Apoptosis and atherosclerosis
Apoptosis constitutes basic characteristic of vessels remodeling that takes place in organogenesis and in pathological situations like injury and atherosclerosis. The significance of apoptosis in atherosclerosis depends on the stage of the plaque, localization and the cell types involved. Apoptosis of vascular smooth muscle cells (SMC), endothelial cells and macrophages may promote plaque growth and pro-coagulation and may induce rupture, the major consequence of atherosclerosis in humans. Apoptosis of macrophages is mainly present in regions showing signs of DNA synthesis/repair. SMC apoptosis is mainly present in less cellular regions and is not associated with DNA synthesis/repair. Even in the early stages of atherosclerosis SMC become susceptible to apoptosis since they increase different pro-apoptotic factors. Moreover, recent data indicate that SMC may be killed by activated macrophages. The loss of the SMC can be detrimental for plaque stability since most of the interstitial collagen fibres, which are important for the tensile strength of the fibrous cap, are produced by SMC. Rupture of atherosclerotic plaques is associated with a thinning of the SMC-rich fibrous cap overlying the core. Rupture occurs particularly at the plaque shoulders, which exhibit lack of SMCs and the presence of inflammatory cells. Apoptotic SMCs are evident in advanced human plaques including the shoulders regions, prompting the suggestion that SMC apoptosis may hasten plaque rupture. Indeed, increased SMC apoptosis occurs in unstable versus stable angina lesions. Apoptosis of macrophages could be beneficial for plaque stability if apoptotic bodies were removed. Apoptotic cells that are not scavenged in the plaque activate thrombin, which could further induce intraplaque thrombosis (Kockx & Knaapen, 2000). Most apoptotic cells in advanced lesions are macrophages next to the lipid core. Loss of macrophages from atherosclerotic lesions would be predicted to promote plaque stability rather than rupture, since macrophages can promote SMC apoptosis by both direct interactions and by release of cytokines (Bennett, 2002). It can be concluded that apoptosis in primary atherosclerosis is detrimental since it could lead to plaque rupture and thrombosis.
Apoptosis and ischaemia/infarction
Cardiac myocyte death during ischemic injury has been thought to occur exclusively by necrosis, but recently several studies have demonstrated that large numbers of myocytes undergo apoptosis in response to ischemic disorders (Saraste et al., 1997). In humans, apoptosis seems to occur primarily in the border zone of the ischemic region and, according to some studies, in the remote from ischemia regions. However, in vivo animal studies have demonstrated apoptosis both in the ischemic region and the ischemic border zone. Apoptosis of cardiomyocytes occurs in a temporally and spatially specific manner. The central, unperfused region also manifests apoptosis, particularly within the first six hours, although between 6-24 hours necrosis is more common (Bennett, 2002). In contrast, in some studies ischemia caused apoptosis in the ischemic region alone, whereas reperfusion caused a decrease in apoptotic cells in the ischemic region and an increase in apoptotic cells in the ischemic border zone and the remote from ischemia regions. These differences theoretically could be explained by the different methods of measuring apoptosis that were used (Krijnen et al., 2002). Apoptosis in the remote non-infarcted myocardium may be partly responsible for myocardial remodelling and dilatation after myocardial infarction, and may be amenable to treatment. Apoptosis is a highly regulated process in which several regulatory proteins play a significant part. P53 limits cellular proliferation by inducing cell cycle arrest and apoptosis in response to cellular stresses such as DNA damage, hypoxia, and oncogene activation. P53 mediates apoptosis through a linear pathway involving bax activation, cytochrome c release from mitochondria, and caspase activation (Shen & White, 2001). The Bcl-2 family of proteins constitutes a critical checkpoint in cell death. These proteins contain agonists and antagonists of apoptosis and alterations of their ratio determine the life or death of a cell (Anversa & Kajstura, 1998). Proapoptotic proteins include Bax, Bak, Bad, and Bcl-xs whereas Bcl-2 and Bcl-xL are antiapoptotic. Several studies have demonstrated that Bcl-2 protein is induced in salvaged myocytes surrounding infracted areas in the regions at risk in acute stage of infraction. Bcl-2 positive myocytes were not seen in the infracted myocytes in the heart with acute infraction. Bcl-2 positive immunoreactivity was not evident in salvaged myocytes of hearts with old infraction, in chronic ischemic disease or in normal hearts. P53 and Bax protein expression was rare in salvaged myocytes within the risk area at the acute stage of infraction. P53 and Bax positive immunoractivity was evident in the infracted myocytes. P53 protein is induced in salvaged myocytes at the old stage of infraction and in chronic ischemic disease. P53 positive immunoreactivity of normal control heart tissue was slight in most of myocytes. Myocytes exposed to various stress, such as chronic ischemia (salvaged myocytes at the old stage of infraction, myocytes at chronic ischemic disease) induced the overexpression of p53 protein (Tsipis et al., 2007). Consequently, the expression of bcl-2 or P53 protein in myocytes of human hearts with infarction may play an important role in the protection or the acceleration of cellular damage after infarction (Figure 1, Figure 2).
Apoptosis and heart failure
Congestive heart failure occurs as a late manifestation in diverse cardiovascular diseases characterized by volume or pressure overload and significant loss of contractile muscle mass. Cardiac output is initially maintained in these disorders by the development of compensatory myocardial hypertrophy and dilatation. However, the early mechanical adaptations to growth stimulus soon fall short of adequate compensation. The mechanism by which compensatory response triggered by myocardial failure culminates in myocardial dysfunction is not clear.
Fig. 1. Immunohistochemical staining for Bcl-2 in myocardial infarction (X400).
Fig. 2. Immunohistochemical staining for P53 in myocardial infarction (X400).
In the past few years, several scientists have proposed apoptosis as the basis of the inexorable decline in ventricular systolic function (Narula et al., 2000). Although the initial studies reported unrealistically high levels of apoptosis in failed heart (as much as 35%), recent studies show an apoptosis rates of <1% (TUNEL-positive cells) during heart failure (Kang & Izumo, 2000). Because of the limitations with TUNEL staining and the difficulties in interpreting these findings, the use of TUNEL alone to detect the presence of apoptosis is not sufficient to define the role of apoptosis in heart failure. Later studies have consistently shown that approximately 80-250 heart muscle cells per 105 cardiac nuclei commit suicide at any given time in patients with late-stage dilated cardiomyopathy. In contrast, the base-line rate of apoptosis in healthy human hearts is only one to ten cardiac myocytes per 105 nuclei (Anversa & Kajstura, 1998; Yue et al., 1998). Even though the rate of apoptosis in heart failure is relatively low in absolute numbers, it is significantly higher than that in the normal heart, which has essentially negligible baseline apoptosis. Recently, animal models of heart failure incorporating transgenic technology have confirmed that very low levels of myocyte apoptosis, levels that are four- to tenfold lower than those seen in human heart failure, are sufficient to cause a lethal, dilated cardiomyopathy (Wencker et al., 2003).
It has been long believed that apoptosis does not occur in terminally differentiated cells such as adult cardiomyocytes. However, all mechanisms responsible for induction of apoptosis are operative in myocytes and are particularly activated during heart failure. Actually, the onset of myocardial failure leads to systemic and myocardial neurohumoral alterations and cytokine expression to maintain cardiac output. Upregulation of these adaptive responses also induces growth response and leads to compensatory myocardial hypertrophy and dilatation. Cardiac myocytes differentiate and withdraw from the cell cycle during the neonatal period, and persistent growth stimulus in the adult myocardium (such as that in heart failure) is perceived as a contradictory genetic demand, and programmed cell death occurs (Narula et al., 2000). P53 (tumor suppressor protein) is involved in the regulation of cell cycle progression in response to DNA damage. This p53 typically causes the cell to delay its entry into S phase until the damage has been repaired. P53 also is involved in triggering an apoptotic response in instances in which the damage is too severe to repair. P53 is a transcriptional regulator of the bcl-2 and bax genes. P53 mediates apoptosis through a linear pathway involving bax transactivation, Bax translocation from the cytosol to membranes, cytochrome c release from mitochondria, and caspase-9 activation, followed by the activation of caspase-3, -6, and -7 (Kim et al., 1994; Shen & White, 2001). P53 down-regulates the antiapoptotic gene product Bcl-2 and up-regulates the proapoptotic gene product Bax. Immunohistochemistry of p53 and antiapoptotic Bcl-2 protein demonstrated higher levels of both of these proteins in heart failure as compared with normal hearts (Figure 3, Figure 4). Tsipis et al. have observed that the percentage of p53- and bcl-2 positive samples in the end-stage dilated cardiomyopathy was 100% (20/20 diseased group samples). A 2- and 2.5-fold increase in p53 and bcl-2 positive samples was observed in the diseased group as compared with the control group. The diseased group had a larger number of samples with strong p53 staining as compared with the control group, which demonstrated weak p53 staining. Bcl-2 staining in the positive samples of the diseased group was generally weak as in the control group (Tsipis et al., 2010). Latif and colleagues, in a quantitation of the bcl-2 family of proteins after Western Immunoprobing, demonstrated a 2.9- and 5.35-fold increase in the levels of Bax and of Bcl-2, respectively, in patients with dilated cardiomyopathy (Latif et al., 2000).
Fig. 3. Immunohistochemical staining for p53 protein in dilated cardiomyopathy (X400).
Fig. 4. Immunohistochemical staining for bcl-2 in dilated cardiomyopathy (X400).
Narula and colleagues demonstrated a release of cytochrome c from mitochondria in patients with heart failure (Narula et al., 2000). In the study of Tsipis et .al, increased expression of P53 protein was seen, but p53 up-regulates the proapoptotic gene product Bax (Tsipis et al., 2010). Elevated levels of Bax and Bak may mediate the release of cytochrome c, as it has been demonstrated that Bax and Bak accelerate the opening of the voltage-dependent anion channel (Latif et al., 2000; Shimizu et al., 1999). These results suggest that increased expression of p53 may be associated with apoptosis in heart failure of end-stage dilated cardiomyopathy. Moreover, various factors present in the failing myocardium have been shown to stimulate apoptosis in cardiac myocytes. Such factors include inflammatory cytokines, reactive oxygen species, nitric oxide, hypoxia, reperfusion, growth factors, and mechanical stretch (Foo et al., 2005). Ventricular decompensation and failure impose an elevated diastolic load on myocytes, resulting in stretching of sarcomeres and the stimulation of multiple second messenger systems which have been linked to the initiation of myocyte reactive hypertrophy in the pathologic heart. Abnormal levels of resting tension may lead to the local release of angiotensin II (Ang II) and the induction of programmed cell death in the myocardium. Sarcomere elongation in vitro results in Ang II release and activation of p53 and p53-dependent genes (Leri et al., 1998). Moreover, overstretching appears to be coupled with oxidant stress, expression of Fas, programmed cell death, architectural rearrangement of myocytes, and impairment in force development of the myocardium (Cheng et al., 1995). Using Western blotting, Olivetti et al. demonstrated a 2.4-fold increase in bcl-2 in patients with heart failure (Olivetti et al., 1997). However, the expression of Bax protein was not altered in the diseased group. This low expression of Bax protein may represent the prevalence of bcl-2 compensatory mechanism. The elevated presence of p53-positive cells, as demonstrated by immunohistochemistry, suggest that apoptosis may be significantly higher in dilated cardiomyopathy than that in the normal heart. On the other hand, increased expression of the antiapoptotic protein bcl-2 in human myocardium with dilated cardiomyopathy may be a compensation for the loss of myocytes and a possible compensatory antiapoptotic mechanism in the diseased group (Tsipis et al., 2010). In conclusion the etiology of heart failure in dilated cardiomyopathy involves multiple agents. The heart failure involves not only the contractile dysfunction, but also the progressive loss of myocytes by apoptosis. The elevated expression of proapoptotic is associated with progressive loss of myocytes in heart failure, and the increased expression of antiapoptotic proteins represent a possible compensatory mechanism. The prevalence of the apoptotic mechanism or this of compensatory antiapoptotic may influence the evolution of heart failure in cardiomyopathy.
Conclusions
Cells are poised between survival and apoptosis, and their fate rests on a balance of powerful intracellular and extracellular forces, whose signals constantly act upon and counteract each other. In many circumstances, apoptosis is a self-protective programmed mechanism that leads to the suicide of a cell when its survival is deemed detrimental to the organism. In other instances, apoptosis is a pathological process that contributes to many disorders. Thus, the pharmacological manipulation of apoptosis represents an active frontier of drug development. Recognition of the inducing mechanisms of apoptosis could open up ways to inhibit cell death in cardiovascular tissues and possibly help to define targets for future drug design. Furthermore, end-stage events of apoptosis, such as the activation of downstream caspases are essentially uniform in all cell types; although some regulatory mechanisms may be unique to cells in cardiovascular tissues. Elucidation of proapoptotic and antiapoptotic mechanisms in cardiomyocytes and vascular smooth muscle cells could delineate potential targets for intervention. In conclusion, various factors present in the diseased myocardium have been shown to stimulate apoptosis in cardiac myocytes. Changes in the induction of genes promoting or opposing apoptosis may modulate the total amount of myocyte damage. There is still a need to clarify the role played by different genetic and environmental factors implicated in cell death or survival.
