OUP user menu

Apoptosis in atherosclerosis: beneficial or detrimental?

Mark M Kockx, Arnold G Herman
DOI: http://dx.doi.org/10.1016/S0008-6363(99)00235-7 736-746 First published online: 1 February 2000

Abstract

Several groups have demonstrated apoptotic cell death in atherosclerotic plaques. The significance of apoptosis in atherosclerosis depends on the stage of the plaque, localization and the cell types involved. Both macrophages and smooth muscle cells undergo apoptosis in atherosclerotic plaques. Apoptosis of macrophages is mainly present in regions showing signs of DNA synthesis/repair. Smooth muscle cell apoptosis is mainly present in less cellular regions and is not associated with DNA synthesis/repair. Even in early stages of atherosclerosis smooth muscle cells become susceptible to undergoing apoptosis since they increase different pro-apoptotic factors. Moreover, recent data indicate that smooth muscle cells may be killed by activated macrophages. The loss of the smooth muscle cells can be detrimental for plaque stability since most of the interstitial collagen fibers, which are important for the tensile strength of the fibrous cap, are produced by SMC. Apoptosis of macrophages could be beneficial for plaque stability if apoptotic bodies are removed. Apoptotic cells that are not scavenged in the plaque activate thrombin which could further induce intraplaque thrombosis. It can be concluded that apoptosis in the primary atherosclerosis is detrimental since it could lead to plaque rupture and thrombosis. Recent data of our group indicate that apoptosis decreases after lipid lowering which could be important in our understanding of the cell biology of plaque stabilization.

Keywords
  • Atherosclerosis
  • Apoptosis
  • Smooth muscle
  • Macrophages
  • Lipid metabolism

Time for primary review 23 days.

1 Introduction

Cell death in atherosclerosis was suggested by Virchow in 1858. The father of cellular pathology stated that atherosclerotic plaques form by cells that replicate and then die. He called this stage fibro–fatty degeneration [1]. Imai and Thomas studied diet-induced lesions in cerebral atherosclerosis in swine some 25 years ago [2]. These authors examined the induced atherosclerotic lesions extensively using transmission electron microscopy and found smooth muscle cell death in the plaques. The authors described these changes as necrosis of the cells although their description of the nuclear and cytoplasmic changes fulfilled the criteria of apoptotic cell death. Kerr, Wyllie and Currie have introduced the term apoptosis to distinguish a special form of cell death different from necrosis [3]. When a cell receives a signal to die an apoptotic death, it goes through a series of morphological changes detectable using light microscopy, starting with shrinkage of the cell membrane and proceeding to condensation of the nuclear chromatin, cellular fragmentation, and, finally, the engulfment of the apoptotic bodies by neighboring cells. Although the term apoptosis was introduced only 25 years ago, typically apoptotic morphology was described by embryologists at the beginning of this century. Embryologists recognized it as a mechanism to counterbalance the excess cellular proliferation during the development of organs and limbs [4]. More recently, apoptosis has also been implicated in the development of the arteries. Cho et al. have studied apoptosis during arterial wall development [5], and Slomp et al. focused on apoptosis and the changes that occur in the ductus arteriosus [6]. Apoptosis, however, is not limited to cell elimination during vascular development. In recent years, apoptosis has been implicated in atherosclerosis [7–21]. Theoretically it would be ideal to induce regression of restenotic lesions and primary atherosclerotic plaques by the induction of apoptosis [22,23]. However, apoptosis could also have negative effects on the stability of atherosclerotic plaques. We will carefully consider this possibility and try to answer the question: is apoptosis in atherosclerosis beneficial or detrimental? It will become clear that human atherosclerotic plaques are very complex structures that are different from restenotic lesions and that the significance of apoptosis depends on numerous factors that are typical for atherosclerotic plaques.

2 Apoptosis in atherosclerotic plaques: quantitative aspects

Recently, the detection of DNA fragmentation using the TUNEL technique or in situ nick translation has become a standard technique for the detection of apoptosis in tissue sections.

Different studies have used this technique to demonstrate that cells can die in atherosclerotic plaques through apoptosis. However, a large variation in the percentage of TUNEL positive nuclei has been found, ranging from less than 2% [7–9] up to 30% [10–13]. The TUNEL technique labels the execution phase of apoptosis which takes in cell cultures in less than 6 h. Some of the reported values would indicate that plaques are in an imminent state of collapse which is certainly not the case as remarked by Newby and George [14]. This suggests that the TUNEL technique is not without pitfalls. In accordance with Hegyi [24] we reported that the technique is very sensitive and needs a careful titration of proteolytic pretreatment and Tdt concentration; otherwise, a high fraction of non-apoptotic nuclei will be labeled. In a recent study a molecular explanation for this phenomenon was found [25]. It was demonstrated in this study that besides apoptotic nuclei, non-apoptotic nuclei that show signs of active gene transcription are labeled using the TUNEL technique. These cells are still active and are transcribing genes that might be related or completely unrelated to the apoptotic cell death pathway. In a true apoptotic cell the nuclear DNA is cleaved in oligonucleosomal-sized fragments and processes like RNA transcription and splicing are abolished. Moreover, even in the early execution phase of apoptosis caspase 3 cleaves the 70 kDa protein component of splicing factor U1 snRNP [26]. The loss of RNA splicing can be considered as an early step in the execution phase of apoptosis. Therefore, it is evident that nuclei that are TUNEL positive and showing signs of high RNA synthesis and splicing activity are clearly not in the execution phase of apoptosis. These findings were recently confirmed by Kanoh et al. These authors found that TUNEL labeled cardiomyocytes are not showing apoptosis but show signs of RNA and DNA synthesis/repair [27]. The fact that the TUNEL technique labels nuclei with high RNA synthetic activity is not surprising since in the past several groups have employed a modification of the DNA in situ nick translation method to allow the in situ detection of sites of active gene transcription [28,29].

Therefore, the TUNEL technique, although a useful technique to detect the execution phase of apoptosis should always be combined with additional techniques such as markers of transcription and morphological criteria. Using these stringent conditions we and others have still found convincing evidence that cells can die within atherosclerotic plaques through apoptosis. The level of apoptotic cell death is also strongly related to the stage of development of the atherosclerotic plaque [17,30,31]. Therefore, a large variability can be expected when atherosclerotic plaques of different stages are compared. In general, adaptive intimal thickening and fatty streaks show very little apoptosis, whereas advanced atherosclerotic plaques show foci of apoptosis. Most of these foci are associated with regions of macrophage infiltration [17,31].

Isner et al. found evidence for apoptotic cell death in primary atherosclerotic lesions and restenotic lesions [7]. Apoptotic cell death was positively linked to cell replication. Restenotic lesions, showing high replication rates, also demonstrated more apoptotic nuclei. Bennett et al. show, in an in vitro study, that proliferating SMC show more apoptotic cell death than non-proliferating SMC [32]. A similar result was found by Bochaton-Piallat in the intimal thickening induced after endothelial denudation of the rat aorta [33]. Bauriedel et al. [15], however, found that human restenotic intimal thickenings showed less apoptotic cell death than primary advanced atherosclerotic plaques. A major difference could be the presence of replicating foam cells of macrophage origin in advanced human atherosclerotic plaques [17,31]. In a study of vein graft atherosclerosis which is considered a form of accelerated human atherosclerosis a consistent association was found between foam cell accumulation and smooth muscle cell death in the fibrous cap [16]. Recently, Boyle et al. found evidence that the macrophages could induce smooth muscle cell death in culture [34].

In the following paragraphs we will discuss apoptosis of endothelial cells, smooth muscle cells and macrophages of primary atherosclerotic plaques. It will become clear that the significance and the mechanisms of apoptosis of each of these cell types show similarities but also many differences.

3 Apoptosis of endothelial cells in atherosclerotic plaques/role of nitric oxide

Injury of the vascular endothelium is a critical event in the pathogenesis of atherosclerosis. Importantly, endothelial cells (EC) in lesion-prone regions, where atherosclerotic plaques preferentially develop are characterized by increased EC turnover rates, suggesting a mechanistic link between EC turnover and the susceptibility to atherosclerotic plaque development. The enhanced turnover is most likely due to increased apoptosis [35]. Interestingly, classical risk factors for atherosclerosis such as high glucose concentrations [36], oxidized LDL [37], increased oxidative stress and angiotensin II stimulate EC apoptosis [38,39].

Interestingly, in the normal arterial wall the endothelial cells protect themselves against apoptosis using two NO-dependent mechanisms (Fig. 1). The low levels of NO that are formed by the low-output isoform ecNOS protects against apoptosis via cyclic GMP-dependent and cyclic GMP-independent mechanisms. Activation of cyclic GMP-dependent protein kinases is associated with protection against apoptosis [40]. Another possible mechanism is S-nitrosylation of caspases, enzymes involved in the executive steps of apoptotic cell death. Interestingly, all caspases contain an essential cysteine within their active center, which is potentially susceptible to S-nitrosylation. Recently, it has indeed been demonstrated that nitric oxide can nitrosylate the caspases interleukin-β-converting enzyme and cysteine protease protein 32 [41,42], thereby affording protection against TNF-α-induced apoptotic cell death. Endothelial cells exposed to shear stress increase their NO production which could protect the endothelial cells against different apoptotic stimuli via a cGMP-independent mechanism. This indicates that in the normal arterial wall NO is protective for different celltypes.

Fig. 1

Differences between the role of nitric oxide in the normal arterial wall vs. atherosclerosis. In the normal arterial wall is nitric oxide derived from the low-output isoform ecNOS protective against apoptosis. This is in contrast with the situation in atherosclerotic plaques where the high-output isoform iNOS is expressed in an environment with a high oxidative stress. During this condition peroxynitrite will form, which can induce DNA damage.

In atherosclerotic plaques the situation is fundamentally different since the high-output isoform iNOS is expressed [43–46] in an environment with a very high oxidative stress (Fig. 1). In this situation nitric oxide itself or peroxynitrite [47] could induce apoptotic cell death and destabilize the atherosclerotic plaque. Recently, apoptotic cell death was studied in different stages of atherosclerosis. Apoptosis was only found in the advanced atherosclerotic plaques in regions that contain numerous foam cells of macrophage origin [17]. Further studies of our group revealed that these macrophages express the high-output isoform iNOS and showed nitrotyrosine which is considered as a footprint of peroxynitrite formation [48]. Interestingly, these iNOS expressing macrophages also showed signs of DNA synthesis/repair.

4 Apoptosis of smooth muscle cells in primary atherosclerotic plaques

Atherosclerotic plaques are complex structures that consists of both smooth muscle cells, macrophages, lymphocytes, microvessels and different collagen types. The plaques often contain a central necrotic core that is separated from the vascular lumen by a fibrous cap. The fibrous cap is composed of smooth muscle cells and interstitial collagen fibers. Plaque rupture occurs when the mechanical stresses in the fibrous cap exceed a critical level that the cap tissue can withstand. Factors increasing the stress are thinning of the fibrous cap, a large lipid-rich necrotic core, a relatively small stenosis, and the fluidity of the lipid pool [49–52].

In addition, a number of biological factors may be weakening the fibrous cap. These include infiltration of the shoulder region of the cap with macrophages, T lymphocytes and a loss of smooth muscle cells [53]. The macrophages can promote local expression and/or activation of matrix metalloproteinases, which decrease the strength of the cap by degrading interstitial collagen fibers [54,55]. Furthermore, loss of the smooth muscle cells will decrease the biosynthesis of interstitial collagen fibers. We have demonstrated that smooth muscle cells can disappear in the plaque via apoptosis [17]. The consequences of smooth muscle cell apoptosis depend on the stage and the location in the plaque of the smooth muscle cell death. Smooth muscle cell apoptosis will lead to a loss of the cells that are responsible for the synthesis of the interstitial collagen fibers [56]. Collagen synthesis in vitro is strongly associated with cell replication. Indeed, it was demonstrated that in primary cultures of adult rat and rabbit aortic smooth muscle cells, the transition into a synthetic phenotype was found to be accompanied by an increase in collagen secretion [57]. However, the situation in the atherosclerotic case is clearly different. Rekhter et al. [58] have demonstrated that although proliferation and type I collagen expression could occur in the same cell, this is a rare event and the majority of collagen-producing cells do not show proliferative activity. We have also arguments that smooth muscle cells in human and experimental atherosclerotic plaques show very low values of cell replication [17]. This indicates that a slight increase in the levels of SMC apoptosis in the plaque will rapidly lead to a drastic decrease of the smooth muscle content having a major influence on the collagen type I synthesis and plaque stability.

4.1 Do smooth muscle cells of atherosclerotic plaques show an increased susceptibility for undergoing apoptosis?

SMC derived from atherosclerotic plaques but not from the media die when brought into culture. Recently, we have studied Bax/Bcl-2 in different stages of atherosclerosis [17]. The smooth muscle cells that were present in the different stages showed striking differences in their expression of the pro-apoptotic protein Bax. We could distinguish three types (Fig. 2) of SMC based on their Bax expression and morphology.

Fig. 2

Smooth muscle cells in the normal media and atherosclerotic plaques can be classified in three types based on their expression of pro-apoptotic factors. Smooth muscle cells in the fatty streaks show lipid accumulation and an increased expression of the pro-apoptotic factor Bax and caspase 3 which increase the susceptibility of the SMC to undergoing apoptosis.

4.1.1 SMC of Type A

These were mainly present in the normal media and the adaptive intimal thickening. These SMC showed no Bax expression and were also negative for caspase 3. Ultrastructurally these SMC showed the classical aspect of SMC of the contractile phenotype without lipid vacuoles but containing numerous microfilaments.

4.1.2 SMC of Type B

These were lipid-laden SMC. These SMC showed a strong Bax expression. These SMC were already present in the early atherosclerotic plaques like the fatty streaks. However, the execution phase of apoptosis, as detected by the TUNEL technique was very rare in these early plaques. What could be the reason for the lipid-laden SMC increasing their Bax expression? Bax can be upregulated via a p53-dependent pathway. Indeed, it was demonstrated that p53 is a direct transcriptional activator of the human Bax gene. [59]. Moreover, p53 is overexpressed but not mutated in atherosclerotic plaques [60]. The reason for the p53 upregulation is not clear but increased oxidative stress and DNA damage has to be considered. Current evidence suggests that DNA damage is sensed by kinases such as DNA-dependent protein kinase (DNA-PK) leading to the phosphorylation and activation of p53 [61]. Therefore, it is intriguing to speculate that the smooth muscle cells show an upregulation of Bax via a p53-dependent mechanism as a response to an increase in oxidative DNA damage in the plaques [17]. Bax is also upregulated in the SMC of experimental atherosclerotic plaques [62]. Interestingly, this upregulation could be reversed after a period of lipid lowering. This indicates that the susceptibility of smooth muscle cells in plaques can possibly be reversed by lipid lowering.

4.1.3 SMC of Type C

These correspond to SMC that were completely disintegrated into cytoplasmic fragments. The SMC origin could still be demonstrated because these cytoplasmic fragments were enclosed by cages of thickened basal laminae. Russel Ross called these SMC pancake-like smooth muscle cells [63]. However, it is clear that these SMC are often fragmented in numerous cytoplasmic fragments. This particular form of SMC cell death was already described by Stehbens as granulovesicular degeneration of the SMC [64]. Recent work by our group revealed that this form of cell death has numerous characteristics that point to apoptotic cell death [17]. We could demonstrate recently that the cytoplasmic fragments of the third type of SMC showed a very high caspase 3 and Bax expression. In a fraction of this Type C SMC it was possible to detect DNA fragmentation using the TUNEL technique. Interestingly, these Type C SMC were completely similar to those that we have found in unstable plaques of saphenous vein grafts [16]. In the vein grafts it was obvious that most of the Type C SMC were found adjacent to foam cells of macrophage origin. This suggested that smooth muscle in plaques can be killed by factors derived from the macrophages (foam cell-derived killing factors). Recently, it was demonstrated that SMC can be killed by macrophage-derived factors [34]. Induced macrophages and T lymphocytes cytokines can induce apoptosis of human and rat vascular SMC in culture and can sensitize SMC to Fas-mediated apoptosis via upregulation of cell surface Fas [65]. Human SMC express Fas whereas T lymphocytes, macrophages, endothelial cells and SMC themselves express the Fas ligand so that Fas/Fas ligand-mediated apoptosis of SMC may occur at sites of high inflammatory cell density [66]. Interestingly, Dong et al. found that in transplant coronary arteriopathy nearly all vascular cells show Fas expression whereas the typical atherosclerotic plaque and normal controls show low levels of Fas [67]. This finding supports the hypothesis that the significance and the molecular mechanisms of apoptosis in different coronary pathologies (primary atherosclerosis, vein graft atherosclerosis, restenosis and transplant arteriopathies) can be different [68].

4.1.4 Conclusion

Apoptosis of smooth muscle cells will lead to a loss of collagen type I which could lead to unstable plaques that are prone to rupture. Moreover, apoptotic smooth muscle cells in the plaques are often not scavenged and could be the main source of the calcifying matrix vesicles [16,17]. These matrix vesicles could lead to plaque calcification. Apoptotic smooth muscle cells can also increase the plaque thrombogenicity [69]. Indeed, plaque SMCs undergoing apoptosis have the same potency to generate thrombin as platelets. This is due to the fact that apoptotic cells expose phosphatidylserine on the surface early in the process [69]. In the presence of factors V and VII, exposed PS can then act as a substrate to thrombin generation. Thus, apoptosis of smooth muscle cells in primary atherosclerotic plaque could be detrimental for plaque stability and increase the risk for thrombosis.

5 Apoptosis of macrophages

Apoptosis of macrophages is mainly found in cellular macrophage-rich regions that also show signs of DNA synthesis. This was demonstrated with Ki-67, PCNA and BrdU incorporation. Mitotic figures are rarely found in these regions. This finding could indicate that DNA synthesis in these cells points to DNA repair. Recently, we have found that the macrophages express inducible nitric oxide synthase, nitrotyrosine and show oxidized lipids [48]. This points to high levels of oxidative stress and peroxynitrite. The high levels of DNA synthesis/repair could be a response to oxidative DNA damage (Fig. 3). In very early studies it was noted that NO targets naked DNA [70,71] and induces oxidative damage in activated macrophages. Both deamination (abasic site formation) and oxidative modification have been described in DNA as a consequence of NO damage [72]. Extending beyond these observations it is believed that NO-damaged DNA elicits DNA repair mechanisms in mammalian cells. In this concept NO-induced apoptotic cell death can be considered as a mechanism that is used by the cell if DNA repair mechanisms fail. Two repair mechanisms have been studied in this context: (1) the p53/p21 and NO-induced DNA repair and apoptosis; (2) the PARP pathway.

Fig. 3

Differences between smooth muscle cells (SMC) and macrophages present in atherosclerotic plaques. Plaque SMC show apoptosis but low levels of DNA synthesis/repair. This is in contrast with macrophages that both show apoptosis and high levels of DNA synthesis/repair. Moreover, plaque macrophages express iNOS and it is plausible that they inactivate their caspases by nitrosylation. This inactivation mechanism and the DNA synthesis/repair could explain why macrophages do not undergo apoptosis despite the high levels of nitric oxide they produce.

5.1 p53/p21 and NO-induced DNA repair and apoptosis

The tumor suppressor gene p53 has come to be known as a master guardian of the genoma and a member of the DNA damage-response pathway [73]. Therefore, the NO-induced DNA damage can upregulate p53 which will arrest the cell cycle in G1 via p21 (WAF1/Cip 1), inhibitor of cyclin-dependent kinases. Recently, Ihling et al. demonstrated p53 and p21 in human atherosclerotic plaques [74]. Induction of apoptosis via p53/p21 is less well understood but can be activated both by transactivation and transactivation-independent pathways. Recent evidence indicates that the Bcl-2 protein family can be involved. It was demonstrated that the pro-apoptotic protein Bax is upregulated in close association with NO [75].

The promotor site of the Bax gene contains a p53 binding domain [59]. Therefore, p53 could act as a sensor for DNA damage that could arrest the cell cycle for DNA repair or upregulating pro-apoptotic factors resulting in increased susceptibility for apoptosis. A recent in vivo study in apoE knockout mice demonstrated that p53 is essential for the cell cycle control (G1 arrest) but not for the induction of apoptotic cell death in atherosclerosis [76]. In this study it was demonstrated that the increased plaque size in apoE-/-p53-/-mice compared to apoE-/-mice was mainly a consequence of increased cell replication of the macrophages in the plaques. The accelerated proliferation rate in the plaques of these apoE-/-p53-/-mice may be mediated at least partly by the loss of the p53-dependent G1 checkpoint control and the ongoing apoptotic cell death points to p53-independent apoptosis pathways in atherosclerosis (Fig. 3).

5.2 PARP and NO-induced DNA repair and apoptosis

DNA damage will induce attachment of poly(ADP ribose) polymerase (PARP) to the strand breaks and extensive synthesis of short-lived polymers by the bound polymerase [77,78]. Although PARP has no direct role in DNA excision repair, the enzyme binds tightly to the DNA strand breaks and repair can be suppressed if PARP synthesis is prevented [78]. Massive PARP activation following extensive DNA damage upon exposure to peroxynitrite leads to depletion of NAD+, the ADP ribose donor [79]. In an effort to resynthesize NAD+, ATP becomes depleted which ultimately leads to cell death due to energy deprivation. Moreover, inhibition of mitochondrial respiration (thereby affecting ATP synthesis) via destruction of Fe–S clusters has been noted [80]. Initially it was stated that it is unlikely that energy depletion due to PARP activation is a general pathway of this NO-mediated apoptotic cell death, since apoptotic cell death is an energy-requiring process and PARP is cleaved by caspases in the early execution phase of apoptotic cell death. However, recent findings indicate that caspases, a family of cysteine proteases that execute the cell death program, can be inactivated by a nitrosylation step [81–83]. This was demonstrated for endothelial cells [41,42] but more recently it was demonstrated that it could be a feature of NOS expressing cells [83]. This could indicate that NOS expressing cells, like macrophages in atherosclerotic plaques, do not cleave PARP and other DNA repair proteins since their caspases are inactivated (Fig. 3). Therefore, the macrophages continuously repair their DNA damage. This could be an explanation for the presence of the high levels of DNA synthesis in macrophages in human and experimental atherosclerotic plaques. Moreover, it was demonstrated that NOS expressing cells can denitrosylate and reactivate their caspases when their Fas apoptotic pathway is activated [83]. If the nitrosylation–denitrosylation hypothesis can be demonstrated in vivo in atherosclerotic plaques new pharmacological tools can be developed to modulate apoptosis in the plaques.

5.3 Apoptosis of macrophages in the plaques: beneficial or detrimental?

As already explained in the previous paragraph macrophages are responsible for collagen breakdown in the plaque. Loss of macrophages will lead to less metalloproteinase activity and to decreased collagen breakdown. This could lead to plaque stabilization and, therefore, decrease the risk for plaque rupture. However, the situation is not that simple. In the normal development and in C. elegans apoptosis is followed by engulfment of the apoptotic bodies by neighbouring cells. A loss of macrophages will lead to a decrease in scavenging of apoptotic bodies. Accumulation of apoptotic bodies could lead to complement and thrombin activation. The importance of scavenging will be discussed in the next section.

6 Scavenging of apoptotic bodies in atherosclerotic plaques

Recognition and uptake of dying cells is an important component of the apoptotic process, preventing the release of toxic intracellular contents and the formation of an inflammatory infiltrate. The demonstration of apoptosis in atherosclerotic plaques might reflect a failure of the uptake mechanism, which could contribute to the progressive accumulation of inflammatory cells in the lesions. The mechanism by which apoptotic cells are recognized and phagocytized are incompletely understood and appear to vary according to the cell type.

6.1 Which cells and factors are involved in the uptake of apoptotic bodies in the plaque?

Although most of the apoptotic bodies are probably phagocytized by macrophages, it is possible that the SMC can also ingest apoptotic bodies. For the macrophages it was demonstrated that the scavenger receptors and CD-36 are involved in the recognition and uptake of apoptotic bodies [84,85]. Besides their high affinity for acetylated and oxidized LDL [86], it was demonstrated that the scavenger receptor might recognize any damaged cell by virtue of its oxidized cell membranes, which might contain domains analogues to those found in Ox-LDL [86,87]. In a global sense, scavenger receptors might represent a means of removing oxidatively damaged components which could otherwise injure surrounding tissues [88]. There is evidence that oxidative damage is a component of the apoptotic program [89]. Krieger et al. [90] have reviewed the structure and binding properties of macrophage scavenger receptors. In addition to Ox-LDL and Ac-LDL, the receptors have been shown to bind polyribonucleotides, polysaccharides, anionic phospholipids including phosphatidylserine. This is important since it has been shown that apoptotic cells expose PS on the external leaflet of their plasma membrane. This PS, which is normally sequestered in the internal leaflet of the plasma, may be recognized by the macrophages via the scavenger receptors. As illustrated in Fig. 4 if the apoptotic bodies are cleared before damage to the plasma membranes is present, a stable fibrous plaque will form with a low thrombogenicity. However, if apoptotic SMC or macrophages are not engulfed a necrotic core with high thrombogenicity will form. This indicates that scavenging of apoptotic bodies in atherosclerotic plaques could determine if a plaque will be stable or unstable [88].

Fig. 4

Scavenging of apoptotic bodies is crucial for understanding the development of necrotic cores in atherosclerosis. Apoptotic bodies of SMC and macrophages expose phosphatidylserine (PS) on their surface which is recognized by scavenger receptor A (SRA) or CD 36. Increased apoptosis of SMC or macrophages or a decrease in the scavenging activity could lead to thrombin activation via the exposed PS.

Lack of scavenging of apoptotic bodies in atherosclerotic plaques could also be important for the further influx of mononuclear cells in the plaque. This is suggested by the finding that during tissue remodeling in embryonic development regions with excessive apoptosis show upregulation of the Endothelial Monocyte-activating Polypeptide II (EMAP-II). Mature EMAP-II can attract mono and polymorphonuclear cells to the site of inflammation and can induce tissue factor in endothelial cells [91,92]. This is another argument that excessive apoptosis could transform the stable plaque in an unstable plaque with high thrombogenicity.

7 Decreased scavenging activity of the macrophages in plaques?

It was demonstrated that macrophages that have phagocytized erythrocytes become deficient for their phagocytotic activity [93]. This is relevant for understanding the significance of apoptosis in atherosclerosis since we have recently demonstrated that macrophages in advanced human atherosclerotic plaques show signs of platelet and erythrophagocytosis [48]. This could indicate that macrophages in advanced human atherosclerotic plaques become deficient for the scavenging of apoptotic bodies. Therefore, apoptotic bodies in these regions are not adequately removed leading to the accumulation of necrotic debris in some regions of the plaque. This is exactly what is seen in some regions of atherosclerotic plaques. The formation of the necrotic core could be the result of both an increased apoptosis and a lack of scavenging by the remaining cells (Fig. 4).

7.1 Apoptosis and thrombin activation/tissue factor activity

It was demonstrated that induction of apoptosis in endothelial cells is associated with increased expression phosphatidylserine and the loss of anticoagulant membrane components [94].

Moreover, apoptotic cells show an increased tissue factor activity. [95]. These findings indicate that increased apoptosis in the plaques could be responsible for the increased pro-coagulant status. It was indeed demonstrated that the tissue factor is mainly localized in the necrotic core of advanced atherosclerotic plaques [96]. Interestingly, Mallat et al. [97] have found that most of the extracellular tissue factor expression is located around apoptotic cells in the necrotic core of human atherosclerotic plaques. This suggests that tissue factor may be shed from apoptotic cells via apoptotic microparticles. It was also demonstrated that high levels of shed membrane particles of monocytic and lymphocytic origins are produced in atherosclerotic plaques that are associated with increased tissue factor activity. Therefore, increased apoptosis in the plaque could lead to an increase in the thrombogenicity in the plaques which could lead to plaque complications.

8 Effects of lipid lowering on apoptosis in atherosclerotic plaques

It was demonstrated in the past that the thickness of atherosclerotic plaques induced by giving rabbits a cholesterol supplement did not decrease after a period of cholesterol withdrawal. However, most of the macrophages disappear from the plaques after a period of cholesterol withdrawal and the plaques are transformed in fibrous plaques. These changes were also associated with a strong decrease in the metalloproteinase activity [98]. However, it is not clear if the macrophages after this aggressive lipid lowering disappear from the plaques by increased apoptosis or decreased accumulation, cell replication. This information is important for understanding the effects of aggressive lipid lowering since induction of excessive apoptosis could increase the thrombogenicity of the plaques, as discussed in the previous paragraphs.

In a recent study, we demonstrated that apoptotic cell death shows a steady decline after lipid lowering [67]. Moreover, the pro-apoptotic protein Bax, which is upregulated in both human and experimental atherosclerotic plaques, strongly decreased after lipid lowering. These changes indicate that apoptosis, and, also, the susceptibility of cells in the plaque to undergoing apoptosis, did not increase after lipid lowering, but showed a steady decline. Furthermore, it was demonstrated that the changes in cell composition of the plaques were mainly a consequence of a strong decrease of DNA synthesis of the macrophages. DNA synthesis of the smooth muscle cells did not decrease after lipid lowering. The smooth muscle remained present and lost its lipid accumulation, whereas the macrophages almost disappeared from the plaques. Interestingly, the atherocsclerotic plaques of the animals that remained on the cholesterol-rich diet showed the same characteristics as the plaques at 26 weeks, which excludes age-associated changes in the plaques. These changes indicated that lipid lowering did not decrease the thickness but increased the fibrous component of the plaques without increasing the thrombogenicity.

9 Conclusion: is apoptosis beneficial or detrimental in primary atherosclerosis?

Apoptosis of endothelial cells and smooth muscle cells is detrimental for plaque stability. Apoptosis of macrophages could be beneficial for plaque stability. Recent data, however, indicate that non-scavenged apoptotic macrophages remain in the plaque as shed microparticles which could be a potent source for tissue factor. Therefore, it is not clear if induction of macrophage apoptosis would be beneficial for plaque stability. Lipid lowering does not induce an increase of macrophage apoptosis but a decreased macrophage accumulation in the plaque. More studies are necessary to find out if selective induction of macrophage apoptosis could lead to the same result on plaque stability. This information is essential for understanding the effects of new apoptosis-modifying drugs on plaque stability.

Acknowledgements

This work was supported by a grant from the Flemish Fund for Scientific Research (FWO). M. Kockx received a fund for fundamental clinical research from the Flemish Fund for Scientific Research (FWO).

References

  1. [1]
  2. [2]
  3. [3]
  4. [4]
  5. [5]
  6. [6]
  7. [7]
  8. [8]
  9. [9]
  10. [10]
  11. [11]
  12. [12]
  13. [13]
  14. [14]
  15. [15]
  16. [16]
  17. [17]
  18. [18]
  19. [19]
  20. [20]
  21. [21]
  22. [22]
  23. [23]
  24. [24]
  25. [25]
  26. [26]
  27. [27]
  28. [28]
  29. [29]
  30. [30]
  31. [31]
  32. [32]
  33. [33]
  34. [34]
  35. [35]
  36. [36]
  37. [37]
  38. [38]
  39. [39]
  40. [40]
  41. [41]
  42. [42]
  43. [43]
  44. [44]
  45. [45]
  46. [46]
  47. [47]
  48. [48]
  49. [49]
  50. [50]
  51. [51]
  52. [52]
  53. [53]
  54. [54]
  55. [55]
  56. [56]
  57. [57]
  58. [58]
  59. [59]
  60. [60]
  61. [61]
  62. [62]
  63. [63]
  64. [64]
  65. [65]
  66. [66]
  67. [67]
  68. [68]
  69. [69]
  70. [70]
  71. [71]
  72. [72]
  73. [73]
  74. [74]
  75. [75]
  76. [76]
  77. [77]
  78. [78]
  79. [79]
  80. [80]
  81. [81]
  82. [82]
  83. [83]
  84. [84]
  85. [85]
  86. [86]
  87. [87]
  88. [88]
  89. [89]
  90. [90]
  91. [91]
  92. [92]
  93. [93]
  94. [94]
  95. [95]
  96. [96]
  97. [97]
  98. [98]
View Abstract