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Circulation Research. 2000;87:856-864

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(Circulation Research. 2000;87:856.)
© 2000 American Heart Association, Inc.

Review

Apoptosis During Cardiovascular Development

Steven A. Fisher1, B. Lowell Langille1, Deepak Srivastava1

From the Departments of Medicine and Physiology, Case Western Reserve School of Medicine (S.A.F.), Cleveland, Ohio; Department of Laboratory Medicine and Pathobiology (B.L.L.), University of Toronto and Toronto General Hospital, Toronto, Canada; and Departments of Pediatric Cardiology and Molecular Biology (D.S.), University of Texas Southwestern Medical Center, Dallas, Tex.

Correspondence to B. Lowell Langille, Toronto General Hospital, University Health Network, 200 Elizabeth St, CCRW 1-836, Toronto, M5G 2C4, Canada. E-mail langille{at}uhnres.utoronto.ca


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowApoptosis in the Developing...
 down arrowVascular Cell Apoptosis During...
 down arrowConclusions
 down arrowReferences
 
Abstract—Morphogenesis and developmental remodeling of cardiovascular tissues involve coordinated regulation of cell proliferation and apoptosis. In the heart, clear evidence points toward focal apoptosis as a contributor to development of the embryonic outflow tract, cardiac valves, conducting system, and the developing coronary vasculature. Apoptosis in the heart is likely regulated by survival and death signals that are also present in many other tissues. Cell type–specific regulation may be superimposed on general cell death/survival machinery through tissue-specific transcriptional pathways. In the vasculature, apoptosis almost certainly contributes to developmental vessel regression, and it is of proven importance in remodeling of arterial structure in response to local changes in hemodynamics. Physical forces, growth factors, and extracellular matrix drive vascular cell survival pathways, and considerable evidence points to local nitric oxide production as an important but complex regulator of vascular cell death. In both the heart and vasculature, progress has been impeded by inadequate information concerning the incidence of apoptosis, its relative importance compared with the diverse cell behaviors that remodel developing tissues, and by our primitive knowledge concerning regulation of cell death in these tissues. However, tools are now available to better understand apoptosis in normal and abnormal development of cardiovascular structures, and a framework has been established that should lead to considerable progress in the coming years.


Key Words: apoptosis • development • myocardium • smooth muscle • endothelium


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowApoptosis in the Developing...
 down arrowVascular Cell Apoptosis During...
 down arrowConclusions
 down arrowReferences
 
The role of spontaneous cell death in normal embryonic development was appreciated a century ago (for an early review, see Glucksmann1 ). Histological studies by Kerr et al2 led them to propose in 1972 that the cell death that occurs in some developmental and pathological contexts is distinct from necrotic cell death. They suggested that this form of cell death be termed "apoptosis," for the controlled cell deletion that reminded them of its Greek derivation, "falling off," as of leaves from a tree.3 The 1990s witnessed an explosion in the study of apoptosis that led to the identification of the components of a programmed cell death (PCD) pathway. Critical discoveries included the caspases, a family of highly conserved proteases that executes the program of cell death4 5 ; receptor (tumor necrosis factor [TNF] superfamily, bone morphogenetic protein [BMP]) and nonreceptor (hypoxia, genotoxic stress, and growth factor withdrawal) means of activation of the caspase cascade6 7 ; the Bcl2 family of proteins, which reside within the mitochondria and regulate cytochrome c release and caspase activation8 ; and a family of inhibitor of apoptosis proteins (IAPs) that inhibit caspase activity9 and thus prevent the fortuitous activation of the pathway.

This review describes what is known of the role of apoptosis in the development of the cardiovascular system, focusing on three major questions.

1. What cells undergo PCD? Identification of the cell type undergoing PCD in vivo can be problematic since the process of apoptosis, as well as a commonly used detection method, terminal deoxynucleotide transferase–mediated dUTP-biotin nick end-labeling (TUNEL), destroys protein epitopes. This problem may be compounded by the undifferentiated or partially differentiated state of cells in developing organs. Evidence of apoptotic cell death includes chromosomal fragmentation detected by the in situ TUNEL labeling or by DNA laddering on gel electrophoresis, vacuolation and nuclear condensation by electron microscopy, eversion of phosphatidylserine residues of the cell membrane, and activation of caspases. Reliance on a single measure is often misleading.10

2. What is the significance of PCD with respect to the formation or the malformation of cardiovascular structures? PCD may have a number of functions in development, including reducing cell numbers, eliminating abnormal or mislocated cells, sculpting tissues, and eliminating vestigial structures.11 The role of the elimination of a cell or group of cells may be surmised by its coincidence with a morphogenic process, but proof of its role requires targeted perturbations of the PCD process. The significance of the elimination of cells must also be considered in the context of the rates of proliferation of the cells within a tissue.12

3. What are the molecular mechanisms? Over the last 5 years, our knowledge of basic genetic pathways that regulate PCD has grown exponentially; however, the role of most apoptotic regulators during cardiovascular development is unclear despite their great importance. Molecular pathways involving such regulators may be the targets for a variety of teratogens, as has been shown in the case of fetal alcohol syndrome.13


*    Apoptosis in the Developing Heart
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Apoptosis in the Developing...
down arrowVascular Cell Apoptosis During...
 down arrowConclusions
 down arrowReferences
 
Early studies of cell death in the cardiovascular system used vital dyes and electron microscopy to identify dead cells in avian, rodent, and human embryos, using what were essentially random sampling methods.14 15 These studies provided a foundation for future investigations of apoptosis in the developing cardiovascular system.

What Cells Undergo Apoptosis and What Is the Significance?
The ventricular and atrial compartments of the developing heart enlarge throughout development; consequently, it is not surprising that high levels of PCD have not been observed in the cardiomyocytes of these chambers. Some apoptotic myocytes were identified by TUNEL and transmission electron microscopy (TEM) in the compact and trabecular zones of the embryonic day (ED) 11 to 16 mouse ventricle,16 and TUNEL-positive cells have been identified in the trabeculae and compact zones of the mouse ventricles from ED13 to day 2 after birth.17 A study of the neonatal rat heart used incorporation of biotinylated dUTP as a marker for DNA strand breaks.18 Cardiomyocytes of the 1-day-old right ventricle (RV) were positive with a prevalence of 0.1%. The prevalence declined during the first 2 weeks of life and was 4- to 8-fold higher in the RV than the left ventricle (LV), and the authors proposed that this process might contribute to the thinning of the RV after birth. However, the prevalence of apoptosis was well below that of cell division; therefore, it is uncertain whether apoptosis serves a specific morphogenic purpose in these instances.

In contrast to the growth of the atrial and ventricular compartments, the embryonic outflow tract (OFT) shortens at specific stages of rodent,19 human,20 and avian21 22 23 development. Early studies identified dying cardiomyocytes by TEM in the conal (myocardial) portion of the chick OFT.24 25 26 Subsequent recombinant adenoviral cardiomyocyte tagging delimited the shortening of the chick OFT to ED6 to ED8 of development23 and demonstrated a coincidence of OFT shortening with OFT cardiomyocyte PCD as evidenced by TUNEL, annexin V staining, and caspase activity (see Figure 1Down). The prevalence of apoptosis was stage-dependent and reached a peak of nearly 50% of cardiomyocytes. The authors suggested that the role of the OFT cardiomyocyte PCD is to shorten and rotate the myocardial conus, to form the subpulmonic infundibular connection of the RV to the pulmonary artery (PA) anteriorly, during the transition of the embryonic heart from a single to dual circulation. It is not known whether a similar mechanism is operative in the remodeling of the mammalian OFT, where other cellular mechanisms have been proposed.19



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  		Figure 1. Figure 1Up. Shortening and rotation of the myocardial portion of the OFT coincident with cardiomyocyte apoptosis. The OFT myocardium labeled with AdCMVGFP undergoes shortening and rotation (compare A and B) in the transition from the single- to dual-circulation heart. At the same time, the atria and ventricles considerably increase in size (A and B are not to scale). Coincident with the OFT remodeling is cardiomyocyte apoptosis. C, Myocardium is delimited by an antimyosin antibody (fluorescein). D, Same section, showing dense foci of TUNEL-positive cardiomyocytes in the OFT (single arrows). There are also uncharacterized TUNEL-positive cells in the OFT and AV cushions (double arrows), which will be sculpted to form the valves of the heart. Approximate embryonic day (ED) is shown. VENT indicates ventricle, AO, aorta; LA, left atria; and RA, right atria. Other abbreviations are defined in the text.

During cardiac valve formation, cardiac cushions form as localized expansions of an extracellular matrix, known as the cardiac jelly, at the sites of atrioventricular and ventriculoarterial connections. Endothelial cells invade the cushions and transform into a mesenchymal cell type,27 28 and the cushions are sculpted to form the fine inflow (mitral, tricuspid) and outflow (aortic and pulmonary) valves and portions of the atrial and ventricular septa. It would appear that this occurs in part by apoptosis. Significant levels of apoptosis have been observed in the mesenchyme of the bulbar and the atrioventricular cushions of birds and mammals and may contribute to the morphogenesis of these structures.15 17 23

Cells originating in the neural crest migrate widely throughout the cardiovascular system and are critical to the formation of a number of cardiac structures, including the aorticopulmonary septum and the media of the great arteries.29 Retroviral labeling of the chick neural crest with lacZ indicated that neural crest cells, in addition to incorporating into these structures, also undergo apoptosis. LacZ-labeled, TUNEL-positive neural crest cells were identified in the developing OFT septum, predominantly beneath the level of the forming semilunar valves and in the endocardial cushions of later-stage (ED5 to ED12) chick embryos.30 LacZ-positive, TUNEL-positive cells were also observed at the sites of the prospective electrical conduction system of later-stage30 31 32 33 34 35 36 37 38 chicken embryos. These regions include the sites of formation of the atrioventricular (AV) node and the bundle of His and right and left bundle branches, ie, the superior aspect of the ventricular septum.31 Other investigators have also observed a high incidence of cell death at this site15 (also Watanabe M, unpublished observations, 2000) without identifying the cell type.

Why neural crest cells would migrate these long distances only to die is not clear. Apoptosis of these cells at the site of the mesenchymal septum may facilitate its replacement by myocardium in a process termed "myocardialization."32 In the region of the prospective conduction system, it has been proposed that cell death may serve as a signal to myocardial cells to differentiate into specialized conduction fibers.31 Retroviral labeling has demonstrated that Purkinje fibers originate from cardiomyocytes in response to endothelin-1 signaling from adjacent coronary arteries.33 34 35 It has also been suggested that apoptosis is involved in the normal postnatal parsing of the human AV node and His bundle.36

Apoptosis has also been observed in the developing atrial septum, the blood islands of the forming epicardium, and at the site of formation of the coronary artery orifices.37 The cell types and morphogenetic roles in these areas are yet to be characterized. Given the rapid clearance of apoptotic cells, it is likely that the incidence of apoptosis is underestimated. More sophisticated techniques for identifying apoptosis, combined with cell fate studies, will likely lead to a greater appreciation of the role of apoptosis in the development of cardiovascular structures.

Role of Apoptosis in Cardiac Malformations
The pathogenesis of most congenital heart defects (CHDs) is unknown; however, CHDs may represent developmental arrest of regional aspects of cardiogenesis, some of which may be due to insufficient numbers of cellular precursors. Studies conducted before the identification of the PCD pathway indicated that teratogens such as cyclophosphamide and glucocorticoids, as well as hemodynamic abnormalities, may cause alterations in the timing or levels of cell death in the embryonic chick OFT cushions.15 Exposure to such agents was often associated with ventricular septal defects and malalignment of the great vessels. Because these agents may affect many cellular processes, it remains to be determined whether the morphological defects were due to an effect on apoptosis.

Apoptosis has also been suggested to play a role in pathologies of the cardiac conduction system and the RV. Histological examinations of autopsy hearts of two young brothers from a family of five brothers, all of whom had isolated idiopathic AV block and arrhythmias, revealed absent or significantly reduced AV nodes, sinoatrial (SA) nodes, and internodal conduction pathways.38 TUNEL-positive cells were evident in myocytes and nonmyocytes at the sites of the AV and SA nodes from these specimens, as well as in the heart of a young woman from an unrelated family who had a similar clinical presentation and histological findings.

A primary disorder of the RV, arrhythmogenic right ventricular cardiomyopathy (ARVC), is characterized by the progressive replacement of the RV myocardium with fibro-fatty tissue in the young39 and is commonly associated with heart block.40 41 Examination of the hearts of patients at autopsy or by endomyocardial biopsy demonstrated abnormal numbers of TUNEL-positive myocytes selectively in the RV of affected patients.38 42 43 In a series of 20 patients with ARVC who were biopsied, the presence of TUNEL-positive cells in the RV biopsy material was more common in those with an acute presentation (5 of 6 patients) than those with a more insidious onset (2 of 12 patients).43

Demonstration of TUNEL-positive cells, if indicative of apoptosis, does not mean that direct activation of the PCD pathway is the primary cause of these diseases. As discussed below, there may be considerable interplay between the molecular pathways of cell differentiation and cell death so that the latter in some instances could reflect a failure of the former, rather than a primary activation of the PCD pathway. In this regard, several families have been identified with an autosomal-dominant form of congenital heart block, frequently associated with atrial septal defects and occasionally with other congenital heart defects.44 44 The patients have mutations in the gene encoding the transcription factor NKX2.5, a homeodomain protein that regulates the differentiation of cardiomyocytes in mice and flies.45 46 47 The molecular mechanisms by which NKX2.5 mutations cause disease is not known, but there is no evidence that NKX2.5 directly regulates the PCD pathway.

The identification of the molecular components of the PCD pathway (discussed below) facilitates a targeted analysis of the role of this pathway in cardiac malformations. Experiments have recently been described in which chick OFT cardiomyocyte PCD is specifically affected via recombinant adenoviral-mediated expression of activators or inhibitors of the pathway. The results suggest that changes in the levels of OFT cardiomyocyte apoptosis may lead to malalignment of the great vessels, ie, cardiac OFT defects, with associated ventricular septal defects (S.A.F., unpublished data, 2000). In mice with deletions of genes in the PCD pathway, FADD (Fas-associated death domain protein, also known as Mort-1)48 and caspase-849 die before ED11.5 and display a dilated cardiac phenotype that results in hemodynamic insufficiency. Whether this is due to an effect on the incidence of apoptosis, where in the cardiovascular system this effect occurs, and whether the dilated cardiac phenotype is primary or secondary to hemodynamic or other alterations remains to be determined.

Molecular Mechanisms
The first molecular evidence of cell death/survival machinery came in the form of the Bcl2 gene. Human Bcl2 could prevent PCD in the worm, Caenorhabditis elegans, indicating a high degree of conservation in apoptotic pathways throughout evolution. More recent studies have revealed a remarkable conservation of most members of the apoptotic pathway (reviewed by Vaux and Korsmeyer50 ). Briefly, numerous death signals and death receptors, including TNF, its receptor, and many growth factors, culminate on a pathway regulated by proapoptotic (eg, Bax, Bad) and antiapoptotic members of the Bcl2 family (eg, Bcl2, BclX).51 Relative balances of the two classes of Bcl2 proteins affect interaction of adaptor proteins, such as APAF-1 and FADD, with caspases,52 which exist as inactive zymogens but become activated on interaction with proteins such as APAF-1. Caspase activation often leads to a feedback loop resulting in amplification of cell death signals.53 A class of IAPs is thought to function in part by inhibiting caspases.54 Caspase-mediated cleavage of numerous essential proteins ultimately results in cellular demise. Regulation of cell death is also mediated through overlapping pathways involving the tumor suppressor p53.55

Within the heart, little is known regarding the molecular basis for PCD. As with most organs, appropriate apoptosis is necessary for tissue remodeling, particularly within the inflow and outflow tracts of the heart, as described above. Surprisingly, most components of the apoptotic pathways described above are expressed in the developing heart, although their expression has not been examined in detail and their role remains unclear. Which factors play important roles in development is unknown, but it is likely that numerous divergent apoptotic pathways converge on a final execution pathway.

Numerous components of the apoptotic pathway have been mutated in mice but, as indicated above, only null mutation of caspase-8 or FADD affects cardiac development.48 49 The trabeculae in both mouse models were disorganized and hypoplastic. Oddly, the phenotype was opposite what one might expect after disruption of proapoptotic pathways, with fewer rather than more cells present. These findings may suggest that the function of proteins involved in apoptosis depends in part on the cellular environment and interactions with other death and survival pathways.

It is possible that the spatiotemporal specificity of cell death during cardiac development is achieved through cell-specific regulatory pathways. Disruption of the retinoic acid pathway by gene targeting of RXR{alpha} and RARß in combination results in increased apoptosis of the OFT mesenchyme and subsequent conotruncal defects.56 57 Similarly, deletion of the signaling peptide, endothelin-1, or its receptor, ETA, causes a variety of OFT and aortic arch defects.58 59 The neural crest–derived pharyngeal arches display higher than normal levels of apoptosis in endothelin-1/ETA mutants, suggesting that the endothelin pathway in part regulates survival of cardiac neural crest cells. dHAND, a tissue-specific basic helix-loop-helix transcription factor, is downstream of the endothelin-1 signaling cascade and is necessary for survival of neural crest–derived mesenchyme, possibly through regulation of the homeobox gene Msx1.60 Mice lacking dHAND also display hypoplasia of the right ventricular segment.61 Our recent studies indicate that dHAND is necessary for survival of cells after they have been specified to the right ventricular lineage. The proapoptotic Bcl2 binding factor Nip3 was found to be upregulated in the dHAND mutant heart in a screen for mediators of dHAND function (A. Aiyer and D. Srivastava, unpublished observations, 2000). These examples of tissue-specific signaling and transcriptional pathways that regulate cell survival suggest that general and specific pathways converge to regulate decisions of cell death and cell survival.


*    Vascular Cell Apoptosis During Development
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowApoptosis in the Developing...
 *Vascular Cell Apoptosis During...
down arrowConclusions
 down arrowReferences
 
The vascular system, like the heart, continuously remodels throughout development, first as primitive vessels form and reorganize, then as the circulation accommodates the demands of organ development and adaptation to ex utero life after parturition. Although tissue growth is the most obvious feature of vascular development, many embryonic and later vessels regress or are totally deleted. Examples of the latter include the paired first, second, and fifth aortic arches and one of the fourth arches (right in mammals, left in birds), as well as vessels in the microvasculature, especially at sites of bone formation.62 63 These processes may be dominated by cell death, although cell migration or transdifferentiation to mesenchyme,62 matrix degradation, and internal division of vessels to form multiple smaller channels (intussusception)64 65 may also occur. In addition to vessel deletion, the vasculature undergoes continuous reorganization during development that occasionally is very dramatic. For example, the aortic origin of the embryonic vessel destined to become the left subclavian artery migrates from the dorsal aorta to the left aortic arch and bypasses other aortic branches, including the ductus arteriosus, before achieving its final position proximal to the latter vessel.64 The mechanisms underlying such reorganization of vessels have received little study but likely involve highly coordinated regulation of cell migration, cell proliferation, and/or cell death. Apoptotic cell death even contributes to developmental vascular remodeling that is dominated by tissue growth.66 67 Apoptosis in these vessels probably reflects the demands for independent adjustment of vessel diameter, wall thickness, and length in accord with the demands of hemodynamics and growth of contiguous tissues. Even in quiescent mature vessels, a low rate of cell death prevails.68

A role for cell death in the development of the vasculature was first recognized decades ago. In 1918, Clark69 had argued that all endothelial cells of regressing embryonic vessels retracted into neighboring vessels; however, he later described degeneration and "disappearance" of smooth muscle in these vessels.70 Early descriptions of ultrastructurally identifiable apoptosis of endothelial cells were based on TEM showing vascular changes in the regressing corpus luteum71 ; however, there has subsequently been surprisingly few studies of the role of cell death in vascular development.

Hemodynamics and Apoptosis
The mechanical forces imposed on arterial tissue are important stimuli for developmental vascular remodeling.72 73 Chronic changes in blood flow rates cause corresponding changes in arterial diameters, whereas alterations in blood pressure affect wall thickness. By these means, vascular structures continually adapt to changes in hemodynamic loads. Fluid shear stress in the case of flow and circumferential tensile stress in the case of pressure elicit this remodeling. Modulation of these hemodynamic loads in developing arteries affects extracellular matrix accumulation and remodeling, and it influences accumulation of vascular cells in the vessel wall.72 Recent work indicates that sensitivity of apoptosis of vascular cells to mechanical forces is important in vascular growth regulation.

Cho et al66 focused on the immediate perinatal period because of the profound arterial remodeling that accompanies physiological adjustments to parturition. Examination of vascular cell kinetics in this period, using a Monte Carlo analysis, demonstrated that arterial cell proliferation rates in the lower aorta much overestimated cell accumulation in this vessel. Additional experiments demonstrated that this disparity was due to high rates of apoptosis postpartum. Apoptosis in this vessel contributes to profound narrowing and tissue growth arrest in the lower aorta after birth, in concert with a 95% reduction in blood flow rate that is largely due to closure of the downstream umbilical arteries. Subsequent studies confirmed that experimental changes in arterial blood flow rates could both initiate apoptosis and suppress cell proliferation rates in developing arteries.67 Indeed, application of techniques that permitted assessments of daily cell death rates indicated that apoptosis rates exceeded cell proliferation rates in the first days after 70% flow reduction in young rabbit carotid arteries.

An elegant series of experiments by Dimmeler et al74 75 demonstrated that both production of nitric oxide (NO) and activation of the phosphatidyl inositol (PI) 3'-kinase pathway by shear stress are antiapoptotic for endothelium (Figure 2Down). A link between these two signals was provided by demonstrations that one of the downstream targets of PI 3-kinase–dependent kinase signaling, AKT, phosphorylates and activates endothelial nitric oxide synthase (eNOS),76 although chronic shear stress also upregulates expression of eNOS.77 78 79 80 We recently found that in vivo inhibition of the PI 3-kinase pathway suppresses flow sensitivity of apoptosis in developing arteries (Yazer M, Cho A, and Langille BL, unpublished data, 1999). Subsequent in vivo81 82 and in vitro83 studies have shown that reductions in blood pressure/wall tension also upregulate arterial cell apoptosis; however, the developmental implications of these observations have not been elucidated. The dramatic decline in pulmonary arterial pressures at parturition, in concert with much increased pulmonary blood flows, could provide an intriguing model for the study of hemodynamic influences on vascular cell death.



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  		Figure 2. Figure 2Up. Regulation of apoptosis in the developing vascular system. Red arrows indicate proapoptotic, green arrows prosurvival, pathways. Hemodynamic shear stress inhibits apoptosis through activation of PI 3'-kinase, phosphorylation of eNOS by AKT, upregulation of eNOS gene expression, and possibly by activation of the VEGF receptor Flk-1. NO suppresses apoptosis in ECs but may induce death of smooth muscle cells, probably via peroxynitrite production (see text). Shear stress also upregulates endothelial expression of PDGF and TGF-ß, which are high in developing arteries. These regulators of cell proliferation and matrix elaboration also control vascular cell apoptosis rates. IGF-1 (not shown) is also a potent survival factor for SMCs. ANG-1 is developmentally expressed and inhibits endothelial cell apoptosis, at least in part, by activating the PI 3'-kinase pathway. Extracellular matrix, or matrix degradation products, also can provide survival signals to vascular cells via integrin activation that can be suppressed by a noncatalytic breakdown product of MMP-2 and PEX. EC indicates endothelial cell; SMC, smooth muscle cell. Other abbreviations are defined in the text.

Not surprisingly, extensive postnatal apoptosis occurs in vessels that regress after birth, ie, the umbilical arteries66 84 and ductus arteriosus.84 The extent to which death of these highly specialized cells is linked to hemodynamics is unknown. Coincident upregulation of proapoptotic members of the Bcl2 family (Bax and the short form of BclX) was observed in umbilical arteries.84 Importantly, Kim et al84 also observed apoptosis near large arterial branch sites postpartum, a finding consistent with a role for cell death in remodeling of arterial bifurcations during perinatal development.

Studies of the influence of hemodynamics on vascular cell apoptosis have focused on large arteries in later development. Their roles in early embryology and in the developing microcirculation have received less study, despite observations that hemodynamic perturbations dramatically affect remodeling of these vessels.85

Apoptosis in the Developing Microvasculature
Meeson et al86 87 have examined the transient vasculature of the pupillary membrane of the eye to study apoptosis during developmental vascular regression. They propose a model of macrophage-dependent initiating apoptosis that induces flow stasis, followed by further, stasis-dependent secondary apoptosis. Vascular endothelial growth factor (VEGF) inhibited apoptosis in this system via its Flk-1 receptor, a finding consistent with previous inferences that VEGF is a survival factor for endothelium.88 89 VEGF, like shear stress, promotes endothelial cell survival activation of the PI 3-kinase pathway.88 Interestingly, Chen et al90 report that shear stress can activate Flk-1 signaling, so there may be overlap between these survival pathways. VEGF also upregulates expression of the caspase inhibitor survivin.91 Drake et al and Brooks et al92 93 have found that matrix interactions with endothelial {alpha}vß3 integrin, which is upregulated during angiogenesis, promote cell survival. This finding is consistent with other reports of matrix-mediated survival of endothelium94 95 as well as smooth muscle.83 96 There also appears to be a role for matrix degradation in endothelial cell survival. Cryptic RGD sequences in native collagen are made accessible to {alpha}vß3 integrin after cleavage by matrix metalloproteinase (MMP).97 Thus, the matrix degradation that facilitates endothelial cell migration during angiogenesis may promote survival of these vessels. Under other circumstances, it is possible that extensive matrix degradation, which often accompanies apoptosis, may ultimately deprive vascular cells of matrix-related survival signals and thereby promote transition to an apoptotic pathway. Also, there is now evidence that a noncatalytic fragment of MMP-2 (hemopexin-like domain, PEX) can block matrix-integrin interactions during angiogenesis98 and thereby inhibit cell survival signals. Given that matrix remodeling and cell death often are coordinately regulated during development, it is likely that further matrix-receptor interactions will prove important in regulation of apoptosis in the developing vasculature.

Regulation of Vascular Cell Apoptosis During Development
Control of apoptosis in the vasculature has been extensively reviewed99 100 101 102 ; therefore, we focus on those aspects that are potentially most important in vascular development.

Interestingly, much of the progress that has been made in understanding apoptosis in vascular (and other) tissues has focused on its inhibition through production of survival factors, including both soluble factors and extracellular matrix, probably because many cells appear poised for apoptosis that must be suppressed for their survival. For example, both endothelial cells and vascular smooth muscle normally express the proapoptotic receptor Fas, and endothelial cells express the Fas ligand (FasL),103 but autocrine induction of apoptosis appears to be suppressed by the inhibitor of downstream signaling, FLICE-inhibitory protein (FLIP).104 FLIP is highly regulated in smooth muscle in response to vascular injury,105 which elicits partial reversion of these cells to a developmental phenotype,106 so a role in normal vascular development is an attractive, but unproven, hypothesis. Fas-mediated vascular smooth muscle cell death appears particularly interesting given that different subpopulations of these cells display different susceptibilities.107

Survival of vascular and other cells during development appears to be controlled by mitogens that are also important in regulating proliferation (Figure 1Up). Such a role was cited above for VEGF and this inference is consistent with the underdevelopment of the aorta and reduced vascular density reported for mice that are heterozygous for null mutation of VEGF.108 VEGF homozygotes display extreme defects in vasculogenesis that probably have multiple origins. It is noteworthy that modest VEGF overexpression in embryos results in selective enlargement of epicardial coronary vessels,109 where significant apoptosis normally occurs,37 so a role for control of local cell death may be particularly important to the growth of this vascular system. The sensitivity of vascular development to both heterozygous mutation and modest overexpression of VEGF underscore the importance of tight control of VEGF expression to normal vascular development.

Fibroblast growth factors (FGFs) also promote survival of endothelium and smooth muscle,110 111 whereas the suppressor of endothelial cell growth, transforming growth factor-ß (TGF-ß), promotes endothelial apoptosis while providing a survival stimulus for smooth muscle.112 Similarly, platelet-derived growth factor (PDGF) and insulin-like growth factor-1 are potent survival factors for smooth muscle.113 The angiopoietins appear to play a primary role in assembly of the blood vessel wall and in regulating angiogenesis,114 115 but angiopoietin-1 (ANG-1) also promotes endothelial cell survival,116 apparently through activation of the PI 3'-kinase pathway.117 This finding is consistent with observations that mice with null mutation of the receptor for ANG-1, Tek (Tie-2), display subnormal populations of endothelium.118 It is unclear whether apoptosis is also related to the abnormal vascular branching patterns seen in these mice,119 which die at mid-gestation.

NO is a bifunctional regulator of apoptosis (for review, see Kim et al120 ) that very often induces cell death, including death of smooth muscle cells,121 most often through formation of peroxynitrite that may induce DNA damage and increase p53 activity. However, NO inhibits endothelial apoptosis74 120 through inhibition of caspases, particularly caspase-3, and this mechanism likely participates in shear stress–related endothelial survival that was described above. Given the highly modulated expression of nitric oxide synthase isoforms during development of blood vessels,122 NO production is potentially an important regulator of developmental vascular apoptosis.

Cell-matrix interactions are potent modulators of vascular cell proliferation,123 migration,124 125 and survival.126 The {alpha}vß3 integrins were cited above as being particularly important in vascular cell survival during development, although other integrins are also important. The promiscuous {alpha}vß3 integrins interact with multiple matrix constituents, but tenascin-C is of proven importance in developmental control of vascular smooth muscle cell apoptosis as well as epidermal growth factor–mediated proliferation.127 Interaction of vitronectin with {alpha}vß3 and/or {alpha}vß3 integrins regulates endothelial cell survival,95 and survival is also promoted by interaction with antibodies that recognize ß1 integrins.126


*    Conclusions
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowApoptosis in the Developing...
 up arrowVascular Cell Apoptosis During...
 *Conclusions
down arrowReferences
 
During morphogenesis and subsequent development, cells of the cardiovascular system differentiate, proliferate, migrate over large distances, and they elaborate, degrade, and remodel extracellular matrix. It is now clear that tightly controlled apoptosis is an important addition to this repertoire of remodeling modalities. Apoptosis appears to be particularly important in reshaping of cardiac and vascular structures in early morphogenesis, and new links between aberrant control of apoptosis and congenital defects point to some exciting avenues for future work. During later development, apoptosis contributes to regulation of growth of established cardiovascular tissues in accord with changing hemodynamic demands imposed on them and with changing demands of the tissues they perfuse. Our current knowledge concerning regulation of apoptosis in the developing cardiovascular system is primitive. Intriguing recent findings indicate important roles in the heart for the retinoic acid pathway and for dHAND-mediated transcriptional control under the regulation of endothelin. In the vasculature, attention has focused on hemodynamic loads, mitogenic survival factors, and extracellular matrix as regulators of apoptosis, and some aspects of downstream signaling have been elucidated. Much more work is needed, especially to determine whether specific cells of the cardiovascular system have a genetically determined susceptibility to apoptosis that is regulated by the expression of pro- and antiapoptotic factors. It is likely that continuous variations in cardiovascular cell phenotype during development will be associated with different modes of control of cell death and with variable susceptibility to apoptosis during normal and pathological development of the cardiovascular structures.


*    Acknowledgments
 

S.A.F. received support from the National Institutes of Health (NIH) and the American Heart Association and thanks Drs Michiko Watanabe and Florence Rothenberg for help with the preparation of the manuscript. B.L.L. is a Career Investigator of the Heart and Stroke Foundation of Ontario. D.S. acknowledges support from the NIH and the March of Dimes.


*    Footnotes
 
1 All authors contributed equally to this work. Back

Received September 5, 2000; revision received September 29, 2000; accepted September 29, 2000.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowApoptosis in the Developing...
 up arrowVascular Cell Apoptosis During...
 up arrowConclusions
 *References
 

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