected in macrophages and T cells within atherosclerotic lesions, and macrophages have been shown to contain apoptotic bodies [5]. Apoptosis may be more pronounced in advanced atherosclerotic lesions compared to early intimal thickening and/or fatty streaks [6]. A preponderance of VSMC apoptosis in advanced lesions is likely to contribute to diminished plaque cellularity. Consistent with this notion, VSMCs cultured from atherosclerotic coronary atherectomy specimens proliferate more slowly and demonstrate higher frequencies of apoptosis than VSMCs cultured from normal vessels [1,7]. VSMC apoptosis has been described in the fibrous cap and underlying media of non-ulcerated lesions from human thoracic aorta and coronary arteries, suggesting that this process could contribute to plaque destabilization and rupture [5]. Apoptotic and necrotic cells have also been detected in atherosclerotic plaques with a recent history of rupture [8].

Apoptotic cells have also been detected in atherectomy specimens from patients with in-stent restenosis [9]. Compared to atherosclerotic and restenotic plaques, the lesions that develop within stents are remarkably cellular, and comprised of VSMCs, macrophages and leukocytes. A relatively high fraction of VSMCs within these lesions are positive for the cell cycle marker proteins PCNA, cdk2 and cyclin E. These lesions also display high frequencies of cellular apoptosis as assessed by TUNEL labeling, indicating that in-stent restenosis is associated with high rates of VSMC turnover.

High frequencies of apoptosis within vascular lesions have also been reported in animal models of atherosclerosis. For example, apoptotic vascular cells have been identified in the atherosclerotic plaques of cholesterol-fed rabbits [10]. In this model, 6 months of cholesterol withdrawal results in a marked reductions in the frequencies of cell replication and apoptosis [11]. It has also been shown in rabbit models of atherosclerosis that lesion size can be reduced by treatments that can promote apoptosis of vascular cells. For example, atherosclerotic lesion size in Watanabe hyperlipidemic rabbits can be reduced by treatment with granulocyte-macrophage colony-stimulating factor, presumably by inducing VSMC apoptosis [12]. Activation of the L-arginine/nitric oxide pathway also induces atheroma regression in hypercholesterolemic rabbits through the induction of macrophage apoptosis [13]. It should also be noted that vascular cell apoptosis has been described in mouse models of atherosclerosis including the advanced vascular lesions of APOE*3-Leiden transgenic mice [14] and in the foam cells and VSMCs of the aorta in hyperlipidemic ApoE- and LDL receptor-deficient mice [15]. These reports of apoptosis in mouse models of vascular disease suggest that manipulation of apoptosis regulatory genes by transgenic and knock-out techniques may prove useful for understanding the role of cell death in vascular pathologies.


   2 Apoptosis resulting from acute vascular injury
Top
 Abstract
 1 Apoptosis in chronic...
 2 Apoptosis resulting from...
3 Death receptor/ligand...
 4 Participation of Bcl-2...
 5 Closing remarks
 Acknowledgments
 References
 
Apoptotic VSMC death has been well documented in animal models of acute vascular injury. Clowes and co-workers provided early insights regarding cell turnover [16,17]. Serial analyses of neointima in balloon-injured rat carotid arteries revealed that total accumulation of VSMCs in the neointima reaches a maximum at 2 weeks post-injury, despite the fact that continuous cellular proliferation occurs for up to 12 weeks with no increase in VSMC number. Thus, the death of neointimal VSMCs was inferred to account for the lack of VSMC accumulation at later time points. Consistent with this concept, it has been demonstrated that neointimal VSMCs undergo apoptosis from 7 to 30 days post-injury [18,19]. In another study, in situ end labeling (ISEL), a technique similar to TUNEL, revealed up to 14% apoptotic cell death in neointimal VSMCs at 20 days post-injury [18]. In these developed lesions, apoptosis appeared to be largely confined to the most luminal cell layers of the neointima, and little or no apoptosis was observed in the VSMCs of the media.

Balloon denudation has also been shown to induce a rapid wave of medial VSMC apoptosis in the rat carotid artery [20]. Uninjured vessels, and vessels harvested immediately after balloon-injury, do not display evidence of apoptosis. However, at 30 min and 1 h post-injury, as many as 70% of medial VSMCs appeared apoptotic by TUNEL staining. Many of these TUNEL-positive cells display nuclear condensation, an independent indicator of apoptosis, and transmission electron microscopic analyses of sections prepared from the injured vessels also showed cells with morphological characteristics of apoptosis. Of note, these markers of apoptosis were no longer evident by 4 h post-injury. By this time, a 65% loss of cellular density was observed, in good agreement with the percentage of TUNEL-positive nuclei detected at the earlier time points. These data suggest that the VSMCs of the rat carotid artery undergo apoptosis rapidly and synchronously following balloon injury, leading to substantial decreases in vessel wall cellularity prior to the initiation of proliferative activity.

Rapid-onset apoptosis has also been observed in single- and double-injury models of balloon angioplasty in rabbit iliac arteries [20–23]. Evidence for angioplasty-induced apoptosis in rabbit vessels includes positive TUNEL staining, the presence of pyknotic nuclei in Hoechst-stained tissues, and the appearance of cells in electron micrographs exhibiting morphological features characteristic of apoptosis. Greater cell loss occurs with the higher balloon-to-artery ratios, and this correlates with greater frequencies of TUNEL-positive cells [22]. In these more complex models, necrotic cell death is likely to also contribute to early cell loss after angioplasty, particularly when high balloon-to-artery ratios are employed. These data suggest that increased apoptosis is responsible, at least in part, for the large reductions in vessel wall cellularity seen when large balloon-to-artery ratios are employed. Surprisingly, in the double-injury model, neointimal VSMCs are less sensitive to elimination by balloon angioplasty compared to medial VSMCs, suggesting that modulation of the VSMC phenotype influences angioplasty-induced apoptosis [21,22]. These findings have important implications for strategies of arterial gene transfer which employ balloon catheter delivery for transfection of vascular smooth muscle cells [22]. Furthermore, Pollman et al. have shown that angioplasty-induced VSMC apoptosis is regulated by a redox-sensitive signaling pathway and that the local administration of antioxidants can minimize loss of vessel wall cellularity [23].

Angioplasty in porcine coronary arteries also produces rapid-onset apoptosis. In this model system, early VSMC apoptosis peaks at 6 h post-injury, and is followed by a proliferative response in succeeding days [24]. This investigation noted that the majority of apoptotic VSMCs were located at sites of obvious trauma at 1 h post-injury. At later times, apoptosis appeared to spread circumferentially into deeper layers of the media suggesting a progressive recruitment of VSMCs.


   3 Death receptor/ligand interactions
Top
 Abstract
 1 Apoptosis in chronic...
 2 Apoptosis resulting from...
 3 Death receptor/ligand...
4 Participation of Bcl-2...
 5 Closing remarks
 Acknowledgments
 References
 
Death receptors comprise a family of structurally related proteins that are expressed on the cell's surface and function to transmit apoptosis-inducing signals when activated by a death ligand. Of this family, the tumor necrosis factor receptor-1 (TNFR) and Fas (also referred to as CD95 and APO-1) have been examined most extensively in vascular cells. The actions of these two receptors may be particularly relevant in the genesis of vascular lesions through their ability to regulate inflammation. Other death receptor systems have also been identified [25], but their roles in the vasculature, if any, have not been elucidated.

Engagement of Fas by its ligand transmits an apoptotic signal to the cell. This death receptor is expressed on numerous cell types including inflammatory and vascular cells [26]. Fas ligand (FasL) is a membrane-bound death factor whose expression is typically confined to inflammatory cells and tissues that routinely encounter inflammatory cells [27,28]. For example, FasL is expressed on cytotoxic T lymphocytes where it contributes to their cytotoxic function and mediates the elimination of autoreactive and peripheral T cells [29]. Other inflammatory cells such as monocytes and macrophages also express both Fas and FasL [30,31]. Spontaneous monocyte apoptosis appears to be mediated by the interaction of Fas and FasL on the cell surface, whereas monocyte-derived macrophages are less susceptible to spontaneous apoptosis and are highly resistant to Fas-mediated death signals. Constitutive FasL expression has also been detected at sites of ‘immune privilege’ such as eye and testis [32,33]. It has been proposed that FasL expression by these tissues prevents inflammatory leukocyte infiltration by inducing death in Fas-expressing immune cells when they attempt to enter the tissue. Similarly, some tumor cells express FasL constitutively and this may contribute to their ability to evade the immune response [34–36]. As discussed in greater detail below, FasL has also been detected on the vascular endothelium where it may have a role in controlling inflammatory cell extravasation [37]. The gld and lpr strains of mice are null for functional FasL and Fas expression, respectively, and suffer from autoimmune disorders [38,39]. Of note, the lpr mice display vasculitis that results from neutrophilic and mononuclear cell infiltrates [40–42]. It is important to note that while endogenous FasL largely functions to downregulate inflammatory reactions, non-physiological expression of this protein can induce an inflammatory response characterized by neutrophil infiltration [43]. This inflammatory response is a consequence of the release of IL-1β, a neutrophil chemoattractant, following Fas engagement.

Fas contains a cytoplasmic domain, referred to as the death domain, that is required to transmit an apoptotic signal [44]. The death domain of Fas interacts with the linker protein FADD (Fas-associated death domain) [45,46] (Fig. 1). A distinct domain in FADD binds to pro-caspase 8 (also referred to as FLICE and MORT1). Clustering of ligated Fas leads to activation of caspase 8 via auto-proteolysis, and caspase 8 then proteolytically activates other caspase family members. Both caspase 8 and FADD are essential for Fas-mediated death signals [47]. Caspase 8 activation, and Fas-mediated apoptosis, can be blocked by endogenous inhibitory molecules such as FLIPs (FLICE-like inhibitory proteins) that are also referred to as I-FLICE, FLAME, CASH, CLARP, MRIT and Usurpin [48–53]. Depending upon the cell type, Fas-mediated death signaling can involve activation of the mitochondria and, in such cases, is sensitive to the anti-apoptotic actions of Bcl-2 and Bcl-XL proteins [54]. Mitochondrial activation can result from caspase 8-mediated cleavage of Bid, a Bcl-2 homologous protein, which then translocates to the mitochondrial membrane and induces cytochrome c release and mitochondrial dysfunction [55,56]. Cytoplasmic cytochrome c activates caspase-9 in the presence of Apaf-1 (for apoptosis activating factor-1) and dATP, leading to the proteolytic activation of caspase-3 (CPP32-like protease). The caspase activation cascades ultimately trigger changes in morphology that accompany apoptosis such as cytoskeletal rearrangements and chromatin fragmentation [57].


Figure 1
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  		Fig. 1 Fas-mediated death signaling. Fas ligand induces oligomerization of Fas, which recruits caspase-8 via the FADD adaptor protein. FADD interaction with caspase-8 leading to an activation of proteolytic activity. In some cell types, activated caspase-8 also cleaves the Bcl-2 homologous protein Bid, which then translocates to the mitochondrial membrane and induces mitochondrial dysfunction and cytochrome c release. Cytosolic cytochrome c binds to Apaf-1 and dATP, leading to the activation of caspase-9. FLIP is a homolog of caspase-8 that lacks the active-center cysteine residue in a caspase-like domain. Thus, FLIP may function as an inhibitor of caspase-8, blocking the Fas death signal.

 
The TNF{alpha} receptor (TNFR) is structurally similar to Fas. However, the ability of TNF{alpha} stimulation to activate either cytotoxic or pro-inflammatory responses results in the TNF{alpha}/TNFR system being more complex than the FasL/Fas system. In some situations TNF{alpha}-stimulation will induce caspase 8 activation and apoptotic cell death via the TRADD and FADD linker proteins (Fig. 2). However, in several instances this death signal is blocked by the TNF{alpha}-induced activation of the NF-{kappa}B transcriptional regulatory protein [58]. Activation of NF-{kappa}B by TNFR involves the recruitment of the proteins RIP, TRAF-2 and NIK, and ultimately leads to the phosphorylation of the NF-{kappa}B inhibitory subunit, I{kappa}B. Phosphorylation of I{kappa}B promotes its degradation, allowing NF-{kappa}B to translocate to the nucleus where it activates the expression of anti-apoptotic factors such as IEX-1L [59], the inhibitor-of-apoptosis proteins (IAPs) c-IAP1 and c-IAP2 which block caspase activity [60,61], and the Bcl-2 homolog A1 [62].


Figure 2
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  		Fig. 2 TNF{alpha} stimulation can have pro- or anti-apoptotic actions. Like Fas, engagement of the TNF{alpha} receptor can induce apoptotic cell death by activating caspase 8. However, in many instances this death signal is blocked by the TNF{alpha}-induced activation of the NF-{kappa}B, which activates a transcriptional program that suppresses apoptosis.

 
It is well established that NF-{kappa}B also activates the transcription of proinflammatory genes [63,64]. TNF{alpha} is expressed by macrophages and smooth muscle cells at the sites of vascular injury, and it is thought to play an important role in the progression of atherosclerotic lesions [65,66]. This cytokine can promote atherogenic processes through its ability to upregulate surface adhesion molecule expression on the endothelial cell surface and recruit inflammatory cells to the vessel wall. TNF{alpha} can also induce endothelial cell apoptosis, which may serve to compromise the integrity of the endothelium and promote atherogenesis [67–69]. TNF{alpha} is also thought to play a key role in transplant arteriosclerosis since it has been shown that a blockade of TNF{alpha} production reduces coronary artery neointimal formation in a rabbit model of cardiac transplant arteriosclerosis while having no effect on the degree of myocardial rejection [70]. However, in an apparent contradiction to this simple paradigm, mice lacking TNFR display accelerated atherosclerosis when fed an atherogenic diet [71].

Fas and FasL expression have been detected on the normal and diseased vessel wall, and it has been proposed that Fas-mediated apoptosis is a feature of atherogenesis [72–75], allograft arteriopathy [76] and the acute inflammatory response to cytokines [37]. Since VSMCs express Fas and inflammatory cells express FasL, it has been proposed that Fas-mediated apoptosis may contribute to atherosclerotic plaque instability [75]. Numerous recent studies have examined the susceptibility of VSMCs to Fas-mediated cell death in vitro and in vivo. It is reported that cultured rat VSMCs express Fas and readily undergo apoptosis when they express cell surface FasL following infection with an adenoviral vector that expresses FasL (Adeno-FasL) [77]. This treatment appears to induce cell death in a paracrine manner since Adeno-FasL-infected cells can induce apoptosis in non-infected smooth muscle cells in co-culture experiments. Local delivery of Adeno-FasL to balloon-injured rat carotid arteries also induces apoptosis in proliferating smooth muscle cells [77,78]. In contrast, it is reported that cultured human smooth muscle cells do not undergo Fas-mediated cell death when exposed to an agonist anti-Fas antibody, except when these cells are also stimulated by TNF{alpha}, Interleukin-1β or IFN{gamma} [75]. The insufficiency of anti-Fas antibody to induce VSMC apoptosis in the absence of cytokine stimulation might result from the inability of this apoptotic agent to efficiently cluster Fas receptor molecules under these conditions [79,80]. In contrast, infection with an adenovirus encoding FasL leads to presentation of membrane-bound FasL, and this situation may lead to more efficient transmission of the death signal [80]. Consistent with this interpretation, sensitization of VSMCs to anti-Fas antibody-induced apoptosis by IFN{gamma} may result from the upregulation Fas expression by this cytokine, thereby favor receptor clustering [75,81].

In striking contrast to VSMCs, vascular endothelial cells are remarkably resistant to Fas-mediated apoptosis under normal conditions. Endothelial cells express Fas receptor on their cell surface, but they do not undergo apoptosis when exposed to agonist anti-Fas antibody [82], or when cell surface Fas ligand is increased by adenovirus-mediated Fas ligand gene delivery [72]. Of note, endothelial cells remain resistant to Fas-mediated cell death even when activated by interferon-{gamma} which markedly increases Fas expression [81,82]. Presumably, endothelial cells are resistant to Fas-mediated cell death because they normally express low levels of cell surface FasL [37,72], and thus it is reasonable to assume that they would also express endogenous inhibitors of the Fas-initiated death signaling pathway to ensure survival. The factors that confer resistance to Fas-mediated apoptosis in endothelial cells are currently unknown. These protective mechanism could involve FLIPs [48], which are expressed in endothelial cells [83]. Other protective mechanisms could involve the participation of Bcl-2 family of proteins which function as positive and negative regulators of apoptosis, including Fas-mediated apoptosis in some [84,85] but not all cell types [86,87]. Other modulators of Fas-mediated cell death that may participate in this regulatory scenario include sentrin [88], GD3 ganglioside [89] and nitric oxide [90,91]. In particular, nitric oxide production by endothelial cells may promote the nitrosylation and inactivation of caspases that are essential for Fas-mediated cell death [92].

The expression of endogenous FasL on the endothelial cell surface may be significant for the pathogenesis of inflammatory–fibroproliferative disorders of the vessel wall. Local administration of TNF{alpha} to arteries also downregulates endothelial FasL expression and induces robust leukocyte infiltration of the vessel wall [37]. This TNF{alpha}-induced macrophage and T cell infiltration is markedly attenuated when FasL is constitutively expressed on the vascular endothelium by adenovirus-mediated gene transfer. Consistent with these findings, ectopic expression of FasL by the VSMCs of endothelium-denuded rat carotid arteries can inhibit T cell-mediated inflammatory responses to adenovirus infection [78]. Since chronic, localized immune response is an important feature of atherogenesis, the observation that FasL can inhibit inflammatory responses within the vessel wall suggests that it may serve an atheroprotective function under some conditions.

Alternatively, it is possible that alterations in the expression of FasL, Fas, or Fas-mediated death signaling components may be a feature of the endothelial cell dysfunction that contributes to atherogenesis. For example, oxidized low density lipoproteins (Ox-LDL) may alter the sensitivity of endothelial cells to Fas-mediated apoptosis [72,83]. Ox-LDL and its lipid constituents have numerous detrimental effects on endothelial cell function including the induction of apoptosis [93–97]. Apoptotic endothelial cells have been detected in human atherosclerotic lesions [74] which accumulate Ox-LDL [98–100]. Ox-LDL-induced apoptosis of cultured endothelial cells can be inhibited by neutralizing antibodies to FasL, and Ox-LDL-induced cell death is also reduced in cultured aortic endothelium derived from gld and lpr mice which lack a functional Fas pathway [72]. Ox-LDL upregulates Fas expression and sensitizes endothelial cells to Fas-mediated death signaling [72,101]. Evidence for a sensitization mechanism comes from the observation that endothelial cells are normally resistant to cell death in response to stimulation with Fas agonists, but Fas agonists can induce apoptosis in the presence of Ox-LDL when endogenous FasL is inactivated with neutralizing antibodies. Consistent with these observations, Suhara et al. have shown that oxidative stresses can upregulate Fas on endothelial cells and also sensitize these cells to Fas-mediated death signaling pathways [102]. The sensitization of endothelial cell Fas-mediated apoptosis by Ox-LDL correlates with the downregulation of FLIP suggesting that this caspase inhibitor may contribute to the natural resistance of endothelial cells to Fas stimulation [83]. Collectively, these data indicate that the Fas/FasL pathway participates in the apoptosis of endothelial cell induced by acute exposure to oxidized LDL. Thus, Fas-mediated endothelial cells death may be a feature that contributes to the accelerated atherosclerosis seen in patients with hyperlipidemia.


   4 Participation of Bcl-2 family proteins and p53
Top
 Abstract
 1 Apoptosis in chronic...
 2 Apoptosis resulting from...
 3 Death receptor/ligand...
 4 Participation of Bcl-2...
5 Closing remarks
 Acknowledgments
 References
 
In many situations, cell viability is controlled by modulations in the levels of Bcl-2 family proteins. Some of these proteins function as inhibitors of apoptosis, whereas others are apoptosis accelerators. Inhibitors of apoptosis include Bcl-2, Bcl-XL and A1, whereas Bax, Bad and Bid function as apoptotic accelerators. The relative stoichiometries of apoptotic accelerator and inhibitory proteins are believed to function as molecular rheostats that control cell survival. Factors that modulate the expression of individual Bcl-2 family proteins include genotoxic and mechanical stresses, aberrant cell cycle activity and the absence of survival factors.

Generally, the Bcl-2 family proteins function at the level of the mitochondria to control cell viability (Fig. 3). X-ray crystallographic and NMR analyses of Bcl-XL structure show that it resembles the membrane proteins diphtheria toxin and colicin, which are thought to form pH-dependent membrane pores [103]. Bcl-XL was shown to form an ion channel in synthetic lipid membranes, suggesting that it might function to regulate mitochondrial function and membrane permeability [104]. Bcl-2 family proteins control the relocalization and actions of cytochrome c, a step that precipitates apoptotic cell death [105]. When release from the mitochondria is triggered by an apoptotic accelerator protein, cytochrome c binds to Apaf-1 and procaspase 9, leading to proteolytic activation of caspase 9. Caspase 9 then proteolytically activates downstream caspases and cell death ensues.


Figure 3
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  		Fig. 3 Bcl-2 family proteins regulate cell viability by altering mitochondrial function. Apoptotic stimuli alter the balance of pro- and anti-apoptotic Bcl-2 family proteins. Pro-apoptotic proteins like Bax promote cytochrome c release from the mitochondria. Liberated cytochrome c then forms a complex with Apaf-1, dATP and pro-caspase 9, leading to caspase 9 activation and apoptosis. Bcl-2 inhibits apoptosis by preserving mitochondrial function. Bcl-2 also prevents apoptosis following cytochrome c release, suggesting that Bcl-2 can function on multiple levels to promote cell survival.

 
A number of studies have examined the role of Bcl-2 family proteins in controlling the viability of vascular cells. The expression of the apoptotic accelerator Bax is elevated in the VSMCs of human atherosclerotic plaques [6] where apoptosis occurs at a high rate. Conversely, the apoptosis protective factor Bcl-XL is expressed in the normal medial VSMCs of rat carotid arteries, and this expression is rapidly downregulated following balloon injury [20]. This decrease in Bcl-XL occurs at 1 hour post-injury in the most luminal layers of the media. Since these luminal layers of the media also exhibit the greatest number of TUNEL-positive nuclei, these data suggest that modulations in the level of Bcl-XL may be a feature of the apoptotic response to acute vascular injury. Indeed, Pollman et al. have recently demonstrated elevated levels of Bcl-XL within atheromatous human and rabbit intimal lesions compared to medial cells [21]. When an anti-sense oligonucleotide directed against Bcl-X was introduced into balloon-injured rabbit carotid arteries at four weeks following injury, Bcl-XL expression was down-regulated and apoptosis of intimal cells occurred at an enhanced rate. This resulted in a 56% decrease in intimal thickness. This study demonstrates that Bcl-2 family proteins are functionally significant within the vessel wall and that the differential expression of Bcl-XL may contribute to the differential sensitivity of medial and neointimal VSMCs to rapid-onset apoptosis [21,22].

It may be of significance for vessel wall disease that the transcription factor p53 activates the expression of Bax [106] and inhibits the expression of Bcl-2 [107], leading to conditions that favor apoptosis. Activation of p53 also increases cell surface Fas expression on human VSMCs by promoting transport from the Golgi apparatus [126]. Anti-proliferative activities of p53 are mediated by its ability to induce the cyclin-dependent kinase inhibitor p21 [108]. p53 can induce VSMC apoptosis in vitro and inhibits VSMC proliferation in vitro and in vivo [7,109]. p53 accumulates in atherosclerotic lesions, and its ability to induce apoptosis in these lesions may be dependent on its interaction with MDM2 [110], a regulator of p53 stability [111]. It has also been reported that the cytomegalovirus immediate early gene IE2-84 antagonizes p53 induced VSMC apoptosis [112]. Recently, it was shown that p53–/–, ApoE–/– mice developed accelerated aortic lesions compared with p53+/+, ApoE–/– mice when fed a high-fat diet [113] However, the increased lesion size resulting from p53-deficiency appeared to result from an increase in cell proliferation rate rather than a decrease in apoptosis. Thus p53-independent apoptotic mechanisms may predominate in the atherogenic vessel wall [114].

Nitric oxide, a key regulator of vascular tone and remodeling, is functionally similar to p53 in that it can induce growth arrest [115] and apoptosis [116] in VSMCs. Recent data in hypercholesterolemic rabbits show that activation of the L-arginine/nitric oxide pathway induces regression of atheroma, perhaps through the induction of macrophage apoptosis [13]. Of note, nitric oxide upregulates Fas expression on VSMCs and this may contribute to the enhanced VSMC apoptosis under these conditions [117].

Alterations in the expression patterns of endogenous vessel wall regulatory proteins can also lead to VSMC apoptosis through a process dependent upon Bcl-2 family proteins. For example, the Gax transcription factor is downregulated in VSMCs under conditions that promote VSMC proliferation in vitro and in vivo [118,119]. Constitutive expression of Gax in proliferative VSMCs will induce apoptosis through a process that involves the p53-independent upregulation of Bax [120,121]. Along these same lines, it has been shown that the antiproliferative factor BTG1 is highly expressed in the apoptotic cells of vascular lesions that develop in Watanabe hyperlipidemic rabbits [122].

Bcl-2 family proteins also function to regulate the viability of endothelial cells. For example, endothelial cell survival is promoted by vascular endothelial growth factor (VEGF) and is associated with the upregulation of Bcl-2 and A1 [123,124]. Related experiments have shown that activation of the protein kinase Akt (also referred to as PKB) is essential for VEGF-mediated survival of endothelial cells and for VEGF-mediated induction of Bcl-2 [124,125]. Finally, TNF{alpha}-induced death of endothelial cells involves the ubiquitin-dependent degradation of Bcl-2 [67].


   5 Closing remarks
Top
 Abstract
 1 Apoptosis in chronic...
 2 Apoptosis resulting from...
 3 Death receptor/ligand...
 4 Participation of Bcl-2...
 5 Closing remarks
Acknowledgments
 References
 
Apoptotic cell death is widespread in the inflammatory–fibroproliferative lesions of the vessel wall. Apoptotic cell death can influence lesion mass, plaque stability, inflammation and the integrity of the endothelium. Recent studies have documented numerous regulatory pathways that can potentially control vascular cell viability. Understanding these regulatory networks at a detailed molecular level should provide insight about the pathogenesis of vascular diseases and may lead to the development of novel therapies to treat these disorders.

Time for primary review 13 days.


   Acknowledgments
Top
 Abstract
 1 Apoptosis in chronic...
 2 Apoptosis resulting from...
 3 Death receptor/ligand...
 4 Participation of Bcl-2...
 5 Closing remarks
 Acknowledgments
References
 
We thank Linda Whittaker for aid in preparing this manuscript, Roy C. Smith and Harris Perlman for helpful discussions and Matthew Findley for preparation of artwork.


   References
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 Abstract
 1 Apoptosis in chronic...
 2 Apoptosis resulting from...
 3 Death receptor/ligand...
 4 Participation of Bcl-2...
 5 Closing remarks
 Acknowledgments
 References
 

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