Control of apoptosis of cardiovascular fibroblasts: A novel drug target

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Control of Apoptosis of Cardiovascular Fibroblasts: A Novel Drug Target Heinz Rupp, Bernhard Maisch I

Abstract: Adequate control of survival or programmed cell death (apoptosis) of cardiovascular cells appears as an important drug target. While prevention of apoptotic death of cardiomyocytes has been assessed in detail, selective induction of apoptosis of vascular smooth muscle cells or fibroblasts could also be of relevance. Thus, induction of apoptosis of vascular smooth muscle cells by p65 NF-vd3 and Bcl-x L antisense oligonucleotides or p53 overexpression could be useful for limiting vascular lesions associated with restenosis.

Although fibroblasts represent the majority of cardiac cells, few attempts were made to induce fibroblast apoptosis in disorders associated with excessive collagen deposition and fibrosis. It is hypothesized that early interference with fibroblast proliferation after myocardial infarction or inflammatory heart disease limits fibrosis which further impairs cardiac performance. A candidate approach could involve growth factor analogues which ate known to induce fibroblast apoptosis when an incomplete growth stimulus persists.

Key Words: Apoptosis 9Fibroblasts 9Gene transfer 9Heart failure Kontrolle der Apoptose des kardiovaskuliiren Fibroblasten: Eine neue Zielgriige fª die Pharmakotherapie Zusammenfassung: Die Funktion auch des kardiovasle Untersuchungen zu ihrer gezielten Apoptose noch aus. kul~iren Systems wird durch das Gleichge.wicht zwischen Ein solcher Ansatz k6nnte vor allem bei Erkrankungen mit programmiertem Zelltod (Apoptose) und Uberleben einer einer exzessiven Kollagensynthese und Fibrose von InteresZelle bestimmt. W~ihrend in den letzten Jahren die Verhinse sein. So kommt es nach Myokardinfarkt nicht nur im derung des apoptotischen Zelltodes von Kardiozyten im Bereich der Narbe, sondern auch im intakten Myokard zu Vordergrund der Untersuchungen stand, gibt es zunehmend einer verst~rkten Kollagenablagerung. Auch bei entzª Hinweise, dal3 auch die selektive AuslOsung einer Apoptose chen Herzerkrankungen (Myokarditis, Perikarditis) kann es bei glatten GefN3muskelzellen und kardialen Fibroblasten zu einer exzessiven Kollagensynthese kommen. Es sollte von therapeutischem Wert sein kann. Eine weitgehend daher die Hypothese geprª werden, ob eine selektive selektive Apoptose konnte bei glatten GeffiBmuskelzellen Apoptose von kardialen Fibroblasten bei einer neuroendodurch eine Behandlung mit p65 NF-~:B oder B.cl-xL-Antikrin bedingten Stimulierung der Fibroblastenproliferation sense-Oligonukleotiden und durch eine p53-Uberexpresvon therapeutischem Wert ist. Vor allem sollten Analoga sion ausgel{5st werden. Die verrninderte Zahl von glatten ron Wachstumsfaktoren (zum Beispiel PDGF-BB) auf ihre GeffiBmuskelzellen k6nnte bei der Verhinderung einer selektiv apoptoseausl6sende Wirkung bei kardialen FibroRestenose nach PTCA wichtig sein. Obwohl Fibroblasten blasten untersucht werden. die h~ufigsten Zellen im Herzen sind, stehen interventionelSchlª

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ell death arising from necrosis has been known for decades. Since it was assumed that energy deficit of the cardiomyocyte is the main cause for this catastrophic type of cell loss, few drug interventions were targeted at controlling cardiomyocyte death in disorders which are n o t a priori associated with hypoxia. The recent interest in apoptosis [4] is timely taking into account that apoptosis is a well recognized determinant of various diseases [21]. Control of apoptosis of cardiomyocytes, vascular smooth muscle cells, endothelial cells and fibroblasts could provide a novel target for

interfering with the progression of cardiovascular diseases. Of particular importance could-.be cardiac fibroblasts which represent the majority of cells of the heart muscle and which have been neglected due to their apparent resistance to various triggers of apoptosis. In view of the often encountered excessive proliferation of fibroblasts and the associated adverse remodeling of the extracellular matrix, apoptosis of fibroblasts could provide a novel approach for limiting fibrosis of the heart. In the present overview interventions are described which have been used for controlling

~Department of Internal Medicine and Cardiology, Philipps-University of Marburg, Germany. Herz 1999;24:225-31(Nr. 3)

225

Rupp H, et aL Control of Apoptosis of Cardiovascular Fibroblasts apoptosis in various cardiovascular cells. Furthermore, the hypothesis is examined that selective induction of fibroblast apoptosis is possible. Although the events controlling apoptosis remain illdefined, there is increasing evidence that cells can monitor irreversible defects in mitochondrial energy metabolism or DNA strand breaks. Under these conditions, the cellular ATP is used for executing the energyrequiring apoptosis program. It is, however, possible that initial steps of the apoptotic program can be performed, whereas due to immediate lack of ATP, the program cannot be completed leading to cell rupture with ensuing inflammation [25]. The view that apoptosis and necrosis are always 2 distinct forros of cell death is thus not justified. In the case of mitochondrial damage, cytochrome c is released independently of the breakdown of the mitochondrial membrane potential ~gm (Figure 1). Through interaction with cytoplasmic "apoptotic protease activating factors" (Apaf), cytochrome c can initiate the cell death program via activation of caspases [8]. The loss of a component of the mitochondrial electron transport chain also triggers superoxide generation. As a consequence of the "mitochondrial permeability transition", the apoptotic protease "apoptosis inducing factor" (AIF) is also released which can trigger caspase activation. Anti-apoptotic Bcl-2 family proteins which antagonize each other via formation of heterodimers function as gatekeepers to prevent the release of cyto-

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In an alternative control mechanism, the integrity of the cell is monitored by the transcription factor p53. This tumor suppressor plays a major role in the defense against tumors and can be activated by a range of stresses including DNA damage, hypoxia, cytokines, metabolic changes, viral infection, and activated oncogenes [29]. Activated p53 arrests cellular growth prior to entry into either S phase or mitosis and triggers apoptosis. There is increasing evidence that activated p53 induces the expression of redox-related genes leading to the formation of cytosolic reactive oxygen species followed by oxidative degradation of mitochondrial components [32] thus leading to propagation of apoptotic cell death. In addition to apoptotic stimuli arising from a severely perturbed integrity of mitochondrial metabolism or DNA damage, external mechanisms exist which can

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chrome c and AIF [8]. In addition to their stabilizing effect on the mitochondrial outer membrane, Bcl-2 proteins may also be involved in the direct binding of Apaf molecules [8]. Bax is a pro-apoptotic member of the Bcl-2 family. The Bax protein shares highly conserved domains with Bcl-2, some of which are required for the formation of Bax-Bcl-2 heterodimers [5]. Bax can forro ion conducting channels in the mitochondrial membrane which may be responsible for its proapoptotic action. Bax can also induce mitochondrial release of cytochrome c which occurs independently of opening of the mitochondrial permeability transition pore (PTP) [12].

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Figure 1. Schematic representation of key events involved in apoptosis (TNF-alpha = tumor necrosis factor-alpha; FasL = Fasligand; T N F R = tumor necrosis factor receptor; F A D D = Fas and Fas-associated death domain; T R A D D = tumor necrosis factoralpha receptor associated death domain; T R A F = tumor necrosis factor receptorassociated factor; c-IAP = inhibitor of apoptosis protein; A I F = apoptosis inducing factor; Apaf = apoptotic protease activating factor; SR = sarcoplasmic reticulum; E R = endoplasmic reticulum). Abbildung 1. Ausgew~ihlte Reaktionen, die an der Kontrolle des programmierten Zelltodes (Apoptose) beteiligt sind. Herz 1999;24:225-31 (Nr. 3)

Rupp H, et al. Control of Apoptos& of Cardiovascular Fibroblasts trigger apoptosis. By this mechanism involving e. g. cytotoxic T lymphocytes, virus-infected cells can be eliminated. Cytotoxic T lymphocytes express the Fas (CD95) ligand (FasL), which binds to Fas on the target cell and thereby initiates apoptosis which occurs independentty of the perforin-granzyme pathway [20]. Downstream of Fas is the adapter molecule FADD (also known as Mort-1) [41]. Apoptosis induced by Fas activates caspase-8 (MACH/FLICE/Mch5), which contains ah N-terminus with FADD (Fas-associating protein with death domain)-like death effector domains, so providing a direct link between cell death receptors and caspases [10]. Following triggering of the Fas receptor, the apoptotic program involves a hierarchy of caspases, with caspase-8 and possibly caspase-10 being at of near the apex of the caspase cascade [10]. Activation of caspases which encompass at least 10 related cysteine proteases through proteolytic degradation of the inactive proenzymes results in cleavage of a great number of intracellular proteins including poly(ADP-ribose) polymerase and lamins leading to the characteristic morphological picture of apoptosis. In addition to the FasL-Fas pathway, additional death receptors exist which can trigger apoptosis. Of particular relevance for the heart is tumor necrosis factor-alpha (TNF-alpha). This cytokine is increased in viral infections, in myocarditis but also in congestive heart failure. A major effect of TNF alpha involves the TNF receptor 1 (TNF-R1) and the adapter protein T R A D D (TNF-Rl-associated protein) with ensuing caspase activation. An independent signaling pathway linked to various cytokine receptors involves sphingomyelinases leading to the formation of ceramide [1]. Thus, there exist various possibilities of controlling survival or apoptotic death of cardiovascular cells.

Coronary Artery Disease In the initial stage of atherosclerosis during fatty streak formation, monocytes migrate into the subendothelium and become transformed into macrophages. Macrophages can be turned into foam cells by bacterial infection with e. g. Chlamydia pneumoniae or uptake of oxidized LDL. Any accumulation of activated macrophages is associated with inflammation arising from the release of matrix degrading enzymes and cytokines. Macrophages have a crucial role not only during the development of atherosclerosis but also at late stages. Of particular interest could be the destabilization of a plaque involving thinning of the fibrous cap leading to plaque rupture. Again macrophages are involved Herz 1999;24:225-31 (Nr. 3)

which release matrix degrading enzymes. Cytokines released from macrophages could also contribute to an inappropriate apoptotic death of neighboring vascular smooth muscle cells. The possibility arises thus that timely elimination of activated macrophages might prove advantageous. The feasibility of an approach targeted at termination of inflammatory reactions is exemplified by FasL gene transfer in the case of collagen-induced arthritis [43]. This approach mimics a principle present in organs exhibiting immune privilege [14]. The eye is such a privileged site that cannot tolerate destructive inflammatory responses. Because the cells of the anterior eye express FasL, infiltrating cells are apoptotically killed. Comparable mechanisms may occur in joints [13] (Figure 2). It was concluded that gene transfer of FasL may representa new therapeutic strategy for autoimmunity caused by FasL dysfunction [17] (Figure 2). The proliferative growth of vascular smooth muscle cells during restenosis could provide another target for drugs that selectively induce apoptosis. There is increasing evidence that the balance between the survival factor Bcl-x L and the cell death promoting factor Bax is perturbed during restenosis. A downregulation of intimal cell Bcl-x L with antisense oligonucleotides induced apoptosis of intimal vascular smooth muscle cells and regressed the vascular lesion [31] (Figure 3). Furthermore, overexpression of the tumor suppressor p53 gene in the injured arterial wall inhibited the proliferation of vascular smooth muscle cells in vitro and in vivo [42]. In ah alternative approach, the "senescent ceIl-derived inhibitor (sdi)-i protein" (p21 product) which is a downstream mediator of p53 in the regulation of cell cycle progression through a G1 phase checkpoint has been overexpressed [28]. Growth of vascular smooth muscle cells was inhibited by overexpression of the p21 gene which was accompanied by induction of apoptosis [28]. There is also increasing evidence that a cross-talk between sympathetic overactivity and cAMP-mediated apoptosis exists. Thus, activation of the "cAMP responsive element modulator" (CREM) induced the expression of the "inducible cAMP early repressor" (ICER) which can block cAMP-induced apoptosis [34] (see Figure 2). The complexity of cAMP influences on apoptosis is also demonstrated by the finding that the p53 dependent pathway can be inhibited via "cAMPresponse element" (CRE) mediated gene expression [40] and that CRE-mediated signals can even function as survival factor for melanoma cells [18]. 227

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Hypertensive Heart Disease It remains an intriguing observation that the myocardial collagen concentration can markedly increase in hypertensive heart disease [7, 15]. The interstitial and perivascular fibrosis in hypertensive heart disease has often been attributed to replacement of lost cardiomyocytes or to an excessive angiotensin II or aldosterone stimulation of cardiac fibroblasts. Although both processes are likely to occur, there is increasing evidence for the involvement of additional factors. Thus, the progressive 228

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Figure 3. Schematic representation of apoptosis-inducing interventions for preventing excessive proliferation of vascular smooth muscle cells. Abbildung 3. M0gliche lnterventionen zur Ausl0sung einer Apoptose von glatten Geffil3muskelzellen.

accumulation of collagen in the absence of overt heart failure can hardly be accounted for by neuro-endocrine activation involving the renin-angiotensin II-aldosterone system. Ir rather appears that a progressive loss of cardiomyocytes occurs which are replaced by collageneous tissue. If one accepts the view that fibroblasts can excessively be stimulated to proliferate and produce collagen, the possibility should also be examined whether apoptosis can selectively be induced in fibroblasts (Figure 4). This approach would not rely on an adequate reduction of neuro-endocrine stimulation of fibroblasts. Herz 1999;24:225-31(Nr. 3)

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Progression of Heart Failure A key feature of an overloaded heart is the expression of genes that are expressed only in the fetal period. A1though the signals responsible for this reorganization of gene expression of the cardiomyocyte remain ill-defined, there is increasing evidence that the altered gene expression contributes to an impaired pump performance of the overloaded heart. Thus, in addition to a possible apoptotic loss of cardiomyocytes, the function of the individual cardiomyocyte is depressed. Recent approaches targeted at improving glucose oxidation of heart muscle partially prevented the fetal gene expression and improved pump performance [38]. One of the consequences of the fetal phenotype and the associated depressed function is activation of the renin-angiotensin II-aldosterone system. An enhanced angiotensin II influence is expected to promote apoptosis of cardiomyocytes [11] and the consecutive replacement fibrosis. Induction of fibroblast apoptosis may be useful for limiting fibrosis of failing heart. This approach could be of particular relevance when inflammatory reactions prevail as exemptified by myocarditis. In acute myocarditis, focal myocytolysis is a common event and is followed by replacement with fibrous tissue [16, 27]. Although apoptosis could contribute to the focal loss of cardiomyocytes, such an event is unlikely to account for the pronounced diffuse interstitial fibrosis of patients with dilated cardiomyopathy or the devetopment of excessive endocardial fibrosis [16, 27]. Again the possibility that persistence of fibroblast proHerz 1999;24:225-31 (Nr. 3)

liferation contributes to excessive fibrosis should be examined. Since the pro-inflammatory gene expression mainly involves' the transcription factor NF-rJ3, the question also arises whether apoptosis could be controlled via NF-r,B signaling (see Figure 1). TNFalpha is one of the proteins which become amplified by NF-~B transactivation. However, TNF-alpha not only induces apoptosis via the above mentioned well-characterized TNF-R1 receptor but can also inhibit apoptosis by "inhibitor of apoptosis protein" (IAP), whereby the TNF-R2 receptor appears to be involved (Figure 1). The c-IAPs do not directly contact TNFR2, but rather associate with "tumor necrosis factor receptor-associated factor" TRAF1 and TRAF2 [33]. The c-IAPs inhibit caspase-8 and thus also the apoptotic program. Despite this complexity of TNF-alpha signaling, there is convincing evidence that administration of p65 antisense NF-~cB can inhibit neointima formation in balloon angioplasty treated rat carotid arteries [3].

Myocardial Infarction Ischemia-induced injury of cardiomyocytes is associated with severe reduction of high-energy phosphates which precludes a regular ATP-dependent apoptotic program. In the periphery of the infarcted region significant apoptosis might, however, occur thereby expanding the infarct zone [9]. Also the reperfusion-induced generation of oxygen radicals [19, 35] is expected to cause additional apoptosis of cardiomyocytes. Apoptosis initiated by infiltrated macrophages [2] has also to be taken into account. Any loss of viable cardiomyocytes requires replacement by connective tissue leading to scar formation. The question arises nonetheless whether proliferation of fibroblasts is adequately controlled after myocardial infarction. There is increasing evidence that in the non-infarcted distant areas of the heart collagen synthesis of fibroblasts is stimulated. Although this detrimental process has been attributed to stimulation of fibroblasts by angiotensin II and aldosterone, an inappropriate apoptosis of fibroblasts could contribute to the observed fibrosis. Fibroblast-mediated fibrosis could not only be limited by reducing stimulatory influences but also by inducing selective apoptosis of fibroblasts. In this respect it should be noted that cardiomyocytes and fibroblasts differ in their response to various established triggers for apoptosis. Thus, hypoxia induces expression of the tumor suppressor p53 in cultured cardiomyocytes but not in fibroblasts [26, 36]. Since p53 can trigger apopto229

Rupp H, et aL Control of Apoptosis of Cardiovascular Fibroblasts sis, it appears that fibroblasts are more resistant to oxygen deprivation than cardiomyocytes. This altered oxygen sensitivity is required taking into account the role of fibroblasts in the repair of infarcted heart muscle of depressed oxygen supply.

Interventions Targeted at Fibroblast Apoptosis Selective control of apoptosis of cardiovascular cells represents a drug target which has not been explored adequately. A l t h o u g h the concept is still hypothetical in m a n y respects, it appears that therapy of cardiovascular diseases could particularly benefit from drugs which selectively induce apoptosis of fibroblasts. Such an approach does not necessarily depend on gene transfer since also growth factor analogues can be used for inducing apoptosis. It is has been assumed that fibroblasts are resistant to apoptosis. Recent findings argue, however, against this assumption. Thus, in situ end labeling of fragmented D N A has detected apoptotic cells in cultured h u m a n lung fibroblasts and it was concluded that fibroblasts behave as predicted by classic models of cell cycle progression and differentiation [39]. Furthermore, although neonatal of normal adult skin fibroblasts did not express p53 or bcl-2, both proteins were induced by exposure to adriamycin [24]. Also the lectin concanavalin A induced apoptosis in h u m a n fibroblasts similarly to other cell types [23]. The lectin-induced apoptosis was associated with b r e a k d o w n of the mitochondrial m e m b r a n e potential and an inadequate protective response of Bcl-2 [23]. Of particular interest is the finding that keloid lesions and keloid fibroblasts were found to have lower rates of apoptosis than normal controls. A h excessive proliferation of skin fibroblasts resulting in keloid lesions was attributed to a focal dysregulation of p53 combined with upregulation of bcl-2 [24]. However, keloid fibroblasts displayed enhanced apoptosis rates in response to hydrocortisone, gamma-interferon, and hypoxia treatment as c o m p a r e d with normal adult fibroblasts [24]. The question thus arises whether activated fibroblasts of e. g. the infarcted heart exhibit properties similar to keloid fibroblasts which render them more resistant to apoptosis. Of particular relevance could be an approach involving the selective deprivation of trophic factors. Polypeptide growth factors in vivo m a y signal cell late positively or negatively in settings that limit the potential of cetls to completely transit the cell cycle. Thus, while platelet230

derived growth factor ( P D G F ) and epidermal growth factor are potent mitogens they can also induce apoptosis in kidney fibroblast cells when they are serumdeprived [22]. In the case of growing h u m a n cardiac fibroblasts, a cyclic peptide analogue of P D G F - B B induced apoptosis [6]. Epidermal growth factor, fibroblast growth factor, thrombin and fetal bovine serum were n o t a b l e to rescue the cells from the effects of the P D G F - B B analogue. These data ate important since P D G F - B B represents a mediator of interstitial hyperplasia and fibrosis [37]. The complexity of the action of growth factors on the differential induction of apoptosis is d e m o n s t r a t e d by the observation that P D G F - B B induced apoptosis in vascular smooth muscle cells while P D G F - A A prevented the apoptosis [30]. Taken together, there is increasing evidence that selective apoptosis of cardiovascular fibroblasts represents a useful drug target. Ideally, drug interventions should prevent cardiomyocyte apoptosis while favoring apoptosis of fibroblasts. Further progress in this promising field requires, however, the characterization of apoptotic checkpoints which are specific for a given cardiovascular cell.

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Address for Correspondence: Prof Dr. Heinz Rupp, Molecular Cardiology Laboratory, Department of Internal Medicine and Cardiology, Karl-von-Frisch-Strafle 1, D-35033 Marburg, Germany, Fon (+49/6421)28-5032, Fax-8964, e-mail: [email protected]

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