The Development of the Embryonic Outflow Tract Provides Novel Insights into Cardiac Differentiation and Remodeling



The Development of the Embryonic Outflow Tract Provides Novel Insights into Cardiac Differentiation and Remodeling Yasuyuki Sugishita, Michiko Watanabe, and Steven A. Fisher*

The embryonic cardiac outflow tract (OFT) connects the developing ventricles with the aortic sac. In birds and mammals, OFT cardiomyocytes are generated from a bsecondary (anterior),Q heartforming field well after the formation of the primitive heart tube. The OFT cardiomyocytes have unique properties and developmental fates as compared with the myocytes of the atrial and ventricular chambers. Many of the OFT cardiomyocytes of the avian embryo are eliminated by programmed cell death (PCD) during OFT remodeling in the transition from a single- to a dual-series circulation. Targeted PCD gain and loss-of-function studies indicate that PCD drives the shortening and rotation of the OFT required for the aorta and pulmonary artery to connect with the left and right ventricles, respectively. Defects in this process model aspects of the relatively common and often life-threatening congenital human conotruncal heart defects. Using indicators of tissue hypoxia, we suggest that OFT myocardial hypoxia may be the trigger for the PCD-dependent remodeling of the OFT. This review discusses these aspects of the formation and remodeling of the embryonic OFT in the context of the broader questions of cardiac muscle biology. (Trends Cardiovasc Med 2004;14:235–241) D 2004, Elsevier Inc. Recent investigations have demonstrated tremendous and unanticipated plasticity in the heart muscle cell lineage. Whereas not long ago the dogma was that cardiomyocytes enter a terminally differentiated state in the neonatal period, there is now evidence that all Yasuyuki Sugishita and Steven A. Fisher are at the Department of Medicine and Michiko Watanabe is at the Department of Pediatrics, Case Western Reserve University, Cleveland, Ohio, USA. * Address correspondence to: Steven A. Fisher, MD, Department of Medicine, Case Western Reserve University, 422 Biomedical Research Building, 2109 Adelbert Road, Cleveland, OH 44106-4958, USA. Tel.: (+1) 216-368-0488; fax: (+1) 216-368-0507 e-mail: [email protected]. D 2004, Elsevier Inc. All rights reserved. 1050-1738/04/$-see front matter

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cardiomyocytes, to varying degrees, are capable of the full repertoire of cell behaviors, including proliferation, migration, programmed cell death (PCD), and renewal from stem cell populations (reviewed in Nadal-Ginard et al. 2003). The highly dynamic growth and remodeling of the embryonic heart provides an excellent system in which to study the diverse behaviors of cells of the heart muscle lineage. This review focuses on cardiomyocytes of the outflow tract (OFT) compartment of the embryonic heart. These cells have unique properties and developmental fates and are subject to unique environmental influences. Elucidation of the behaviors of these cells may have broader implications for understanding the remodeling of the mature heart in disease states.

The Unique Origin of OFT Cardiomyocyte, the bAnterior or Secondary Heart-Forming FieldQ

Histologic analyses of heart development prior to the era of molecular biology identified a region of the mesoderm that gives rise to differentiated cardiomyocytes and thus is termed the bcardiac crescentQ or the bprimary heart-forming fieldQ (reviewed in Fishman and Chien 1997). In this field, mesenchymal cells differentiate into cells that have the features of cardiomyocytes, including spontaneous depolarization and conducted action potentials; myofibrils and force generation; and the expression of transcription factors such as Nkx, GATA, HAND, and SRF proteins that drive the muscle gene program (reviewed in Srivastava and Olson 2000). The differentiation of cardiomyocytes in the anterolateral mesoderm occurs very early in avian (D1-2) development and later in mammalian (mouse D7-8, human D20) development, but similar in timing relative to gastrulation. These cells migrate to the midline and fuse to form the primitive heart tube, and contribute to the atria and ventricles of the vertebrate heart. The differentiated cardiomyocytes of the primary heart tube are highly proliferative, resulting in tremendous increases in the size of the primitive heart tube. Histologic analyses of avian and mammalian embryos suggested that mesenchymal cells continue to differentiate into a cardiomyocyte phenotype at the arterial and venous poles of the heart after the formation of the primary heart tube (Argqello et al. 1975, Viragh et al. 1989). Further evidence for the late addition of myocardium to the arterial pole of the heart came from the tissue tagging studies of De la Cruz et al. (1977). Labeled particles placed at the cephalic end of the chicken embryonic heart tube at Hamburger-Hamilton (HH) stage 12 embryonic day 2 (ED2) (Hamburger and Hamilton 1951) were found in the middle of the conal myocardium at HH22 (ED4), consistent with the addition of myocardium to the arterial pole. This portion of the embryonic heart is commonly referred to as the OFT—also termed the conus and truncus by some investigators (see Pexieder 1995)—and connects the ventricle to the aortic sac in the primitive single circulation heart. This embryologic structure received minimal

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attention in the ensuing decades as the role of the neural crest cells in the septation of the cardiac OFT was investigated (reviewed in Creazzo et al. 1998). The OFT myocardium has recently received renewed interest because several laboratories have used molecular techniques to study the formation and fate of this tissue. Groups led by Markwald and Kirby used recombinant adenovirus expressing a reporter protein, or early endogenous markers of the cardiomyocyte lineage, to show that cardiomyocytes were added to the arterial pole of the avian heart from splanchnic mesoderm beneath the caudal pharynx (Mjaatvedt et al. 2001, Waldo et al. 2001). Buckingham and co-workers used the integration of a LacZ reporter into the FGF10 gene locus to come to a similar conclusion in mice (Kelly et al. 2001). The investigators proposed that these cardiomyocytes are generated from a bsecondary or anterior heartforming fieldQ well after the primary myocardium has formed (reviewed in Kelly et al. 2001). These studies have raised a number of new questions regarding cardiomyocyte differentiation: Is a preconditioning stimulus required for the cells to be receptive to potential inducing stimuli? Do bone morphogenetic proteins and fibroblast growth factors, which are released from the adjacent endoderm to induce the cardiomyocyte gene program in the primary heart-forming field (Lough et al. 1996, Schultheiss et al. 1997), play a similar role in the bsecondary/anterior Q field? How much of the OFT myocardium is added from the bsecondary heartforming fieldQ? Could cells be induced at any time to become cardiomyocytes, given the right signals? 

Differentiated Properties of the OFT Cardiomyocytes

Many properties distinguish the OFT cardiomyocytes from atrial or ventricular cardiomyocytes. A complete discussion of the differences between OFT and atrial and ventricular cardiomyocytes is beyond the scope of this review, and is reviewed elsewhere (Franco et al. 1998). The differences can be approximated with the generalizations that OFT cardiomyocytes have lower rates of proliferation, have a more bprimitiveQ contractile phenotype with less well-

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developed sarcoplasmic reticulum and persistent expression of smooth muscle a-actin, and have slower impulse conduction as compared with ventricular myocytes of the same developmental stage (Christoffels et al. 2000, Ruzicka and Schwartz 1988, Thompson et al. 1990). These properties are consistent with the proposed Windkessel and sphincteric functions of the OFT chamber in early development and in lower vertebrates. The molecular bases of the distinct properties have not been studied systematically. Whereas a few transcription factors have been shown to be selectively expressed in the OFT myocardial compartment—for example, the homeodomain protein Pitx2C (Liu et al. 2002)—the extent to which phenotypic differences reflect differences in intrinsic gene regulatory programs or unique external cues (hemodynamics or cell signals) remains unknown. 

OFT Remodeling During the Transition from a Single to a Dual Circulation

The OFT of warm-blooded animals (birds and mammals) undergoes dramatic remodeling in the transition of the embryo from the primitive single- to the dual-series circulation (ontogeny recapitulating phylogeny). In this section, we focus on a model that we have proposed in which myocardial hypoxia is the trigger for the PCD-dependent remodeling of the OFT. For more comprehensive reviews of OFT morphogenesis, see the reviews by Webb et al. (2003) and Ya et al. (1997). The sequence of discoveries is similar to that described above. Histologic and tissue-tagging studies of embryonic avian and mammalian (including human) hearts in the 1970s and 1980s suggested that at subsequent stages of development, approximately co-incident with septation of the heart, the OFT myocardium shortens to as little as onefourth of its maximum length (De La Cruz et al. 1977, Dor and Corone 1985, Goor et al. 1972, Thompson et al. 1987) as the forming aorta rotates to a posterior position to connect to the left ventricle. Histologic studies performed in the 1970s by Hurle and Ojeda (1979) and Pexieder (1975) identified cell death in the OFT myocardium and adjacent mesenchymal cushions, and suggested this as a mechanism for the retraction of the OFT

myocardium, but, again, there was little study of these processes over the subsequent decades. In the mid-1990s, we developed a method of gene delivery to the embryonic avian heart adapted from retroviral fate mapping pioneered by Sanes and Cepko (Cepko 1988, Sanes et al. 1986) and utilized by Mikawa et al. (1992) to show clonal expansion of embryonic ventricular cardiomyocytes. When recombinant, replication-defective, adenovirus-expressing LacZ or green fluorescence protein (GFP) tags under the control of the cytomegalovirus (CMV) enhancer/promoter were injected into the pericardial space of stage 14–18 (ED2.5–4) embryos, expression of the tag was observed only in the OFT cardiomyocytes (Figure 1A) (Watanabe et al. 1998 and 2001). In contrast, when the same experiment was performed with the Rous sarcoma virus promoter/enhancer driving transgene expression, all of the cardiomyocytes of the embryonic heart expressed the tag (Fisher and Watanabe 1996). The reason for the unique pattern of expression from the CMV promoter/ enhancer is still unknown. The CMV enhancer contains many regulatory elements, including nuclear factor nB and retinoic acid binding sites, and is also regulated in transgenic mice (Koedood et. al 1995). The specific expression of the marker protein in the avian OFT cardiomyocytes provided a novel tool to follow the fate of these cells during OFT remodeling over a period of 3 to 4 days, within the window of transient transgene expression achieved with recombinant adenovirus. We showed that the OFT cardiomyocytes reorganize from a tubular structure that connects the forming ventricles to the aortic sac, to a wedge-shaped myocardium connecting the right ventricle to the pulmonary artery in the dual circulation stage 35 (ED9) embryo (Figure 1A) (Watanabe et al. 1998 and 2001). The latter structure is commonly referred to as the subpulmonic infundibulum, whereas the left-sided circulation lacks such a structure, because the aorta connects directly to the left ventricle. The disappearance of many of the GFPlabeled (or LacZ-labeled) cardiomyocytes suggested that they may be eliminated by cell death; by narrowly defining the period when this occurs (~2 days of development; ED5–7) we were able to intensely sample for markers of PCD at TCM Vol. 14, No. 6, 2004

Figure 1. Development and remodeling of the embryonic chick cardiac outflow tract (OFT) (A) The OFT myocardium of the stage 25 embryonic day 5 (ED5) and stage 36 (ED10) chick heart is labeled with recombinant adenoviralexpressed red fluorescent protein. The heart and vessels are filled with blue ink in A and blood in B. Note the absolute reduction in the length of the OFT between these two stages, as well as the relative reduction in relation to the ventricles, which have increased in size by cardiomyocyte proliferation. The OFT has also been divided by septation into pulmonic and systemic outflows, and the aortic root has rotated to a posterior position, where it connects with the left ventricle. The OFT cardiomyocytes constitute the infundibular connection of the right ventricle to the pulmonary artery in the four-chambered heart. (B) Stage 29 chick embryonic heart stained with LysoTracker Red (LTR), a marker for apoptosis. LTR selectively accumulates in cardiomyocytes of the proximal OFT (arrow) with a time course that parallels the shortening and rotation shown in A. (C) Whole-mount staining of stage 30 (ED6) quail embryonic heart with QH1, an antibody directed against an epitope specific for cells of the endothelial cell lineage (in quail only). Proendothelial cells are observed over the surface of the ventricle (arrow) and are more concentrated over the OFT, where they have organized into a vascular network. (D) The nitroimidazole EF5 is used to indicate the relatively hypoxic regions of the stage 30 (ED6) embryonic chick heart. After infusion of EF5, binding was detected with a Cy3-conjugated antiEF5 monoclonal antibody. The section was co-stained with Sr-1, an antibody against sarcomeric a actin, to delineate the myocardium. EF5 was detected in the OFT myocardium (arrowheads), suggesting that the OFT myocardium is relatively hypoxic during the period of programmed celldeath-dependent remodeling of the OFT and vasculogenesis. Bars, 500 Am. Ao, aorta; LA, left atrium; LV, left ventricle; PA, pulmonary artery; RA, right atrium; RV, right ventricle.

Figure 2. Working model for the developmental remodeling of the avian cardiac outflow tract (OFT). OFT myocardial hypoxia results from the high diffusion distance for oxygen from the blood in the lumen through the endocardial cushion mesenchyme to the OFT myocardium. Hypoxia directly or indirectly triggers a number of responses, including cardiomyocyte programmed cell death and recruitment of endothelial progenitor cells and vasculogenesis. Hypoxia signaling through the transcription factor hypoxia-inducible factor (HIF)-1a may induce the expression in cardiomyocytes of survival genes—for example, vascular endothelial growth factor receptor 2 (VEGFR2), which is expressed in the distal OFT myocardium. HIF-1a is also reported to drive the transcription of proapoptotic Bcl2 family members such as BNip3 and Bax, but their patterns of expression in the developing heart are not known. o, apoptotic cardiomyocytes; —, endothelial cells forming vascular network.

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these specific stages. Using a number of markers of PCD—including terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) technique, annexin-V binding, lysosomal dyes, measurements of caspase activity in vitro and cleaved caspase-3 in situ, and histologic analysis by light and transmission electron microscopy—we showed that the prevalence of cardiomyocyte PCD temporally correlated with the shortening and rotation of the avian OFT in the transition to a dual circulation (Figure 1B) (Schaefer et al. 2004, Watanabe et al. 1998). This analysis revealed a broad distribution of PCD in the proximal OFT myocardium early in remodeling, and concentrated PCD in myocardium underneath the forming aorta as it drops down and behind the pulmonary artery to connect to the left ventricle. In sections, we also observed at a very specific stage of development (stage 31; ED6) a focus of cell death in the distal OFT myocardium, with 50% or more of the cardiomyocytes in this region positive for markers of apoptosis. These observations suggested the hypothesis that regulated elimination of OFT cardiomyocytes by PCD may serve specific morphogenic purposes. We showed that adenoviral-mediated forced expression of the X-linked inhibitor of apoptosis protein (an inhibitor of caspase enzymes) targeted to the OFT cardiomyocytes, markedly attenuated OFT shortening and rotation (Watanabe et al. 2001). This resulted in the aorta side-by-side the pulmonary artery and connecting via the myocardial infundibulum to the right ventricle—i.e. double outlet right ventricle (DORV) with transposition of the aorta. Treatment of embryos with soluble peptide inhibitors of caspase enzymes resulted in a similar OFT shortening defect, but with additional valve and trabecular defects (Watanabe et al. 2001). Complementary PCD gain-offunction experiments by forced expression of a death (Fas) ligand also resulted in malrotation of the aorta, as well as absence of the subpulmonic infundibulum (Sallee et al. 2004). These experiments demonstrate that the subpulmonic infundibulum is a remnant of the OFT myocardium, and that the temporally and spatially restricted elimination of OFT cardiomyocytes is necessary for the remodeling of the OFT (shortening and rotation) in the transition to a dual cir-

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culation. Interestingly, experimentally blocking the addition of myocardium to the chick OFT also leads to conotruncal defects (Yelbuz et al. 2002), suggesting that perturbations of formation or remodeling of the OFT myocardium may lead to conotruncal defects. The remodeling of the OFT is highly complex. Other processes shown or implicated include the recruitment of cells from the neural crest that are required for the septation of the OFT (reviewed in Creazzo et al. 1998), the recruitment of endothelial progenitors from the embryonic epicardium to form the coronary arteries (Figure 1C), and the migration of myocardium into the mesenchyme to form the muscular septum (bmyocardializationQ) (van den Hoff et al. 1999). Whether these processes are linked has not received much investigation (but see below). We have shown that the incidence of PCD in the OFT is not reduced in embryos in which the bcardiac neural crestQ has been ablated (Rothenberg et al. 2002), indicating that neural crest cell-dependent septation of the OFT and PCD-dependent OFT shortening and rotation are independent processes. These studies in the chick embryo raise the possibility that PCD-dependent remodeling of the OFT is a target of environmental or genetic teratogens associated with congenital defects in the human cardiac OFT. OFT defects grouped as conotruncal defects account for a significant percentage of congenital human heart defects and include Tetralogy of Fallot, DORV, transposition of great arteries, and persistent truncus arteriosus (reviewed in Liberthson 1989). Common to many of these disorders is the malformation or the malposition of the myocardial infundibular connection between the great vessels and the ventricles (Van Praagh et al. 1983). A key question is whether the remodeling of the mammalian embryonic cardiac OFT also requires cardiomyocyte PCD. PCD has been described in the developing mouse cardiac OFT myocardium and endocardial cushions (Kubalak et al. 2002, Sharma et al. 2004, S Fisher and M Watanabe, unpublished observations), but its significance has not been established. Inactivation of a number of genes in the PCD pathway, including caspase-8, FADD, and FLIP, cause early embryonic lethality in mice (Var-

folomeev et al. 1998, Yeh et al. 1998), but the cell type or organ that is primarily affected has not been established. 

Triggers of OFT remodeling

What is the trigger for the site- and stage-specific PCD of the cardiomyocytes of the OFT? One clue was that PCD-dependent remodeling of the OFT is approximately co-incident with the recruitment of proendothelial cells to the OFT and their organization into a subepicardial vascular network (Figure 1C) (Rothenberg et al. 2002, Sugishita et al. 2004b), which will eventually form the coronary arteries. The co-incidence of PCD and vasculogenesis suggested that myocardial hypoxia could be a trigger. Direct measurements of cellular oxygen concentrations in vivo are problematic. We therefore used the nitroimidazole EF5 [2-(2-nitro-1H-imidazol-1-yl)-N(2,2,3,3,3-pentafluoropropyl) acetamide] as a surrogate marker for cellular oxygen concentrations (Evans et al. 1997). This and related compounds are reduced by nitroreductases under hypoxic conditions and form covalent adducts to macromolecules in cells. EF5 binding is detected with an anti-EF5 monoclonal antibody. EF5 binding was specifically detected in the OFT myocardium from stages 25 to 32, with a peak at stage 30 (Figure 1D), paralleling the prevalence and time course of cardiomyocyte apoptosis and OFT remodeling (Sugishita et al. 2004a). We also detected the hypoxia-inducible transcription factor (HIF)-1a specifically in the nuclei of OFT cardiomyocytes, paralleling EF5 binding (Sugishita et al. 2004b). Incubation of embryos in ovo in hyperoxic or hypoxic air produced the expected decrease or increase in EF5 binding and HIF-1a nuclear accumulation, indicating that these were sensitive to oxygen concentrations. In addition, continuous exposure of embryos to hyperoxia (95% O 2 /5% CO 2 ) significantly attenuated the PCD-dependent remodeling of the OFT (Sugishita et al. 2004a), providing experimental support for the role of OFT myocardial hypoxia in OFT remodeling. The reason for the hypoxia of the OFT myocardium relative to the other compartments is suggested by anatomic features. The ventricles are highly trabeculated, facilitating the diffusion of oxygen at a stage TCM Vol. 14, No. 6, 2004

of development when the oxygen supply is diffusion limited (prior to coronary vasculogenesis). In contrast, the untrabeculated OFT myocardium is adjacent to endocardial mesenchyme (cushions), approximately 300 Am thick (Figure 1D), well above the estimated limit of oxygen diffusion of 100 Am (Popel et al. 2003). What might be the mechanisms by which hypoxia triggers remodeling of the OFT? Oxygen deprivation may cause necrosis or PCD; the response presumably represents the balance between energy demand and energy supply and the adaptations of actively contracting cardiomyocytes (reviewed in Bishopric et al. 2001, Semenza et al. 1999). The developing heart provides a unique model of diffusion-limited myocardial hypoxia, because there is no vascular supply at the early stages of development. What might be the hypoxic responses? We have shown that the hypoxic OFT cardiomyocytes express the vascular endothelial growth factor receptor 2 (VEGFR2), a protein most commonly associated with the endothelial cell angiogenic response. However, VEGFR2 is also expressed in other mature cell types in response to hypoxia/ischemia (Jin et al. 2000, Ogunshola et al. 2002, Rissanen et al. 2002), and cells that express VEGFR2 in the mouse may contribute to muscle lineages (Motoike et al. 2003). We have also shown that the adenoviral-mediated expression of a dominant negative form of the downstream kinase Akt (PKB) increases the prevalence of PCD of the OFT cardiomyocytes (Sugishita et al. 2004b). These findings suggest a model in which VEGF functions in both autocrine signaling through Akt, to protect the OFT cardiomyocytes from hypoxic cell death; and paracrine signaling, to recruit endothelial progenitor cells to the OFT as a vasculogenic response to hypoxia (see model in Figure 2). Other prosurvival signals may be regulated by hypoxia/HIF—for example, insulin-like growth factors and their binding proteins—but their roles in development have not been examined. Many of the OFT cardiomyocytes are eliminated by PCD during the transition to a dual circulation. Hypoxia (or ischemia) may trigger either the intrinsic (mitochondrial) or extrinsic (death liganddependent) (Scarabelli et al. 2001, Yaniv et al. 2002) pathway of PCD. One pathTCM Vol. 14, No. 6, 2004

way that has been identified in vitro is the HIF-1a-dependent induction of expression of the proapoptotic Bcl-2 family members Nip3 and Nix (Bruick 2000, Kubasiak et al. 2002, Regula et al. 2002, Sowter et al. 2001). Hypoxia is also reported to upregulate the expression of death receptors and death ligands in cardiomyocytes in vitro (Tanaka et al. 1994, Yaniv et al. 2002). In many studies, including our own studies of the embryonic heart, hypoxia by itself is insufficient to induce PCD. It has been proposed that additional factors—for example, acidosis or reoxygenation (reperfusion)—are required to activate PCD (Armstrong 2004, Kubasiak et al. 2002). Alternatively, it is possible that hypoxia that is not severe enough to cause necrotic cell death induces adaptive responses that may lead to a mixed pattern of cell survival and PCD. One such scheme could be the invasion of the OFT myocardium by endothelial progenitor cells, which could present death ligands to the OFT cardiomyocytes, causing their demise; while restoring oxygen concentrations through vasculogenesis, causing their survival. The pathway for the execution of the program of cell death in the developing heart is still largely undefined. Mouse models provide an excellent tool for testing the roles of specific genes in cardiac morphogenesis through their targeted inactivation. Inactivation of a number of genes that may function in hypoxic signaling results in demise of the embryo secondary to cardiovascular insufficiency. This includes hypoxic transcriptional regulators such as HIF1a and HIF-1h (ARNT), their target genes such as VEGF, and the VEGF and erythropoietin receptors (reviewed in Simon et al. 2002). Our own experiments have shown that inactivation of Cited2, a transcriptional regulator that is proposed to antagonize HIF-dependent transcriptional responses, causes conotruncal heart defects (Yin et al. 2002). The extent to which the defects observed in these experiments represent an inadequate response to hypoxia in specific tissues at specific stages of development remains to be determined. 

Conclusion

We have proposed a model in avian development in which myocardial hypo-

xia triggers PCD-dependent remodeling of the OFT. In the future, it will be crucial to identify the specific gene targets of hypoxia/ HIF-1 signaling and how the expression of pro- or antiapoptotic genes in the OFT determines whether the OFT cardiomyocytes are eliminated by PCD or survive to form the subpulmonic infundibulum. The study of the remodeling of the embryonic cardiac OFT may also provide insights into cardiac remodeling and the pro- and antiapoptotic responses of the hypoxic myocardium in the adult. Finally, it will also be of interest to determine whether the tissue hypoxia renders the OFT myocardium susceptible to teratogens, perhaps contributing to the relatively high incidence of congenital conotruncal heart defects in the human population. 

Acknowledgments

This research was supported by National Institutes of Health grant R01 HL 6531401 (to S.A.F.) and an American Heart Association (AHA) Ohio Valley Grant-inAid (to M.W.). Y.S. was supported by an AHA Ohio Valley Postdoctoral Fellowship and a Japan Heart Foundation/ Bayer Yakuhin Research Grant Abroad.

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An Emerging Role for Kru¨ppel-Like Factors in Vascular Biology Mark W. Feinberg, Zhiyong Lin, Sudeshna Fisch, and Mukesh K. Jain*

The Kru¨ppel-like family of transcription factors play diverse roles regulating cellular differentiation and tissue development. Accumulating evidence supports an important role for these factors in vascular biology. This review examines the current knowledge of this gene family’s role in key cell types that critically regulate vessel biology under physiologic and pathologic states. (Trends Cardiovasc Med 2004;14:241–241) D 2004, Elsevier Inc.



Introduction to Kru¨ppel-Like Factors

Kru¨ppel-like factors (KLFs) are a subfamily of the zinc-finger class of DNAbinding transcriptional regulators. Although there are a large number of zinc-finger proteins, several features distinguish KLFs from other family members. First, KLFs contain three Cys2/ His2 zinc fingers located at the extreme C terminus of the protein (Bieker 1996 and 2001, Turner and Crossley 1999). Second, KLFs share a highly conserved seven-amino acid interfinger space sequence, TGEKP(Y/F)X (Dang et al. 2000). Third, structural studies have identified three critical residues in each finger that dictate DNA-binding specificity. This may account for the fact that many KLFs are able to bind to similar DNA sequences such as the bGT box Q or bCACCC Q element (Bieker 1996, Dang et al. 2000). In contrast to the zinc-finger

Mark W. Feinberg, Zhiyong Lin, Sudeshna Fisch, and Mukesh K. Jain are at the Cardiovascular Division, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, USA. * Address correspondence to: Mukesh K. Jain, MD, Cardiovascular Division, Brigham and Women’s Hospital, 20 Shattuck Street, Boston, MA 02115, USA. Tel.: (+1) 617-2780175; fax: (+1) 617-732-5132; e-mail: mjain@ rics.bwh.harvard.edu. D 2004, Elsevier Inc. All rights reserved. 1050-1738/04/$-see front matter

regions, the non-DNA-binding regions are highly divergent, and modular activation and repression domains have been identified that regulate transcriptional activity (Figure 1). The nomenclature is based on homology to the DNA-binding domain of the Drosophila protein Kru¨ppel, a german word meaning bcripple.Q This term is derived from the observation that Drosophila embryos homozygous for Kru¨ppel die due to altered thoracic and anterior abdominal segments (Jackle et al. 1985, Nusslein-Volhard and Wieschaus 1980, Preiss et al. 1985). The first mammalian Kru¨ppel, termed erythroid Kru¨ ppel-like factor (KLF1/EKLF), was identified in 1993 as a factor specifically expressed in red blood cells (Miller and Bieker 1993). Gene targeting experiments verified the essential role for this factor in h-globin gene synthesis and erythrocyte development (Nuez et al. 1995, Perkins et al. 1995). As founding members, both Drosophila Kru¨ppel and KLF1/EKLF are elegant examples of the critical role these factors play in differentiation and development. Since the identification of EKLF/ KLF1, a number of experimental approaches ranging from homology screening to database mining have been employed to identify additional Kru¨ppellike factors. To date, 16 mammalian KLFs have been identified and are designated KLF1 through KLF16, based

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