INVITED REVIEW
Regulation of apoptosis by vasoactive peptides

Gerasimos S. Filippatos1, Nupur Gangopadhyay2, Omosalewa Lalude3, Narayanan Parameswaran2, Sami I. Said4,5, William Spielman2, and Bruce D. Uhal2

1 Second Division of Cardiology, Evangelismos General Hospital, GR-11526 Athens, Greece; 2 Department of Physiology, Michigan State University, East Lansing, Michigan 48824; 3 Division of Cardiology, MetroHealth Medical Center/Case Western Reserve University, Cleveland, Ohio 44106; 4 Department of Medicine, University Medical Center, Stony Brook 11794; and 5 Veterans Affairs Medical Center, Northport, New York 11772


    ABSTRACT
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ABSTRACT
INTRODUCTION
APOPTOSIS AND ET
APOPTOSIS AND ANP
APOPTOSIS AND AM
REGULATION OF APOPTOSIS BY...
REGULATION OF APOPTOSIS BY...
SUMMARY
REFERENCES

Although originally discovered because of their ability to affect hemodynamics, vasoactive peptides have been found to function in a variety of capacities including neurotransmission, endocrine functions, and the regulation of cell proliferation. A growing body of evidence describes the ability of vasoactive peptides to regulate cell death by apoptosis in either a positive or negative fashion depending on the peptide and the type of target cell. The available evidence to date is strongest for the peptides endothelin, angiotensin II, vasoactive intestinal peptide, atrial natriuretic peptide, and adrenomedullin. Each of these peptides is discussed, with specific regard to apoptosis, in terms of regulatory activity, target cell specificity, and potential role in pulmonary physiology.

programmed cell death; blood pressure; pulmonary pathophysiology; hypertension; lung fibrosis


    INTRODUCTION
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INTRODUCTION
APOPTOSIS AND ET
APOPTOSIS AND ANP
APOPTOSIS AND AM
REGULATION OF APOPTOSIS BY...
REGULATION OF APOPTOSIS BY...
SUMMARY
REFERENCES

THE VASOACTIVE PEPTIDES were discovered primarily on the basis of their effects on hemodynamics, but the physiological roles of these molecules are now known to extend far beyond the control of vessel tone. The observations many years ago, for example, that angiotensin (ANG) and vasoactive intestinal peptide (VIP) were present in neurons (18, 53) led rapidly to the concept that these and other peptides function in neurotransmission (76). This is but one of the many roles that the vasoactive peptides play outside the vasculature to control a variety of processes under both normal and pathological conditions.

As investigations of the in vivo activities of vasoactive peptides were expanded, evidence suggested that some of the peptides might function as regulators of cell proliferation and/or death in a variety of tissues and cell types. Among these, the roles of ANG and endothelin (ET) in mitogenesis by cardiac fibroblasts and smooth muscle cells, respectively, have been well documented (5, 87). More recently, a growing body of evidence indicates important roles for some vasoactive peptides in the regulation of apoptosis as either a positive or negative regulator depending on the peptide. The evidence to date for the regulation of apoptosis is most strong for ANG, VIP, ET, atrial natriuretic peptide (ANP), and adrenomedullin (AM), each of which is discussed below. For those peptides studied more frequently, the present discussion is limited to cell types of the lung, in particular, vascular endothelial cells, vascular smooth muscle cells (VSMCs), alveolar epithelial cells (AECs), fibroblasts, and certain leukocytes.


    APOPTOSIS AND ET
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INTRODUCTION
APOPTOSIS AND ET
APOPTOSIS AND ANP
APOPTOSIS AND AM
REGULATION OF APOPTOSIS BY...
REGULATION OF APOPTOSIS BY...
SUMMARY
REFERENCES

ET-1 is a vasoconstrictor and growth factor for several cell types, including smooth muscle cells (126, 128). It acts through two G protein-coupled receptor subtypes termed ETA and ETB. VSMCs mainly express ETA receptors that mediate contraction, whereas endothelial cells express ETB receptors that mediate vasodilation via nitric oxide generation.

It has been suggested that ET-1 plays an important role in disorders of cell growth, and ET receptor antagonists can prevent ventricular remodeling and postangioplasty neointima formation (17, 88). On the basis that ET-1 inhibits the growth of hepatic Ito cells through activation of ETB receptors (55), Wu-Wong et al. (125) hypothesized that ET-1 might regulate apoptosis in human smooth muscle cells. These authors induced apoptosis in human pericardial and prostatic smooth muscle cells with the antineoplastic agent paclitaxel, and in both types of cells, ET-1 inhibited apoptosis. The apoptosis was dose dependent, with an EC50 of 1 nM, and was abolished by A-127722, an antagonist selective for the ETA receptor. ET-3 and ANG II had no effect on the apoptosis induced by paclitaxel (125).

Antiapoptotic roles of ET. Shichiri et al. have shown that ET-1 is a survival factor for fibroblasts (98) and endothelial cells (97), an effect that was also mediated by the ETA receptor via mitogen-activated protein kinase (MAPK) activation. In fibroblast cell lines, serum deprivation induced c-Myc-dependent apoptosis that was reduced by ET-1 at concentrations lower than those required for stimulation of DNA synthesis. The protective effect was mediated by the ETA receptor; the ETA receptor antagonist BQ-123 completely blocked the survival effect, but the ETB receptor antagonist BQ-788 did not. Pretreatment with the inhibitor of MAPK kinase, PD-98059, or antisense oligonucleotides against the translation initiation site of rat p42/p44 MAPK mRNA antagonized the survival effect of ET-1 as did transfection of the cells with a dominant negative form of MAPK kinase (MAPKK1 S222A). Together, these data suggested that the MAPK pathway plays a central role in cell survival in response to ET-1.

Related work has shown that ET-1 also modulates apoptosis of rat VSMCs. ET-1 antagonized VSMC apoptosis induced by serum deprivation and nitric oxide; this antiapoptotic effect also was mediated by the ETA receptor through the MAPK pathway (99). A central role for extracellular signal-regulated kinase (ERK) 1/2 as the downstream mediator for the antiapoptotic effect of ET-1 has recently been demonstrated by Wu-Wong et al. (127). In human prostatic smooth muscle cells, ERK1/2 activity decreased and apoptosis was induced after paclitaxel treatment or serum withdrawal, but ERK1/2 activity was maintained at higher levels and DNA fragmentation was attenuated when ET-1 was added. The inhibitor of ERK, PD-98059, induced apoptosis in cells cultured in serum-supplemented medium, potentiated the apoptotic effect of serum withdrawal, and blocked the antiapoptotic effect of ET-1. Recently, it was shown that ET-1 has a protective effect in omega -3 fatty acid-induced apoptosis of VSMCs as evidenced by the inhibition of caspase-3 activation and subsequent DNA fragmentation in late-stage apoptosis (15). Cytosolic ET-1 also prevents nuclear calcium overload in human VSMCs, protecting them from apoptosis (4).

Investigations of the role of ET-1 in models of ischemia and reperfusion are somewhat contradictory. In a model of myocardial stunning, Klainguti et al. (47) have shown that myocardial ET-1 is increased after 20 min of ischemia and have suggested that ET-1 might play a role as an inducer of apoptosis. In agreement with that theory, Shaw et al. (94) have shown that treatment with the ETA/ETB receptor antagonist SB-209670 could reduce the level of apoptosis observed in the lung after ischemia-reperfusion injury in dogs subjected to left lung allotransplantation. Notably, the cells undergoing apoptosis were in the airway epithelium and parenchyma, suggesting that ET-1 might be proapoptotic for epithelial cells but antiapoptotic for smooth muscle cells.

Regardless, the protective effect of ET-1 on apoptosis was also shown in the aorta by Sharifi and Schiffrin (93) in deoxycorticosterone acetate-salt hypertensive rats; treatment with ETA receptor antagonists increased the rate of apoptosis and decreased aortic cross-sectional area. A possible role for the ETB receptor in the protective effects of ET-1 was examined in endothelial cells by Shichiri et al. (96), who showed that ET also suppressed apoptosis of rat aortic endothelial cells in response to serum deprivation. Moreover, the ETB receptor antagonist BQ-788 and the nonselective ETA/ETB receptor antagonists (TAK-044 and PD-145065) enhanced apoptosis caused by serum deprivation, but the ETB receptor agonist BQ-3020 decreased the apoptotic index in a dose-dependent manner.

Proapoptotic roles of ET. However, there are other instances in which ET-1 may be proapoptotic as well as antiapoptotic. Cattaruzza et al. (8) have shown that ET-1 and cyclic strain of VSMCs induce apoptosis that appeared to be ETB receptor mediated; ET-1 (10 nM) promoted apoptosis that was completely suppressed by the ETB receptor antagonist BQ-788 but not by the ETA receptor antagonist BQ-123. Moreover, VSMCs derived from homozygous spotting lethal rats, which lack a functional ETB receptor, showed no signs of apoptosis after exposure to cyclic strain and exogenous ET-1. These results were confirmed in an experimental model in which segments of rabbit carotid artery were subjected to increased intraluminal pressure (49). PreproET-1 mRNA and ET-1 were upregulated predominantly in endothelial cells, and the ETB receptor in smooth muscle cells was significantly increased. An increase in apoptosis in the media of the carotid arteries was detected when arterial segments were exposed to a perfusion pressure of 160 mmHg for 6 h. The pressure-induced increase in apoptosis was prevented by the ETB receptor antagonist BQ-788 but not by the ETA receptor antagonist BQ-123 (49).

ET-1 may also play a role in the pathophysiology of cancers; it has been suggested that ET-1 is associated with prostate cancer because ET-1 is increased in patients with advanced stages of the disease (68, 95). Rat and human colon carcinoma cell lines express Fas and Fas ligand, but these cells are resistant to Fas-induced apoptosis; because ET-1 is implicated as a growth factor in some cell types, Eberl et al. (20) examined the role of ET-1 in cancer cell resistance to apoptosis in response to Fas ligand. The mixed ETA/ETB receptor antagonist bosentan potentiated Fas-induced apoptosis, but low concentrations of exogenous ET-1 (10-13 to 10-10 M) antagonized the bosentan-induced apoptosis. At higher concentrations (10-8 to 10-7 M), the protective effect of ET-1 was absent.

In colon carcinoma cells that express only ETA receptors, the ETA receptor antagonist BQ-123 and the protein kinase (PK) C inhibitor bisindolylmaleimide IX could induce apoptosis, but the ETB receptor-specific antagonist BQ-788 had no effect (19). Egidy et al. (21) found that in human glioblastoma, ETA receptors were highly expressed in glioblastoma vessels, whereas ETB receptors were found mainly in the tumor cells. Those findings were interpreted to suggest that glioblastoma vessels might be a source of ET-1 that acts on cancer cells via the ETB receptor. The possibility that a similar mechanism might be active in cancers of the lung has not been evaluated.

In A375 human melanoma cells, ET-1 induced apoptosis and nuclear accumulation of p53 (70). These effects appeared to be mediated by the ETB receptor because the ratio of ETA to ETB receptors was 1:4. On the basis of this and related works discussed above, the relative abundance of ETA versus ETB receptors expressed by individual cell types is believed to be important for the control of apoptosis. Regardless, the influence of ET-1 on the regulation of apoptosis may have important clinical implications. Imbalanced proliferation and apoptosis are important in tumor progression, in cardiac remodeling, in the pathogenesis of vascular hypertrophy in hypertension, and in restenosis of arteries. Whether ET-1 induces or suppresses apoptosis appears to depend on cell type and the cell-specific expression of ETA versus ETB receptor subtypes; a summary of the available data to date is presented in Fig. 1. The potential roles of ET-1 and its receptors in the control of lung cell apoptosis are yet to be evaluated.


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Fig. 1.   Role of endothelin (ET)-1 in the regulation of apoptosis. In fibroblasts and vascular smooth muscle cells (SMCs), activation of ET receptor type B (ETB), as occurs in cyclic strain and at high vascular perfusion pressure, activates apoptosis (solid lines) in a manner inhibitable by ETB receptor-selective antagonists BQ-788, TAK-044, and PD-145065 (dotted lines). In the same cells, apoptosis in response to serum deprivation, paclitaxel, or nitric oxide (NO) is blocked by activation of ETA receptor, an effect that is mediated through mitogen-activated protein kinase (MAPK) and is inhibitable by ETA receptor-selective antagonists A-127722 and BQ-123. Activation of ETB receptor also induces apoptosis in A375 carcinoma cells and lung epithelial cells during ischemia-reperfusion. In contrast, in endothelial and colon carcinoma cells, activation of ETB receptor blocks apoptosis in response to serum deprivation or Fas-Fas ligand (FasL) interaction; in either case, the inhibition is prevented by ETB receptor-selective antagonists (dotted lines). See text for details.


    APOPTOSIS AND ANP
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ABSTRACT
INTRODUCTION
APOPTOSIS AND ET
APOPTOSIS AND ANP
APOPTOSIS AND AM
REGULATION OF APOPTOSIS BY...
REGULATION OF APOPTOSIS BY...
SUMMARY
REFERENCES

In 1981, de Bold et al. (11) observed that infusion of extracts of atrial tissue into rats caused a copious natriuresis. These observations led to the isolation and cloning of ANP, the first member of a family of peptides with potent natriuretic, diuretic, and vasorelaxant activity (41). It is produced primarily in the cardiac atria, mainly in response to increased atrial wall tension as a result of increased intravascular volume. Several hormones and neurotransmitters such as ET, arginine, vasopressin, and catecholamines directly stimulate the secretion of ANP. Urodilatin [ANP-(95-126)] is the product of alternative ANP gene expression in the kidney (51). Brain natriuretic peptide (BNP), although found in the human brain, is predominant in cardiac ventricles (67). C-type natriuretic peptide (CNP), which possesses vasorelaxant activity but is not natriuretic, is found primarily in the brain and also in the anterior pituitary, the kidney, vascular endothelial cells, and, at low levels, plasma (102).

Three natriuretic peptide receptors (NPR-A, NPR-B, and NPR-C) have been identified in mammalian tissues (48). NPR-A and NPR-B are linked to the cGMP-dependent signaling cascade and mediate most of the cardiovascular and renal effects of the ANPs. NPR-C is thought to be involved in the clearance of the peptides (54). In general, the natriuretic peptides defend against salt and water retention, inhibit the production and action of vasoconstrictor peptides, promote vascular relaxation, and inhibit sympathetic outflow. These peptides have also been shown to possess antimitogenic activity in the cardiovascular and other organ systems (28, 36, 104). Their role in the regulation of apoptosis has been described mainly in the cardiovascular system.

ANP and apoptosis of cardiac myocytes. Myocardial hypertrophy occurs early in the clinical course of heart failure and is an important risk factor for subsequent cardiac morbidity and mortality. Induction of the natriuretic peptide genes is a feature of cardiac hypertrophy in all mammalian species and is a prognostic indicator of clinical severity. Loss of cardiac myocytes as a result of apoptosis has been reported in both experimental and clinical cardiac hypertrophy (35). Increased local levels of ANP may promote the transition from myocardial hypertrophy to heart failure by inducing apoptotic cell loss (31). Apoptotic cell loss has been well demonstrated in failing human hearts (22, 71). Wu et al. (124) examined the effect of ANP on neonatal rat cardiac myocytes. ANP induced apoptosis in a manner that was dose dependent and mediated by NPR-A and NPR-B, both of which are coupled to the generation of cGMP.

That study (124) also showed that apoptosis in response to ANP was myocyte specific and did not occur in fibroblasts and smooth muscle cells despite similar expression patterns of all three isoforms of NPRs in the three cell types. The apoptosis was antagonized by norepinephrine-induced increases in cAMP. Furthermore, ANP inhibited the expression of Mcl-1, an antiapoptotic homolog of Bcl-2. During embryonic development, the ANP gene is expressed in both the atrium and ventricle, but its expression is downregulated in the ventricle shortly after birth. During the progression of heart hypertrophy and, in particular, the transition from hypertrophy to failure, reexpression of ANP in myocytes occurs in the left ventricle (130). The abundance of mRNA for ANP in the left ventricle is thus an important index of this transition.

The recent study by Kang et al. (39) showed that copper deficiency in mice induced a cardiac hypertrophy that was progressive over time and was associated with myocardial cellular apoptosis. That investigation also showed that copper deficiency caused a significant elevation in cardiac ANP mRNA and that this elevation was markedly depressed in the hearts of metallothionein-transgenic mice. The study also showed that ANP induced apoptosis in cardiac myocytes in a dose- and time-dependent fashion and that metallothionein-transgenic cardiomyocytes were significantly resistant to the ANP-induced apoptosis.

ANP and apoptosis of endothelial smooth muscle cells and VSMCs. Several studies have shown that ANP and CNP (36, 78) as well as other cGMP-elevating agents such as nitric oxide, sodium nitroprusside, and the cGMP mimetic 8-bromo-cGMP (77, 131) inhibit vascular smooth muscle hypertrophy and proliferation.

ANP has also been shown to be antimitogenic in cardiac myocytes and fibroblasts (7). The vascular structure is thought to be maintained by the countervailing balance between growth-promoting vasoconstrictive antiapoptotic factors and growth-inhibiting vasodilatory proapoptotic factors. Just as growth promoters can prevent apoptosis, Trindade et al. (107) hypothesized that growth inhibitors might induce apoptosis. Consistent with this hypothesis, exposure of rabbit aortic VSMCs to ANP or CNP (10-8 to 10-6 M) dose dependently increased apoptosis, with a maximal stimulation of over threefold. This effect was shown to be mediated by the cGMP-linked receptors NPR-A and NPR-B. These and other aspects of the signaling of apoptosis are summarized in Fig. 2.


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Fig. 2.   Signaling of apoptosis in response to atrial natriuretic peptides (ANPs). Pathological conditions including congestive heart failure, myocardial infarction, and shock result in increased plasma and tissue ANP and other peptides including brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP). These can induce apoptosis in target cells of the heart and vasculature that express the natriuretic peptide receptor (NPR)-A or NPR-B, both of which act through increased cGMP. In cardiac myocytes, the apoptotic response to ANPs can be inhibited (-) by metallothionein (MT) or norepinephrine. In vascular SMCs (VSMCs) or endothelial cells (ECs) of the rat, the apoptotic response to ANP can be inhibited by ET-1, HS-142-1 (a selective antagonist of NPR-A or NPR-B), or KT-5823 (an inhibitor of cGMP-dependent kinases). See text for details.

In a more recent study, Suenobu et al. (103) demonstrated that ANP, BNP, and CNP dose dependently induced apoptosis of rat endothelial cells with equal potency. ANP-induced apoptosis was blocked by a natriuretic peptide receptor antagonist (HS-142-1) that selectively antagonizes NPR-A and NPR-B (62); it was also blocked by a selective inhibitor of cGMP-dependent kinase (KT-5823) and by the vasoconstrictor ET-1 but not by a selective inhibitor of soluble guanylyl cyclase (1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one). In the same study, pretreatment of endothelial cells caused a marked accumulation of the tumor suppressor gene product p53, whereas the B cell leukemia/lymphoma gene product Bcl-2 was not affected. The physiological and pathophysiological relevance of these in vitro data, however, remains uncertain because supraphysiological concentrations of natriuretic peptides were used. It is possible that supraphysiological concentrations could act on the vascular endothelium under certain pathological conditions such as congestive heart failure (30), myocardial infarction (63), and septic shock (30).

The available data to date suggest that ANP may be important in the transition from myocardial hypertrophy to heart failure through its ability to induce myocyte apoptosis. It is possible, therefore, that drugs that counter the effects of ANP on apoptosis may be useful in the treatment of heart failure. With regard to the vasculature, the countervailing balance between endothelium-derived vasodilators and vasoconstrictors may determine endothelial cell and VSMC apoptosis and survival, which, in turn, may contribute to the development and/or progression of vascular pathology. The regulation of apoptosis by natriuretic peptides may thus play an important role in vascular remodeling during atherosclerosis, angiogenesis, and hypertension.


    APOPTOSIS AND AM
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APOPTOSIS AND ET
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REGULATION OF APOPTOSIS BY...
REGULATION OF APOPTOSIS BY...
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Since the discovery and initial isolation of AM from pheochromocytoma, a role has been suggested for this peptide in important biological processes such as cell proliferation and apoptosis. Nevertheless, the first biological responses observed for AM were potent hypotensive and vasodilatory effects and the ability to raise intracellular cAMP (45, 46, 91). Human AM is a 52-amino acid peptide with a single disulfide bridge between residues 16 and 21 and with an amidated tyrosine carboxy terminus (45, 46). It shows some homology with calcitonin gene-related peptide (CGRP) and is therefore considered a member of the calcitonin, CGRP, amylin peptide family (64, 123). AM is synthesized as part of a larger precursor molecule termed preproAM. In humans, this precursor consists of 185 amino acids. PreproAM contains a 21-amino acid NH2-terminal signal peptide that immediately precedes a 20-amino acid amidated peptide designated proAM NH2-terminal 20 peptide (45, 46). For a discussion of the many biological actions of proAM NH2-terminal 20 peptide, the reader is referred to an excellent review by Samson (90).

AM and CGRP not only belong to the same family of peptides, they also interact with the same receptor. Recent studies have suggested that AM and CGRP both interact with calcitonin receptor-like receptor, which was originally designated as the CGRP receptor. The interaction and affinity of these two ligands to this receptor are regulated by the expression of accessory proteins called receptor activity-modifying proteins (RAMPs) (58). The expression of RAMP-1, RAMP-2, and RAMP-3 affects the affinity of the calcitonin receptor to calcitonin and amylin and that of the calcitonin receptor-like receptor to AM and CGRP (6, 50, 58, 65, 107). This review focuses entirely on the role of AM in apoptosis, and readers are therefore referred to the review by Foord and Marshall (25) for a more complete discussion of RAMPs and their known functions.

The gene encoding preproAM is termed the AM gene and has been mapped and localized to a single locus on chromosome 11. The AM gene is expressed in a wide range of tissues. Evidence from both immunocytochemistry and cultured cell lines reveals that AM is expressed by many different cell types including vascular endothelial cells (32). In addition, many tumor cell lines express the AM mRNA and/or immunoreactive peptide (60). The normal plasma concentration of AM is in the range of 1-10 pM, with most values between 2 and 3.5 pM. In many cardiovascular, renal, and respiratory disorders, plasma AM has been reported to be elevated, suggesting that AM may be a component of the regulation of hemodynamics and might be released to compensate for elevated blood pressure (32).

Antiapoptotic and proapoptotic roles of AM. Since the initial discovery of AM in a tumor (40) the effects of AM on tumor cell lines and other cell types have been widely reported (89). AM has been suggested to be a growth factor for several tumor cell lines including lung tumor cells (60). Antibodies against AM were shown to inhibit the growth of some tumor cells, suggesting that autocrine synthesis of AM confers growth-promoting activity (60). Furthermore, AM has been demonstrated to protect vascular endothelial cells from apoptosis, suggesting a cell survival role for AM in the vasculature (43). A role for AM in the growth of endothelial cells in vivo is also suggested by AM gene knockout mice. The knockout is embryonically lethal due to the absence of placental vascularization (100). Although it is generally thought to promote cell proliferation and to be a survival factor for tumor and endothelial cells, respectively, AM is antiproliferative for VSMCs and glomerular mesangial cells. In addition, Parameswaran et al. (72) and others (42, 59) have shown that AM can induce apoptosis in glomerular mesangial cells.

Signaling of apoptosis by AM. Mechanisms believed to be involved in the signaling of apoptosis by AM are summarized in Fig. 3. cAMP has been shown to be the second messenger for the AM-sensitive receptor in most systems tested to date. However, some of the effects of AM on cell survival, such as the protective effect on endothelial cells, have been shown to be independent of cAMP. Agents capable of elevating or mimicking cAMP, such as forskolin or dibutyryl cAMP, had no effect on apoptosis in endothelial cells, and cAMP antagonists did not affect AM-mediated cell survival (43, 92). In glomerular mesangial cells, however, cAMP and its activators can induce apoptosis (65). Furthermore, AM-induced apoptosis in mesangial cells appears to be dependent on cAMP because a PKA inhibitor could block apoptosis in response to AM (72). Thus the signaling of apoptosis or cell survival by AM appears to be cAMP dependent in renal mesangial cells but cAMP independent in some endothelial cell types.


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Fig. 3.   Signaling pathways of adrenomedullin (AM) action on apoptosis by mesangial and endothelial cells. AM activates apoptosis in mesangial cells (left, solid arrows) but protects against apoptosis of endothelial cells (right, dotted arrows). AM-induced apoptosis of mesangial cells is mediated by cAMP-dependent protein kinase (PK) A. Activation of cAMP-PKA, p38-MAPK, and protein phosphatase 2A (PP2A) is necessary for AM-induced apoptosis. Caspase-3 and caspase-8 activation in response to AM has also been observed. However, the interaction of the caspase pathways with that of MAPK is not currently understood. The protective effect of AM on endothelial cells is independent of cAMP-PKA and cGMP-PKG but is dependent on NO. The receptor complex with which AM interacts in both cell types is represented by a 7-transmembrane-domain G protein-coupled receptor that associates with a single transmembrane receptor activity-modifying protein (RAMP) (106). ERK, extracellular signal-regulated kinase. See text for details.

Definition of the mechanisms by which AM mediates antiapoptotic versus proapoptotic signaling requires further investigation. Although the antiapoptotic effect of AM on endothelial cells is not mediated by either cAMP or cGMP, evidence suggests that nitric oxide may be a mediator of AM-induced cell survival. AM and nitric oxide induced an antiapoptotic effect in endothelial cells through a cGMP-independent mechanism (92) despite the fact that cGMP is a downstream target for nitric oxide in other cell types. In 1999, Shichiri et al. (96) showed that the antiapoptotic action of AM on endothelial cells was mediated through the induction of the antiapoptotic gene max.

In contrast, renal mesangial cell apoptosis in response to AM is dependent on cAMP-PKA activation (66). AM-stimulated apoptosis also is accompanied by activation of caspase-8 and caspase-3 (66, 73); caspase-8 is activated as early as 8 h after treatment, whereas caspase-3 activation reaches a peak by 16 h. AM also activates the c-Jun NH2-terminal kinase (JNK) and p38 MAPK pathways in mesangial cells as well as the activity of protein phosphatase 2A (PP2A) (22, 74). This occurs in conjunction with inhibition of the ERK pathway, which is mediated through the increase in PP2A activity (74). Activation of p38 and PP2A appears to be important in the stimulation of apoptosis because the p38 inhibitor SB-203580 or the PP2A inhibitor okadaic acid could block AM-induced apoptosis (74, 75). The role of the JNK pathway in this process is still unclear as is the possible interaction of the MAPK and caspase-mediated pathways in AM-stimulated apoptosis.

The physiological significance of the antiapoptotic or proapoptotic effects of AM remains to be determined. With regard to endothelial cells, it is hypothesized that the marked increase in AM in various pathological states may function to defend the vascular lining cells from injurious agents in an autocrine or paracrine manner. In light of the knowledge that aberrant proliferation of glomerular mesangial cells is an important event in glomerular disease, AM-stimulated apoptosis of these cells, also in an autocrine or paracrine manner, may serve to hinder pathogenesis. In both instances, although the biological response to AM may be quite different, it is possible that AM could play the beneficial role of retarding disease progression.


    REGULATION OF APOPTOSIS BY VIP AND RELATED PEPTIDES
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Evidence relating to the regulation of apoptosis by VIP and similar peptides is derived from studies of apoptosis of lung parenchymal cells in the course of acute lung injury, of activated T lymphocytes (83) and of neuronal cells subjected to certain neurotoxic agents or trophic deprivation.

VIP and lung injury. After the demonstration that VIP could reduce or prevent lung injury in experimental models of acute respiratory distress syndrome (ARDS), Said and associates (84-86) examined the possibility that this peptide may exert its protective effect, in part, by modulating apoptotic cell death. First, it was necessary to show whether apoptosis was indeed an important pathogenic mechanism in these models of lung injury. This appeared to be the case based on three lines of evidence (85). First, the activity of caspase-3, a key effector of apoptosis, was increased in lungs injured by excitotoxicity, i.e., by overactivation of the N-methyl-D-aspartate subtype of glutamate receptors (86). Second, the inhibition of caspase activity by selective caspase inhibitors prevented or attenuated this injury. Third, lung injury was associated with downregulation of the antiapoptotic protooncogene bcl-2 (80) in lungs injured by N-methyl-D-aspartate or by oxidative stress due to the prooxidant herbicide paraquat or to xanthine oxidase.

Having demonstrated that apoptosis contributes importantly to cell death in these models of lung injury, the same investigators (85) then evaluated the antiapoptotic activity of VIP and its contribution to the prevention or attenuation of acute edematous lung injury. These studies revealed that the reduction in lung injury by VIP was associated with the inhibition of caspase activation and the upregulation of bcl-2, both of which suppress cell death and promote cell survival (26, 85).

Cell types protected by VIP. The basis for the high-permeability edema of ARDS is a loss of alveolar-microvascular barrier function (121). In assessing the importance and regulation of apoptosis in ARDS, therefore, it is useful to examine apoptotic and antiapoptotic events in AEC and pulmonary endothelial cell preparations. Morphological and ultrastructural features of apoptosis were observed in pulmonary endothelium as a part of acute lung injury induced by endotoxin lipopolysaccharide in mice (27). In primary cultures of rat AECs and the corresponding human cell line A549, apoptosis has been induced by tumor necrosis factor (TNF)-alpha (114), Fas ligand (118), ANG II (119), the antineoplastic drug bleomycin (29, 115), and the antiarrhythmic drug amiodarone (2). VIP, tested in the first three of these experimental models, dose dependently inhibited apoptosis (110).

Soluble Fas ligand was present in the bronchoalveolar lavage (BAL) fluid of patients with severe ARDS, and this BAL fluid induced apoptosis of primary human distal lung epithelial cells (57). BAL fluid from ARDS patients, however, had an antiapoptotic effect on neutrophils (56). Widespread, caspase-3-dependent apoptosis of lymphocytes was detected in patients dying of sepsis and multiorgan dysfunction (3) and possibly contributed to the impairment of the immune response (34). The possible modification of apoptosis by VIP in these settings has not been tested.

VIP and the related pituitary adenylate cyclase-activating peptide (PACAP) have been shown to exert major modulatory influences on multiple aspects of the immune response (79). One recently reported action relates to activation-induced apoptosis of T lymphocytes, a process that is mediated through Fas-Fas ligand interactions (12). Both VIP and PACAP inhibit activation-induced apoptosis of T lymphocytes in vitro and in vivo, largely due to peptide-induced reduction of Fas ligand expression (12). The inhibition was dependent on specific VIP/PACAP receptors VPAC, and VPAC2 but not VPAC1. Through this action, VIP and PACAP might allow the survival of a small number of activated T cells to differentiate into memory cells. Furthermore, by inhibiting this form of cell-mediated cytotoxicity, the peptides might protect against the cell death of bystander targets that occurs in autoimmune and inflammatory diseases (12).

Said (82) has found that VIP and PACAP protect a variety of neural cells against apoptotic death and promote their survival. Such protection has been demonstrated for both peptides against glutamate toxicity of cerebral cortical neurons and neuronally differentiated pheochromocytoma (PC12) cells (86) and against apoptotic PC12 cell death due to trophic support withdrawal (14). PACAP protects developing and mature cerebellar granule neurons against apoptotic cell death (112). The neuroprotective effects of VIP and PACAP (see Fig. 4) are mediated, at least in part, via activation of PKA through PKC (112) and cAMP response element binding protein phosphorylation (113). The effector mechanisms include inhibition of caspase-3 activity (13) upregulation of bcl-2, and suppression of cytoplasmic cytochrome c translocation (Antonawich F and Said SI, unpublished observations).


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Fig. 4.   Cell survival pathways involving vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP). VIP and PACAP promote cell survival largely via activation of adenylate cyclase (AC)-dependent PKA (VIP and PACAP-27) and PKC (PACAP-38). MAPK is also activated. The reactions culminate in activation of cAMP response element binding protein (CREB) via its phosphorylation, followed by upregulation of the antiapoptotic protooncogene Bcl-2. PLC, phospholipase C; P, phosphorylation. [Reproduced with permission from Said and Dickman (85).]

The ability of VIP and related peptides to inhibit apoptosis induced by different means and involving diverse mechanisms and pathways suggests a role for these peptides as physiological modulators of programmed cell death. As pharmacological agents, the peptides may have potential therapeutic value in limiting or preventing cell injury and death in the lung, brain, heart (38), and other vital organs.


    REGULATION OF APOPTOSIS BY ANG II
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INTRODUCTION
APOPTOSIS AND ET
APOPTOSIS AND ANP
APOPTOSIS AND AM
REGULATION OF APOPTOSIS BY...
REGULATION OF APOPTOSIS BY...
SUMMARY
REFERENCES

Investigations of the long-term effect of inhibitors of ANG-converting enzyme (ACE) on cardiac and other tissues revealed that ACE inhibitors have a significant influence on cardiac remodeling (105). In light of the knowledge that tissue remodeling involves a coordinated interplay between cell proliferation and death, those findings led to the hypothesis that ANG might be a regulator of cell death by apoptosis. That theory was proved correct by the discovery that ANG II is a potent inducer of apoptosis in freshly isolated primary cultures of cardiac myocytes (9, 37). In subsequent years, this concept was confirmed both in vitro and in vivo through a variety of studies that now comprise a significant body of literature (reviewed in Ref. 23). This discussion, however, focuses on cells of the lung, in particular cells of the capillary endothelium and alveolar epithelium and lung myofibroblasts.

ANG II and endothelial cells. Capillary endothelial cells are considered the most important producers of circulating ANG II by virtue of the ACE activity in the lumen of the pulmonary vascular bed (69, 81). On the other hand, endothelial cells also are known to undergo apoptosis in response to ANG II, albeit at concentrations significantly higher than the normal plasma ANG II level. In a study of cultured human umbilical vein endothelial cells, Dimmeler et al. (16) showed that purified ANG II could induce apoptosis, with an EC50 of 100 nM, a concentration well above the normal plasma ANG II concentration of 5-8 pM (1). The apoptosis could be prevented by simultaneous blockade of the ANG receptor subtypes 1 and 2 (AT1 and AT2, respectively). An investigation (52) in cultured human coronary artery endothelial cells demonstrated that ANG II could potentiate apoptosis induced by TNF-alpha and experimental anoxia-reoxygenation. In that study, apoptosis was blocked by losartan, which suggested that AT1 was the ANG receptor subtype active in mediating this response.

To date, the effect of ANG II on apoptosis has not yet been determined with primary cultures of well-differentiated pulmonary microvascular endothelial cells. Thus the possibility exists that lung endothelial cells in vivo respond differently to ANG II than the human umbilical vein endothelial cell or human coronary artery endothelial cell lines. On the other hand, the EC50 for induction of apoptosis in these cell lines is far above the plasma ANG II concentration, at least under normal conditions. Whether or not the significantly elevated plasma ANG II levels attained in ARDS (122) or other lung injury settings are within a range capable of inducing pulmonary endothelial cell death is an interesting topic for future inquiry.

ANGII and epithelial cells. More recent evidence indicates that several extravascular cell types of the lung undergo apoptosis in response to ANG II at significantly lower concentrations and also have the capacity for inducible expression of ANG II in response to toxins. In 1999, Wang et al. (115) found that ANG II was a potent inducer of apoptosis in AECs, which exhibit an EC50 for ANG II of ~10 nM. Although this concentration is still higher than the normal plasma ANG II level of ~10 pM (1), measurement of the extravascular or interstitial ANG II concentration found it to be as much as 100-fold higher than that in plasma, at least in the heart (111) and eye (10). Although the extravascular ANG II concentration in lung is unknown, plasma and lung tissue ANG II levels are known to increase in lung injury (101, 122).

More importantly, subsequent studies found that endogenous or xenobiotic toxins can evoke an autocrine synthesis of ANG II by AECs. Wang et al. (118) in 1999 found that activation of Fas (APO1, CD95), a receptor previously shown to be expressed and functional in AECs for the induction of apoptosis in vivo (24), stimulates AECs to synthesize ANG II de novo. Moreover, the autocrine synthesis of ANG II was found to be required for the induction of apoptosis by Fas; the apoptosis could be abrogated by ANG receptor antagonists or other blockers of ANG II function (118). This finding provided a mechanism for the earlier observation that the prototype ACE inhibitor captopril was a potent blocker of apoptosis in the AEC-derived cell line A549 independent of its thiol moiety (108).

Autocrine generation of ANG II is also required for the apoptotic death of AECs in response to TNF-alpha , a toxin capable of reducing AEC number in primary culture by >50% over 40 h of exposure (114). Both the induction of apoptosis and the net decrease in cell number could be abrogated by an ANG receptor antagonist, by ACE inhibitors, or by antisense oligonucleotides against angiotensinogen (ANGEN) mRNA. The ability of the same agents to prevent apoptosis of AECs in response to the benzofuran antiarrythmic agent amiodarone (2) or the antineoplastic agent bleomycin (115) suggests that autocrine generation of ANG II may be a constitutive mechanism for the signaling of apoptosis in AECs regardless of the initiating stimulus. Moreover, the successful blockade of bleomycin-induced apoptosis in vivo by an ACE inhibitor (115) suggests that the same mechanism defined in primary cultures of AECs is also active in the intact animal.

Initial investigations of the ability of ANG II to induce apoptosis in AECs revealed that AECs express both major subtypes of the ANG II receptor, AT1 and AT2, at least at the level of mRNA (119). A more recent study (116), however, indicated that AEC apoptosis can be blocked by AT1 receptor-selective antagonists but not by AT2 receptor-selective blockers. These findings are consistent with observations from cultured human coronary artery endothelial cells (see ANG II and endothelial cells) but are in contrast to studies of pheochromocytoma and PC12W cell lines (33, 129), which suggested that AT2 receptor is the mediator of apoptosis. Whether this discrepancy is related to the use of well-differentiated primary cell cultures versus transformed cell lines as model systems or whether the ratio of AT1 to AT2 receptors in AECs is modulated in various injury settings awaits further investigation. Regardless, the finding that autocrine generation of ANG II is required as a mediator of apoptotic cell death in the alveolar epithelium has significant implications for the therapeutic management of acute lung injury and fibrogenesis (23).

ANG II and fibroblasts. An additional local source of ANG II that is independent of circulating ANG II is the myofibroblast. In the heart, cardiac myofibroblasts that emerge after myocardial infarction are known to synthesize ANG II and thereby influence local gene expression and apoptosis of surrounding myocytes (44). The same phenomenon was recently found by Wang et al. (117) to occur in the lungs, at least in patients with some forms of fibrotic lung disease. Primary cultures of myofibroblasts isolated from open lung biopsy specimens from patients with fibrotic lung disorders were found to synthesize ANGEN and to convert a fraction of the proprotein to ANG II. Although the proteolytic conversion was incomplete, primary AECs express the converting enzymes, and thus ANGEN can induce apoptosis of AECs as potently as ANG II (114), at least in vitro. The possibility that the ANGEN synthesized by the myofibroblasts affects the apoptosis and/or phenotype of the fibroblast population itself is currently under investigation.

Together, these findings are regarded as evidence to explain the apoptotic death of AECs in vivo that are immediately adjacent to the foci of myofibroblasts within biopsies of fibrotic human lungs (109). A determination of whether ACE inhibitors or ANG receptor antagonists might have a beneficial effect in patients suffering from fibrotic lung disease or other forms of lung injury will be performed in clinical trials; the positive results of previous experiments with several different animal models suggest that this possibility is worthy of evaluation (61, 115, 120). The known roles of ANG II as a regulator of apoptosis of cells in the lung are summarized in Fig. 5.


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Fig. 5.   Regulation of apoptosis by angiotensin II (ANG II) in the lungs. Myofibroblasts emerging in fibrotic lung tissue (1) synthesize angiotensinogen (ANGEN), which is converted (2) by adjacent alveolar epithelial cells (AECs) to ANG II. Apoptosis is induced through ANG II binding to receptor subtype 1 (AT1; 3), with an EC50 of ~10 nM in primary cultures of AECs; although the AT1 receptor is depicted here as basolateral, the actual orientation of the receptor in AECs is unknown. Induction of apoptosis in AECs by FasL or tumor necrosis factor (TNF)-alpha stimulates autocrine production of ANGEN (4); interruption of steps 2, 3, or 4 prevents AEC apoptosis. In the capillary, ANG II induces apoptosis of endothelial cells (5), with an EC50 of ~100 nM, through pathways involving both AT1 and AT2 receptor subtypes. Endothelial cell survival is ensured by the extremely low intravascular concentration of ANG II ([ANG II]; 6), but this rises significantly in lung injury. The potential contribution of circulating ANG II to the extravascular pool in the lung (7) is unknown, but in other tissues, extravascular ANG II is as much as 100-fold higher than that in plasma. ARDS, acute respiratory distress syndrome. See text for details and references.


    SUMMARY
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ABSTRACT
INTRODUCTION
APOPTOSIS AND ET
APOPTOSIS AND ANP
APOPTOSIS AND AM
REGULATION OF APOPTOSIS BY...
REGULATION OF APOPTOSIS BY...
SUMMARY
REFERENCES

As discussed above, the effects of the vasoactive peptides on the control of apoptosis are many and varied. Early data had suggested a model in which the hemodynamic effects of a given peptide might predict its influence on cell proliferation or death. However, as is often the case, additional data proved that view to be overly optimistic; although the vasodilators VIP and AM have antiapoptotic properties, ANP is vasodilatory but appears to be generally proapoptotic. Moreover, a given peptide can either promote or block apoptosis depending on the cell type and likely other factors yet to be discovered.

Among these is the possibility of "cross talk" between proapoptotic versus antiapoptotic peptides. For example, VIP was shown to prevent apoptosis in response to ANG II (110) and may well be found in future studies to block the proapoptotic stimuli of other peptides. Continued definition of the signaling pathways evoked by the active receptors for each vasoactive peptide will ultimately shed light on the specific mechanisms of interaction. However, the findings that VIP and PACAP exert antiapoptotic effects through a pathway that is dependent on cAMP and PKA (112, 113), whereas the antiapoptotic influence of AM on endothelial cells appears to be cAMP independent (43, 92), offer a glimpse of the complexities likely to be encountered.

Nonetheless, the available data indicate that the control of cell death exerted by vasoactive peptides is of sufficient power to exert significant physiological effects in vivo (85, 86, 115). These findings support the contention that the pharmacological manipulation of apoptosis holds great potential as a means of controlling cell population size and function as well as the progression of disease (26, 31, 35). In some instances, the therapeutic potential of peptide receptor antagonists, inhibitors of peptide synthetic enzymes, or the vasoactive peptides themselves are currently being evaluated through clinical trials. It is the hope of all the authors that the discussion above will stimulate interest and additional effort in this interesting and important area of research.


    ACKNOWLEDGEMENTS

The research reported here was supported by National Heart, Lung, and Blood Institute Grants HL-30450 (to S. I. Said) and HL-45136 (to B. D. Uhal) and the Department of Veterans Affairs (S. I. Said). Portions of the manuscript preparation were supported by Evangelismos General Hospital (Athens, Greece) and the Department of Physiology, Michigan State University (East Lansing, MI).


    FOOTNOTES

Address for reprint requests and other correspondence: B. D. Uhal, Dept. of Physiology, 310 Giltner Hall, Michigan State Univ., East Lansing, MI 48824 (E-mail: uhal{at}msu.edu).


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REFERENCES

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