- Email: [email protected]
Apoptosis: a potential target for discovering novel therapies for cardiovascular diseases
474
Apoptosis: a potential target for discovering novel therapies cardiovascular diseases Tian-Li Yue, Eliot H Ohlstein and Robert R Ruffolo Jr* The realization
that apoptosis
is genetically
programmed
the exciting prospect that modulating apoptosis novel approaches for treatment of cardiovascular which
apoptosis
weight kinases
has been
inhibitors of caspases have been evaluated,
of cardiovascular
apoptotic
demonstrated.
Low
raises
may provide diseases in molecular
and mitogen-activated with promising results
protein in a variety
models.
Addresses Department of Cardiovascular Pharmacology, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406-0939, USA, Biological Sciences, UW-2523, SmithKline Beecham Pharmaceuticals, 709 Swedeland Road, Box 1539, King of Prussia, PA 19406-0939, *e-mail: Robert -- R [email protected]
Current
Opinion
in Chemical
Biology
1999,
USA;
3:474-480
http://biomednet.com/elecref/1367593100300474 0 Elsevier
Science
Ltd
ISSN
1367-5931
Abbreviations CHF congestive cyto
DD DED ERK FADD JNK MAPK TNF
c
heart failure cytochrome c death domain death effector domain extracellular signal regulated kinase Fas-associating protein with DD c-Jun amino-terminal kinase mitogen-activated protein kinase tumor necrosis factor
Introduction Apoptosis is a process of programmed cell death that was initially defined by morphological characteristics, including cell shrinkage, membrane blebbing, chromatin condensation and nuclear fragmentation [I]. The field of apoptosis is unusual in several respects. First, the general importance of apoptosis ha; been widely recognized only in the past decade, and its significance is currently being evaluated in many areas of biology. A substantial body of literature has been generated over the past 10 years, and the rate at which data are accumulating in this area is increasing exponentially. Thus, the number of publications per year related to apoptosis in cardiovascular disease has increased from one in 1991 to over 300 in 1998. Moreover, situations in which apoptosis plays a critical role are diverse, and virtually all fields of biology show a growing interest in apoptosis. Apoptosis is controlled by an evolutionarily conserved program and acts in embryonic development and tissue homeostasis, and can also be induced by various pathological insults. The uniform morphological features observed in apoptotic cells from different organisms suggest that a common mechanism may operate to trigger programmed cell death. Apoptosis in both excessive and reduced
for
amounts may have pathological implications (for example, apoptosis may be involved in a number of neurodegenerative diseases such as Parkinson’s disease; reduced amounts may lead to cancer). Our increased understanding that apoptosis is controlled by genes raises the exciting prospect that modulating apoptosis may provide a novel approach to the discovery of new drugs for the treatment of a variety of diseases [Z’]. In this review, we will focus on the role that apoptosis plays in cardiovascular diseases, and will highlight some mechanisms, mainly the signal transduction network and the execution machinery, that have been reported to be modulated by pharmacological agents.
Biochemical
and genetic
control
of apoptosis
As a general summary, apoptotic pathways involve a sensor that detects a death-inducing signal, a signal transduction network, and the execution machinery that actively carries out the process of cell death. The apoptotic process can be divided into three functionally distinct phases: the induction phase - a change in the cellular environment leading to the cell activating the mechanism of apoptosis through receptor and signal transduction mechanisms; the execution phase - the processes within the cell that result in committal to apoptotic cell death; and the degradation phase - the events associated with the final disposal of the cell corpse, leading the cell past a ‘point of no return’. It is likely that apoptosis will be controlled in a cell-typespecific fashion, but the basic elements of the death machinery may be universal. With many of the regulators of apoptosis identified, it is now possible to begin to define the functional relationship between them.
Signaling pathways that transduce extracellular death signals Death-receptor-mediated
signal
transduction
The best characterized pathways for the initiation of apoptosis involve those initiated by death receptors such as Fas/CD95 and the tumor necrosis factor (TNF) receptor (TNFR) family, most of which contain a death domain (DD) in their cytoplasmic region (Figure 1); [3”]. The death receptors interact via their DD with intracellular DD-containing adapters, such as FADD (Fas-associating protein with DD) and TRADD (TNF receptor-associated death domain), and recruit these adapters to the cell membrane. Thus, binding of Fas ligand to the Fas receptor leads to clustering of the Fas receptor’s DD. The adapter, FADD, then binds through its own DD to the clustered receptor DD. FADD also contains a ‘death effector domain’ (DED) that binds to an analogous domain within the procaspase-8 protein. Upon recruitment by FADD, pro-caspase-8oligomerization drives its own activation (to caspase-8)through self-cleavage (see Figure 1). Caspase-8then activates downstream effector
Apoptosis
and cardiovascular
diseases
Yue,
Ohlstein
and
Ruffolo
475
Figure 1
A simplified schematic diagram of the apoptotic death pathways. Death receptor (e.g. Fas) ligation results in formation of a signaling complex that includes the receptor, adaptor (e.g. FADD) and procaspase-8. The interaction between the intracellular domain of Fas and FADD is mediated via dimerization of two homologous regions in the two proteins, the DD. FADD, in turn, associates with the proenzyme form of caspase-8 through dimerization of a domain known as the DED; a similar DED region occurs in procaspase-8. Upon recruitment by FADD, procaspase oligomerization drives its own activation through self-cleavage (Asp-x indicates the cleavage site in procaspase-8). Caspase-8 then activates downstream effector caspases such as caspase-3. Activation of procaspase-9 requires multiple factors, including adaptor Apaf-1, dATP and cyto c. The cyto c is released from mitochondria in response to a variety of apoptotic stimuli, such as oxidants, calcium, ceramide and
pro-apoptotic Bcl-2 family members (e.g. Bax, Bik), while cyto c release can be inhibited by anti-apoptotic Bcl-2 family members Bcl2 and Bcl-XL, which also inhibit the association of Apaf-1 with procaspaser9, therefore preventing activation of caspase-9. Caspase-9 activates downstream effector caspases (e.g. caspase-3) thereby initiating apoptosis. The MAPK cascades comprising MEKK (MAPK kinase kinase), MKK (MAPK kinase) and MAPK are activated by external stress stimuli and result in phosphorylation of transcription factors (e.g. ATF2, c-Jun) and initiate transcription of downstream effecters. The direct relationship between MAPKs and activation of caspases is not clear. The red arrows indicate the potential targets for pharmacological intervention. Dashed lines indicate that the direct relationship between activation of caspase-3 and activation of transcription factors by MAPKs is not clearly established at this time.
caspases, such as caspase-3, thereby initiating apoptosis. It is believed that this signal transduction process is an important mechanism for Fas-ligandand TNFa-induced activation of caspases, and therefore cell death. The existence of functional TNFRl in the human heart and the elevated levels of TNFcx and soluble Fas in patients with congestive heart failure have been reported recently [4].
serine and threonine residues on certain proteins) that are activated by dual phosphorylation on threonine and tyrosine residues in response to a wide array of extracellular stimuli [5,6”]. The best-characterized subfamilies of the MAPK superfamily are the two ‘stress-responsive’ MAPK subfamilies, namely, c-Jun amino-terminal kinase/stress-activated protein kinase (JNK/SAPK) and ~3%MAPK (Figure l), and the extracellular signal regulated kinases (ERKs). In mammalian cells, the parallel kinase cascades for the three MAPK subfamily members have been described, which comprise a MAPK kinase kinase (MEKl), that phosphorylates and
Mitogen-activated protein kinase pathway Mitogen-activated protein kinases (MAPKs) are prolinedirected serine/threonine kinases (i.e. they phosphorylate
476
Next
generation
therapeutics
activates a MAPK kinase (MKK) which in turn phosphorylams and activates a MAPK (JNK, p38 and ERK). The JNK and p38 pathways are activated by a variety of external stress stimuli, such as reactive oxygen species, UV irradiation, ceramide and cytokines, and the ERK pathway can be activated by growth factors. Signal transduction via MAPKs results in phosphorylation of inducible transcription factors, such as c-Jun and ATFZ, which then act to initiate transcription of downstream effecters. Most recent in vitro and in vtio studieshave demonstratedthat JNK and ~38 are implicated in cardiac remodeling (cardiomyocyte apoptosisand hypertrophy) [7]. Interestingly, cardiomyocyte hypertrophy and apoptosiscan be induced by distinct members of the p38 family, suggestinga significant role for p38 signaling in the pathophysiology of congestive heart failure (CHF) [S]. It is clear from recent studies that MAPK signalingpathways are more complex than previously expected. This complexity provides an opportunity for selectivity within a given cell type or following a particular challenge.
The effector
machinery
of apoptosis
At its simplest, the apoptotic machinery includes three basic components: death proteases (caspases), CED4/Apaf-1 adapter molecules, and Bcl-2 family members. Caspasesrepresent the executionary arm of the apoptotic machinery. CED-4/Apaf-1 appears to play critical roles in the conversion of pro-caspasesinto active caspasesthrough direct physical interactions. The Bcl-2 family includes both pro- and anti-apoptotic members that can regulate each other to shift the balance from a pro-apoptotic environment to an anti-apoptotic environment, and vice versa. Caspases
A family of cysteine proteases known as caspasesis believed to be a critical component of the cell death machinery [9”]. To date, more than 14 caspaseshave been cloned and partially characterized in mammals,and many, but not all, have been implicated in the apoptotic process. They are synthesized in the cell asinactive precursors comprising four distinct domains (i.e. an amino-terminal domain, a large subunit [-20 kDa], a small subunit [-lo kDa] and a linker region between the large and small domains flanked by an aspartate residue). Activation of each caspase is induced by proteolytic cleavage between domains, resulting in the removal of the prodomain and linker regions, and assembly of the large and small subunits into an active enzyme complex. The mammalian cell death caspaseshave been divided into initiators and effecters, and two main caspasecascades have been delineated in mammalian cells (Figure 1) [lo’]. Activation of pro-caspase-8 requires association with FADD through the DED, as described previously, while pro-caspase-9activation requires multiple factors including adapter Apaf-1, cytochrome c and dATP (Figure 1). Different initiator caspasesmediate distinct sets of signals. For example, caspase-8is associated with apoptosis involving Fas receptor, and caspase-9 is involved in death induced by cytotoxic agents. A
pro-apoptotic signal culminates in activation of an initiator caspase which, in turn, activates effector caspases such as caspase-3,resulting in cellular disassembly.This model explains how distinct apoptotic signals induce the same biochemical and morphological changes that are characteristic of apoptosis. One potential mechanism to control activation of these initiator caspasesmay involve inhibition of the interaction between pro-caspasesand their activators. The role of caspases in mammalian apoptosis is complex in that multiple caspasesmay have redundant functions or act in concert to execute the apoptotic process in a cell-specific and stimulus-dependent manner. It has been demonstrated that caspasesare expressed in cardiovascular tissuesand implicated in cardiac apoptotic processes[ ll’,l’Z’]. Cytochrome
c as a regulator
of caspase
activation
The discovery that cytochrome c (cyto c), a key component of the mitochondrial electron transport system, is required for the activation of caspase-3wasunexpected, but provides a new framework in which to understand caspaseactivation in mammaliancells [13”,14]. Cyto c is releasedfrom mitochondria in responseto a variety of apoptotic stimuli, such as reactive oxygen radicals, calcium and ceramide. In the cytosol, cyto c can bind Apaf-1, and in the presenceof dATP or ATP, Apaf-1 adopts a conformation that enablesit to bind to, and thus promote activation of pro-caspase-9,which then processesand activates other caspases to orchestratethe biochemical execution of cells (seeFigure 1). Bcl-2 family
proteins
The Bcl-2 family of proteins, comprising at least 16 in humans, serves ascritical regulators of pathways involved in apoptosis [15]. The family contains both pro-survival members, such as Bcl-2 and B&XL, and pro-apoptotic members, such as Bax and Bik. Pro- and anti-apoptotic family members can heterodimerize and in sodoing, they can regulate the function of each other in order to change the balance between pro-apoptotic and anti-apoptotic environments. Two major mechanisms have been suggested for pro-survival proteins: maintenance of organelle integrity and prevention of the release from mitochondria of cyto r, and inhibition of the associationof Apaf-1 with pro-caspase-9,thereby preventing caspase-9 activation (see Figure 1). Pro-apoptotic members are thought to act through displacing the adapters from the pro-survival proteins. It has recently been suggestedthat Bax and Bax-like proteins may mediate caspase-independent death via channel-forming activity, which could promote the mitochondrial permability transition or puncture the mitochondrial outer membrane, thereby initiating apoptosis.
Apoptosis
in cardiovascular
diseases
Apoptosis in the cardiovascular system has been demonstrated only recently; however, evidence of apoptosis has been reported from diverse aspectsof cardiovascular medicine, ranging from CHF to conduction system defects to
Apoptosis
coronary atherosclerosis [ 16,17”,18’]. Table 1 lists the cardiovascular diseases associated with apoptosis. Studies in cultured cardiomyocytes Apoptosis has been demonstrated in cultured rat cardiomyocytes under conditions similar as those thought to occur in the failing hearts of patients (for example, ischemia, mechanical stretch forces, viral infection, and in the presence of oxygen free radicals and cytokines such as TNFa). Expression of pro-apoptotic genes (e.g. ~53, Fas, Bad and Bax) and anti-apoptotic genes (e.g. Bed-2) have been observed in apoptotic cardiomyocytes [191. Activation of the MAPK signaling pathways and caspases have also been demonstrated in myocardial cells undergoing apoptosis. Of particular interest, manipulation by MAPK or caspase inhibitors enables modulation of cell death induced by a variety of insults [ll’,ZO-221. Experimental studies in animals with heart failure or ischemic injury Several lines of investigation now clearly indicate that apoptosis can be elicited in cardiomyocytes in viva in response to different forms of cellular injury similar to those involved in the pathogenesis and progression of CHE Thus, animal models in which the loss of cardiomyby apoptosis ocytes is caused include multiple intracoronary microembolization-induced heart failure and rapid electrical-pacing-induced dilated cardiomyopathy in the canine, hypertension-induced heart failure in rats, and acute ischemia/reperfusion-induced cardiac injury in the rat and rabbit [16,17”]. In these studies, the induction of cardiomyocyte apoptosis has been clearly demonstrated. The appearance of apoptosis is a significant factor in myocyte death in these models, with a number of common features, such as early onset (1-3 h), a large variation in prevalence and distribution of apoptotic cells, and a clear dissociation between apoptosis and necrosis. The association of myocyte apoptosis with pro-apoptotic genes (Fas and Bax), and activation of stress-responsive MAPKs (JNK and p38), as well as caspases, have been demonstrated. The modulation by pharmacological agents of cardiac apoptosis in animal models with ischemic cardiac injury has also been observed [12’,23].
Apoptosis in patients with congestive heart failure or following myocardial infarction Evidence now also exists to indicate that apoptosis occurs in the hearts of patients with cardiomyopathies [24,25]. The occurrence of apoptosis in patients with CHF suggests that apoptosis may play a role in the progression of the disease and the chronic remodeling of the myocardium that occurs in heart failure. Apoptosis is a relatively infrequent event in hearc failure patients with ischemic cardiomyopathy, but is abundant in idiopathic dilated cardiomyopathy. Several reports have documented the occurrence of apoptosis in human heart specimens obtained from patients who died following myocardial infarction. In all studies, specimens of
and cardiovascular
diseases
Yue, Ohlstein
and Ruffolo
477
Table 1 Cardiovascular Cardiac
development Congenital
atrioventricular
with apoptosis
in humans.
block
overload
Cardiac
and heart failure Dilated cardiomyopathy lschemic cardiomyopathy Arrhythmogenic right ventricular
Acute
dysplasia
myocardiac infarction Myocarditis Cardiac allograft rejection Pre-excitation syndromes
Coronary
See
diseases associated
diseases Atherosclerosis
[17**,18*1.
myocardium following an acute myocardial infarction displayed features consistent with apoptosis. The expression of several genes associated with apoptosis, including Bd-2, Fas and Bax, were observed in human myocardium following myocardial infarction. In addition, it has also been reported that failing human hearts express elevated levels of caspase3 and TNFa. Moreover, the existence of functional TNFRl in human heart has been reported [4].
Apoptosis
in atherosclerosis
Multiple studies in both animals and humans have found apoptosis to occur in atherosclerotic coronary, carotid and aortic arteries, as well as saphenous vein grafts [ 18’1. These studies have observed that smooth muscle cells, principally located in the intimal fibrotic portion of the atherosclerotic plaque, and macrophages located in the intima, especially the lipid-laden core of the atheroma, show increased evidence of apoptosis compared with normal vessels. A significant number of cells undergoing apoptosis have been shown to be immunoreactive with anti-caspase-1 and -3 antiserum. Atherectomy specimens from restenotic lesions also showed evidence of apoptosis which strongly correlated with the presence of intimal hyperplasia. The death receptor, Fas, is expressed on as many as two thirds of the cells in the fibrous cap in human atherosclerotic lesions. Interestingly, isolated vascular smooth muscle cells from human atherosclerotic plaques were shown to have a higher propensity for apoptosis, and cytokines (such as TNFa) markedly sensitize cells to Fas-induced apoptosis. Recent studies have also suggested that oxidative mechanisms play a role in the apoptosis of vascular smooth muscle cells [ 17**]. It is still unclear whether apoptosis is a late finding, occurring only as part of the end stage of this disease, or whether increased apoptosis is associated with the early stages of atherogenesis. The major clinical implication of apoptotic cell death in atherosclerotic lesions is not clear. Apoptosis may result in reduced plaque stability, making the atheroma more susceptible to rupture thereby producing a
478
Next generation
Table
2
Pharmacological
therapeutics
manipulation
Model
Target
NRCM
Caspases ROS
NRCM
~38
NRCM
JNK and ~38 MAPK ERK
NRCM
Caspases
Rat
~38
rabbit
apoptosis. Apootosis
inducer
ZVAD-fmk NAC
lschemia
ET-l,
SB203580 Caspase-3 PE
PD98059
Staurosporine
in viva
Isc/rep
Rat heart
in viva
Isc/rep
IGF
lsclrep
Carvedilol
Rabbit
heart
in viva
JNK
ZVAD-fmk
of CM
I
1191
Apoptosis
of CM
-1
DOI
of CM
Apoptosis of CM no effect
SB203580
heart
Apoptosis
Apoptosis
ZVAD-fmk AC-YVAD-CHO
l&rep
References
El1
ET-l -induced ~38 and CM hypertrophyJ
SB202190
‘4202
MAPK
Results
Agent
TNFn, IL-1 8, IFN-y
MAPK
NRCM
Perfused
of myocardial
Apoptosis Cardiac heart Apoptosis
‘?
[301
I
[l 1‘I
of CM 1 functions
of CM J LVdpldt Infarction size 1
Apoptosis CK Apoptosis JNK
of CM loss 1
myocardial infarction. In addition to proteolysis, loss of smooth muscle cells in the fibrous cap of atherosclerotic lesions is known to predispose the lesions to plaque instability and therefore may increase the risk of unstable angina pectoris and acute myocardial infarction. In contrast, apoptosis may be beneficial by preventing the excessive cellular proliferation that occurs following balloon angioplasty or stent-induced vascular injury. Animal data have demonstrated that the enhanced cell proliferation is paralleled by increased susceptibility to apoptosis in the injured vessels, suggesting that apoptosis appears to be a major determinant of restenosis [18’].
Pharmacological cardiovascular
anti-ojcidant Current
The structure of carvedilol (1-[SH-carbazol-4-yloxyl-3-[(2(methoxyphenoxy)ethyl}aminoI-2-propanol). The chiral centre in the drug.
Opmon
star
,n Chem,cal
indicates
the
B~olagy
only
k-31 WI
manipulation apoptosis
kinase oxygen
l/2, MKKl12) species; SB203580
of
Inasmuch as apoptosis in the cardiovascular system has been demonstrated recently, low molecular weight inhibitors of caspasesand MAPKs have been evaluated in a variety of cardiovascular apoptotic models. Table 2 lists several examples. These studies target early processesof apoptosiswhere the cells have not passed‘the point of no return’. The results obtained to date are promising and indicate that cardiovascular apoptosiscan be modulated by pharmacological agents. Of particular interest is the finding that carvedilol (Figure 2; CoregB, SmithKline Beecham Pharmaceuticals, King of Prussia,Pennsylvania, USA), a new vasodilating P-adrenoceptor antagonist with strong antioxidant properties, has been demonstrated in clinical trials to dramatically reduce mortality in patients with CHF, and can also inhibit ischemia/reperfusioninduced apoptosis in cardiomyocytes in the rabbit. The possible mechanismsof carvedilol include down-regulation of the SAPK signaling pathway and Fas expression, and possibly alsoP-adrenoceptor blockade [26’].
Conclusions
H
[12’1
of CM I, Fas 1 L, Infarct size 1
cardiomyocyte; PD98059, MEKl/2 (MAPK inhibitor; PE, phenylephrine; ROS, reactive and SB202190, ~38 MAPK inhibitors.
2
1‘
1
AC, acetyl; CK, creatine kinase; CM, cardiomyocyte; ET-l, endothelin-1 ; fmk, (C-methyl)-CH,F; IFN-1: interferon-y; IGF, insulin-like growth factor; IL-l, interleukin-1 ; Isclrep, ischemia/reperfusion; LVdp/dt, first derivative of left ventricular pressure; NAC, N-acetylcysteine; NRCM, neonatal rat
Figure
[31'1 ?
and future
perspectives
Although end-stage events of apoptosis are likely to be essentially uniform in all cell types, someregulatory mechanismsmay be unique to the cells in cardiovasculartissues. Further elucidation of pro-apoptotic and anti-apoptotic mechanisms in cardiomyocytes and vascular cells could delineate potential targets for pharmacologicalintervention in a cell-type-specific fashion. This is particularly important
for chronic treatment with apoptotic modulators. For example, chronic treatment with a noncell-type-specific inhibitor of apoptosis may inhibit or slow all apoptosis throughout the body, which could have potentially serious consequences for the physiological processes dependent on apoptosis. Induction of apoptosis in locally proliferating vascular smooth muscle cells could represent a novel therapeutic approach for prevention of restenosis. In order to exploit the pathways involved in apoptosis in the heart pharmacologically, we must rely on a better understanding of the molecular mechanisms specifically responsible for the process of cardiovascular apoptosis. This fundamental information may underpin the basis for new strategies to exploit apoptotic mechanisms as novel drug targets. Initial attempts to reduce neointima formation by inducing smooth muscle cell apoptosis through adenoviral gene transfer have been promising in experimental models of vascular injury [27-291. Low molecular weight caspase inhibitors may represent a new class of drugs for the treatment of acute cardiovascular diseases in which apoptosis is believed to play a significant role. Our knowledge of the role of apoptosis in cardiovascular diseases is still limited. In this respect, several points appear to deserve further investigation: what is the true incidence of apoptosis in cardiovascular diseases; which endogenous and exogenous stimuli induce apoptosis in myocardial and vascular cells; and is apoptosis an early cause or a terminal event that is associated with cardiovascular diseases? To answer these questions, more sensitive and quantitative assays for detecting apoptosis in cardiovascular tissue are necessary. A better understanding of the role of apoptosis in cardiovascular diseases will probably lead to the discovery of additional targets for pharmacological intervention.
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Apoptosis: a potential target for discovering novel therapies cardiovascular diseases Tian-Li Yue, Eliot H Ohlstein and Robert R Ruffolo Jr* The realization
that apoptosis
is genetically
programmed
the exciting prospect that modulating apoptosis novel approaches for treatment of cardiovascular which
apoptosis
weight kinases
has been
inhibitors of caspases have been evaluated,
of cardiovascular
apoptotic
demonstrated.
Low
raises
may provide diseases in molecular
and mitogen-activated with promising results
protein in a variety
models.
Addresses Department of Cardiovascular Pharmacology, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406-0939, USA, Biological Sciences, UW-2523, SmithKline Beecham Pharmaceuticals, 709 Swedeland Road, Box 1539, King of Prussia, PA 19406-0939, *e-mail: Robert -- R [email protected]
Current
Opinion
in Chemical
Biology
1999,
USA;
3:474-480
http://biomednet.com/elecref/1367593100300474 0 Elsevier
Science
Ltd
ISSN
1367-5931
Abbreviations CHF congestive cyto
DD DED ERK FADD JNK MAPK TNF
c
heart failure cytochrome c death domain death effector domain extracellular signal regulated kinase Fas-associating protein with DD c-Jun amino-terminal kinase mitogen-activated protein kinase tumor necrosis factor
Introduction Apoptosis is a process of programmed cell death that was initially defined by morphological characteristics, including cell shrinkage, membrane blebbing, chromatin condensation and nuclear fragmentation [I]. The field of apoptosis is unusual in several respects. First, the general importance of apoptosis ha; been widely recognized only in the past decade, and its significance is currently being evaluated in many areas of biology. A substantial body of literature has been generated over the past 10 years, and the rate at which data are accumulating in this area is increasing exponentially. Thus, the number of publications per year related to apoptosis in cardiovascular disease has increased from one in 1991 to over 300 in 1998. Moreover, situations in which apoptosis plays a critical role are diverse, and virtually all fields of biology show a growing interest in apoptosis. Apoptosis is controlled by an evolutionarily conserved program and acts in embryonic development and tissue homeostasis, and can also be induced by various pathological insults. The uniform morphological features observed in apoptotic cells from different organisms suggest that a common mechanism may operate to trigger programmed cell death. Apoptosis in both excessive and reduced
for
amounts may have pathological implications (for example, apoptosis may be involved in a number of neurodegenerative diseases such as Parkinson’s disease; reduced amounts may lead to cancer). Our increased understanding that apoptosis is controlled by genes raises the exciting prospect that modulating apoptosis may provide a novel approach to the discovery of new drugs for the treatment of a variety of diseases [Z’]. In this review, we will focus on the role that apoptosis plays in cardiovascular diseases, and will highlight some mechanisms, mainly the signal transduction network and the execution machinery, that have been reported to be modulated by pharmacological agents.
Biochemical
and genetic
control
of apoptosis
As a general summary, apoptotic pathways involve a sensor that detects a death-inducing signal, a signal transduction network, and the execution machinery that actively carries out the process of cell death. The apoptotic process can be divided into three functionally distinct phases: the induction phase - a change in the cellular environment leading to the cell activating the mechanism of apoptosis through receptor and signal transduction mechanisms; the execution phase - the processes within the cell that result in committal to apoptotic cell death; and the degradation phase - the events associated with the final disposal of the cell corpse, leading the cell past a ‘point of no return’. It is likely that apoptosis will be controlled in a cell-typespecific fashion, but the basic elements of the death machinery may be universal. With many of the regulators of apoptosis identified, it is now possible to begin to define the functional relationship between them.
Signaling pathways that transduce extracellular death signals Death-receptor-mediated
signal
transduction
The best characterized pathways for the initiation of apoptosis involve those initiated by death receptors such as Fas/CD95 and the tumor necrosis factor (TNF) receptor (TNFR) family, most of which contain a death domain (DD) in their cytoplasmic region (Figure 1); [3”]. The death receptors interact via their DD with intracellular DD-containing adapters, such as FADD (Fas-associating protein with DD) and TRADD (TNF receptor-associated death domain), and recruit these adapters to the cell membrane. Thus, binding of Fas ligand to the Fas receptor leads to clustering of the Fas receptor’s DD. The adapter, FADD, then binds through its own DD to the clustered receptor DD. FADD also contains a ‘death effector domain’ (DED) that binds to an analogous domain within the procaspase-8 protein. Upon recruitment by FADD, pro-caspase-8oligomerization drives its own activation (to caspase-8)through self-cleavage (see Figure 1). Caspase-8then activates downstream effector
Apoptosis
and cardiovascular
diseases
Yue,
Ohlstein
and
Ruffolo
475
Figure 1
A simplified schematic diagram of the apoptotic death pathways. Death receptor (e.g. Fas) ligation results in formation of a signaling complex that includes the receptor, adaptor (e.g. FADD) and procaspase-8. The interaction between the intracellular domain of Fas and FADD is mediated via dimerization of two homologous regions in the two proteins, the DD. FADD, in turn, associates with the proenzyme form of caspase-8 through dimerization of a domain known as the DED; a similar DED region occurs in procaspase-8. Upon recruitment by FADD, procaspase oligomerization drives its own activation through self-cleavage (Asp-x indicates the cleavage site in procaspase-8). Caspase-8 then activates downstream effector caspases such as caspase-3. Activation of procaspase-9 requires multiple factors, including adaptor Apaf-1, dATP and cyto c. The cyto c is released from mitochondria in response to a variety of apoptotic stimuli, such as oxidants, calcium, ceramide and
pro-apoptotic Bcl-2 family members (e.g. Bax, Bik), while cyto c release can be inhibited by anti-apoptotic Bcl-2 family members Bcl2 and Bcl-XL, which also inhibit the association of Apaf-1 with procaspaser9, therefore preventing activation of caspase-9. Caspase-9 activates downstream effector caspases (e.g. caspase-3) thereby initiating apoptosis. The MAPK cascades comprising MEKK (MAPK kinase kinase), MKK (MAPK kinase) and MAPK are activated by external stress stimuli and result in phosphorylation of transcription factors (e.g. ATF2, c-Jun) and initiate transcription of downstream effecters. The direct relationship between MAPKs and activation of caspases is not clear. The red arrows indicate the potential targets for pharmacological intervention. Dashed lines indicate that the direct relationship between activation of caspase-3 and activation of transcription factors by MAPKs is not clearly established at this time.
caspases, such as caspase-3, thereby initiating apoptosis. It is believed that this signal transduction process is an important mechanism for Fas-ligandand TNFa-induced activation of caspases, and therefore cell death. The existence of functional TNFRl in the human heart and the elevated levels of TNFcx and soluble Fas in patients with congestive heart failure have been reported recently [4].
serine and threonine residues on certain proteins) that are activated by dual phosphorylation on threonine and tyrosine residues in response to a wide array of extracellular stimuli [5,6”]. The best-characterized subfamilies of the MAPK superfamily are the two ‘stress-responsive’ MAPK subfamilies, namely, c-Jun amino-terminal kinase/stress-activated protein kinase (JNK/SAPK) and ~3%MAPK (Figure l), and the extracellular signal regulated kinases (ERKs). In mammalian cells, the parallel kinase cascades for the three MAPK subfamily members have been described, which comprise a MAPK kinase kinase (MEKl), that phosphorylates and
Mitogen-activated protein kinase pathway Mitogen-activated protein kinases (MAPKs) are prolinedirected serine/threonine kinases (i.e. they phosphorylate
476
Next
generation
therapeutics
activates a MAPK kinase (MKK) which in turn phosphorylams and activates a MAPK (JNK, p38 and ERK). The JNK and p38 pathways are activated by a variety of external stress stimuli, such as reactive oxygen species, UV irradiation, ceramide and cytokines, and the ERK pathway can be activated by growth factors. Signal transduction via MAPKs results in phosphorylation of inducible transcription factors, such as c-Jun and ATFZ, which then act to initiate transcription of downstream effecters. Most recent in vitro and in vtio studieshave demonstratedthat JNK and ~38 are implicated in cardiac remodeling (cardiomyocyte apoptosisand hypertrophy) [7]. Interestingly, cardiomyocyte hypertrophy and apoptosiscan be induced by distinct members of the p38 family, suggestinga significant role for p38 signaling in the pathophysiology of congestive heart failure (CHF) [S]. It is clear from recent studies that MAPK signalingpathways are more complex than previously expected. This complexity provides an opportunity for selectivity within a given cell type or following a particular challenge.
The effector
machinery
of apoptosis
At its simplest, the apoptotic machinery includes three basic components: death proteases (caspases), CED4/Apaf-1 adapter molecules, and Bcl-2 family members. Caspasesrepresent the executionary arm of the apoptotic machinery. CED-4/Apaf-1 appears to play critical roles in the conversion of pro-caspasesinto active caspasesthrough direct physical interactions. The Bcl-2 family includes both pro- and anti-apoptotic members that can regulate each other to shift the balance from a pro-apoptotic environment to an anti-apoptotic environment, and vice versa. Caspases
A family of cysteine proteases known as caspasesis believed to be a critical component of the cell death machinery [9”]. To date, more than 14 caspaseshave been cloned and partially characterized in mammals,and many, but not all, have been implicated in the apoptotic process. They are synthesized in the cell asinactive precursors comprising four distinct domains (i.e. an amino-terminal domain, a large subunit [-20 kDa], a small subunit [-lo kDa] and a linker region between the large and small domains flanked by an aspartate residue). Activation of each caspase is induced by proteolytic cleavage between domains, resulting in the removal of the prodomain and linker regions, and assembly of the large and small subunits into an active enzyme complex. The mammalian cell death caspaseshave been divided into initiators and effecters, and two main caspasecascades have been delineated in mammalian cells (Figure 1) [lo’]. Activation of pro-caspase-8 requires association with FADD through the DED, as described previously, while pro-caspase-9activation requires multiple factors including adapter Apaf-1, cytochrome c and dATP (Figure 1). Different initiator caspasesmediate distinct sets of signals. For example, caspase-8is associated with apoptosis involving Fas receptor, and caspase-9 is involved in death induced by cytotoxic agents. A
pro-apoptotic signal culminates in activation of an initiator caspase which, in turn, activates effector caspases such as caspase-3,resulting in cellular disassembly.This model explains how distinct apoptotic signals induce the same biochemical and morphological changes that are characteristic of apoptosis. One potential mechanism to control activation of these initiator caspasesmay involve inhibition of the interaction between pro-caspasesand their activators. The role of caspases in mammalian apoptosis is complex in that multiple caspasesmay have redundant functions or act in concert to execute the apoptotic process in a cell-specific and stimulus-dependent manner. It has been demonstrated that caspasesare expressed in cardiovascular tissuesand implicated in cardiac apoptotic processes[ ll’,l’Z’]. Cytochrome
c as a regulator
of caspase
activation
The discovery that cytochrome c (cyto c), a key component of the mitochondrial electron transport system, is required for the activation of caspase-3wasunexpected, but provides a new framework in which to understand caspaseactivation in mammaliancells [13”,14]. Cyto c is releasedfrom mitochondria in responseto a variety of apoptotic stimuli, such as reactive oxygen radicals, calcium and ceramide. In the cytosol, cyto c can bind Apaf-1, and in the presenceof dATP or ATP, Apaf-1 adopts a conformation that enablesit to bind to, and thus promote activation of pro-caspase-9,which then processesand activates other caspases to orchestratethe biochemical execution of cells (seeFigure 1). Bcl-2 family
proteins
The Bcl-2 family of proteins, comprising at least 16 in humans, serves ascritical regulators of pathways involved in apoptosis [15]. The family contains both pro-survival members, such as Bcl-2 and B&XL, and pro-apoptotic members, such as Bax and Bik. Pro- and anti-apoptotic family members can heterodimerize and in sodoing, they can regulate the function of each other in order to change the balance between pro-apoptotic and anti-apoptotic environments. Two major mechanisms have been suggested for pro-survival proteins: maintenance of organelle integrity and prevention of the release from mitochondria of cyto r, and inhibition of the associationof Apaf-1 with pro-caspase-9,thereby preventing caspase-9 activation (see Figure 1). Pro-apoptotic members are thought to act through displacing the adapters from the pro-survival proteins. It has recently been suggestedthat Bax and Bax-like proteins may mediate caspase-independent death via channel-forming activity, which could promote the mitochondrial permability transition or puncture the mitochondrial outer membrane, thereby initiating apoptosis.
Apoptosis
in cardiovascular
diseases
Apoptosis in the cardiovascular system has been demonstrated only recently; however, evidence of apoptosis has been reported from diverse aspectsof cardiovascular medicine, ranging from CHF to conduction system defects to
Apoptosis
coronary atherosclerosis [ 16,17”,18’]. Table 1 lists the cardiovascular diseases associated with apoptosis. Studies in cultured cardiomyocytes Apoptosis has been demonstrated in cultured rat cardiomyocytes under conditions similar as those thought to occur in the failing hearts of patients (for example, ischemia, mechanical stretch forces, viral infection, and in the presence of oxygen free radicals and cytokines such as TNFa). Expression of pro-apoptotic genes (e.g. ~53, Fas, Bad and Bax) and anti-apoptotic genes (e.g. Bed-2) have been observed in apoptotic cardiomyocytes [191. Activation of the MAPK signaling pathways and caspases have also been demonstrated in myocardial cells undergoing apoptosis. Of particular interest, manipulation by MAPK or caspase inhibitors enables modulation of cell death induced by a variety of insults [ll’,ZO-221. Experimental studies in animals with heart failure or ischemic injury Several lines of investigation now clearly indicate that apoptosis can be elicited in cardiomyocytes in viva in response to different forms of cellular injury similar to those involved in the pathogenesis and progression of CHE Thus, animal models in which the loss of cardiomyby apoptosis ocytes is caused include multiple intracoronary microembolization-induced heart failure and rapid electrical-pacing-induced dilated cardiomyopathy in the canine, hypertension-induced heart failure in rats, and acute ischemia/reperfusion-induced cardiac injury in the rat and rabbit [16,17”]. In these studies, the induction of cardiomyocyte apoptosis has been clearly demonstrated. The appearance of apoptosis is a significant factor in myocyte death in these models, with a number of common features, such as early onset (1-3 h), a large variation in prevalence and distribution of apoptotic cells, and a clear dissociation between apoptosis and necrosis. The association of myocyte apoptosis with pro-apoptotic genes (Fas and Bax), and activation of stress-responsive MAPKs (JNK and p38), as well as caspases, have been demonstrated. The modulation by pharmacological agents of cardiac apoptosis in animal models with ischemic cardiac injury has also been observed [12’,23].
Apoptosis in patients with congestive heart failure or following myocardial infarction Evidence now also exists to indicate that apoptosis occurs in the hearts of patients with cardiomyopathies [24,25]. The occurrence of apoptosis in patients with CHF suggests that apoptosis may play a role in the progression of the disease and the chronic remodeling of the myocardium that occurs in heart failure. Apoptosis is a relatively infrequent event in hearc failure patients with ischemic cardiomyopathy, but is abundant in idiopathic dilated cardiomyopathy. Several reports have documented the occurrence of apoptosis in human heart specimens obtained from patients who died following myocardial infarction. In all studies, specimens of
and cardiovascular
diseases
Yue, Ohlstein
and Ruffolo
477
Table 1 Cardiovascular Cardiac
development Congenital
atrioventricular
with apoptosis
in humans.
block
overload
Cardiac
and heart failure Dilated cardiomyopathy lschemic cardiomyopathy Arrhythmogenic right ventricular
Acute
dysplasia
myocardiac infarction Myocarditis Cardiac allograft rejection Pre-excitation syndromes
Coronary
See
diseases associated
diseases Atherosclerosis
[17**,18*1.
myocardium following an acute myocardial infarction displayed features consistent with apoptosis. The expression of several genes associated with apoptosis, including Bd-2, Fas and Bax, were observed in human myocardium following myocardial infarction. In addition, it has also been reported that failing human hearts express elevated levels of caspase3 and TNFa. Moreover, the existence of functional TNFRl in human heart has been reported [4].
Apoptosis
in atherosclerosis
Multiple studies in both animals and humans have found apoptosis to occur in atherosclerotic coronary, carotid and aortic arteries, as well as saphenous vein grafts [ 18’1. These studies have observed that smooth muscle cells, principally located in the intimal fibrotic portion of the atherosclerotic plaque, and macrophages located in the intima, especially the lipid-laden core of the atheroma, show increased evidence of apoptosis compared with normal vessels. A significant number of cells undergoing apoptosis have been shown to be immunoreactive with anti-caspase-1 and -3 antiserum. Atherectomy specimens from restenotic lesions also showed evidence of apoptosis which strongly correlated with the presence of intimal hyperplasia. The death receptor, Fas, is expressed on as many as two thirds of the cells in the fibrous cap in human atherosclerotic lesions. Interestingly, isolated vascular smooth muscle cells from human atherosclerotic plaques were shown to have a higher propensity for apoptosis, and cytokines (such as TNFa) markedly sensitize cells to Fas-induced apoptosis. Recent studies have also suggested that oxidative mechanisms play a role in the apoptosis of vascular smooth muscle cells [ 17**]. It is still unclear whether apoptosis is a late finding, occurring only as part of the end stage of this disease, or whether increased apoptosis is associated with the early stages of atherogenesis. The major clinical implication of apoptotic cell death in atherosclerotic lesions is not clear. Apoptosis may result in reduced plaque stability, making the atheroma more susceptible to rupture thereby producing a
478
Next generation
Table
2
Pharmacological
therapeutics
manipulation
Model
Target
NRCM
Caspases ROS
NRCM
~38
NRCM
JNK and ~38 MAPK ERK
NRCM
Caspases
Rat
~38
rabbit
apoptosis. Apootosis
inducer
ZVAD-fmk NAC
lschemia
ET-l,
SB203580 Caspase-3 PE
PD98059
Staurosporine
in viva
Isc/rep
Rat heart
in viva
Isc/rep
IGF
lsclrep
Carvedilol
Rabbit
heart
in viva
JNK
ZVAD-fmk
of CM
I
1191
Apoptosis
of CM
-1
DOI
of CM
Apoptosis of CM no effect
SB203580
heart
Apoptosis
Apoptosis
ZVAD-fmk AC-YVAD-CHO
l&rep
References
El1
ET-l -induced ~38 and CM hypertrophyJ
SB202190
‘4202
MAPK
Results
Agent
TNFn, IL-1 8, IFN-y
MAPK
NRCM
Perfused
of myocardial
Apoptosis Cardiac heart Apoptosis
‘?
[301
I
[l 1‘I
of CM 1 functions
of CM J LVdpldt Infarction size 1
Apoptosis CK Apoptosis JNK
of CM loss 1
myocardial infarction. In addition to proteolysis, loss of smooth muscle cells in the fibrous cap of atherosclerotic lesions is known to predispose the lesions to plaque instability and therefore may increase the risk of unstable angina pectoris and acute myocardial infarction. In contrast, apoptosis may be beneficial by preventing the excessive cellular proliferation that occurs following balloon angioplasty or stent-induced vascular injury. Animal data have demonstrated that the enhanced cell proliferation is paralleled by increased susceptibility to apoptosis in the injured vessels, suggesting that apoptosis appears to be a major determinant of restenosis [18’].
Pharmacological cardiovascular
anti-ojcidant Current
The structure of carvedilol (1-[SH-carbazol-4-yloxyl-3-[(2(methoxyphenoxy)ethyl}aminoI-2-propanol). The chiral centre in the drug.
Opmon
star
,n Chem,cal
indicates
the
B~olagy
only
k-31 WI
manipulation apoptosis
kinase oxygen
l/2, MKKl12) species; SB203580
of
Inasmuch as apoptosis in the cardiovascular system has been demonstrated recently, low molecular weight inhibitors of caspasesand MAPKs have been evaluated in a variety of cardiovascular apoptotic models. Table 2 lists several examples. These studies target early processesof apoptosiswhere the cells have not passed‘the point of no return’. The results obtained to date are promising and indicate that cardiovascular apoptosiscan be modulated by pharmacological agents. Of particular interest is the finding that carvedilol (Figure 2; CoregB, SmithKline Beecham Pharmaceuticals, King of Prussia,Pennsylvania, USA), a new vasodilating P-adrenoceptor antagonist with strong antioxidant properties, has been demonstrated in clinical trials to dramatically reduce mortality in patients with CHF, and can also inhibit ischemia/reperfusioninduced apoptosis in cardiomyocytes in the rabbit. The possible mechanismsof carvedilol include down-regulation of the SAPK signaling pathway and Fas expression, and possibly alsoP-adrenoceptor blockade [26’].
Conclusions
H
[12’1
of CM I, Fas 1 L, Infarct size 1
cardiomyocyte; PD98059, MEKl/2 (MAPK inhibitor; PE, phenylephrine; ROS, reactive and SB202190, ~38 MAPK inhibitors.
2
1‘
1
AC, acetyl; CK, creatine kinase; CM, cardiomyocyte; ET-l, endothelin-1 ; fmk, (C-methyl)-CH,F; IFN-1: interferon-y; IGF, insulin-like growth factor; IL-l, interleukin-1 ; Isclrep, ischemia/reperfusion; LVdp/dt, first derivative of left ventricular pressure; NAC, N-acetylcysteine; NRCM, neonatal rat
Figure
[31'1 ?
and future
perspectives
Although end-stage events of apoptosis are likely to be essentially uniform in all cell types, someregulatory mechanismsmay be unique to the cells in cardiovasculartissues. Further elucidation of pro-apoptotic and anti-apoptotic mechanisms in cardiomyocytes and vascular cells could delineate potential targets for pharmacologicalintervention in a cell-type-specific fashion. This is particularly important
for chronic treatment with apoptotic modulators. For example, chronic treatment with a noncell-type-specific inhibitor of apoptosis may inhibit or slow all apoptosis throughout the body, which could have potentially serious consequences for the physiological processes dependent on apoptosis. Induction of apoptosis in locally proliferating vascular smooth muscle cells could represent a novel therapeutic approach for prevention of restenosis. In order to exploit the pathways involved in apoptosis in the heart pharmacologically, we must rely on a better understanding of the molecular mechanisms specifically responsible for the process of cardiovascular apoptosis. This fundamental information may underpin the basis for new strategies to exploit apoptotic mechanisms as novel drug targets. Initial attempts to reduce neointima formation by inducing smooth muscle cell apoptosis through adenoviral gene transfer have been promising in experimental models of vascular injury [27-291. Low molecular weight caspase inhibitors may represent a new class of drugs for the treatment of acute cardiovascular diseases in which apoptosis is believed to play a significant role. Our knowledge of the role of apoptosis in cardiovascular diseases is still limited. In this respect, several points appear to deserve further investigation: what is the true incidence of apoptosis in cardiovascular diseases; which endogenous and exogenous stimuli induce apoptosis in myocardial and vascular cells; and is apoptosis an early cause or a terminal event that is associated with cardiovascular diseases? To answer these questions, more sensitive and quantitative assays for detecting apoptosis in cardiovascular tissue are necessary. A better understanding of the role of apoptosis in cardiovascular diseases will probably lead to the discovery of additional targets for pharmacological intervention.
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published
within
period
of review,
of special interest **of outstanding interest
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Yue TL, Wang C, Romanic AM, Kikly K, Keller P, DeWolf WE, Hart TK, Thomas HC, Storer B, Gu JL et al.: Staurosporine-induced apoptosis in cardiomyocytes: a potential role of caspase-3. J MO/ Cell Cardiol 1998, 30:495-507. This is the first documentation of activation of caspase-3 in cultured rat neonatal cardiomyocytes undergoing apoptosis. 12. .
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