Sculpting the cardiac outflow tract

The cardiac outflow tract is the site of anomalies that affect a substantial proportion of individuals with congenital heart defects. The morphogenesis of this site is complex, and requires coordinated development of many cell types and tissues. It is therefore not surprising that developmental mistakes arise here, and that the steps and mechanisms of morphogenesis are still controversial and poorly understood, despite advances in molecular techniques. Recent findings have provided new insight into mechanisms of outflow tract morphogenesis, including clarification of its origins and the fate of cardiomyocytes, as well as invading cell populations. Application of new and old techniques and a wide range of approaches to tackle the unanswered questions about the outflow tract calls for collaboration among investigators from different disciplines including anatomists, physiologists, and molecular biologists.     

Myocardialization of the cardiac outflow tract.

During development, the single-circuited cardiac tube transforms into a double-circuited four-chambered heart by a complex process of remodeling, differential growth, and septation. In this process the endocardial cushion tissues of the atrioventricular junction and outflow tract (OFT) play a crucial role as they contribute to the mesenchymal components of the developing septa and valves in the developing heart. After fusion, the endocardial ridges in the proximal portion of the OFT initially form a mesenchymal outlet septum. In the adult heart, however, this outlet septum is basically a muscular structure. Hence, the mesenchyme of the proximal outlet septum has to be replaced by cardiomyocytes. We have dubbed this process "myocardialization." Our immunohistochemical analysis of staged chicken hearts demonstrates that myocardialization takes place by ingrowth of existing myocardium into the mesenchymal outlet septum. Compared to other events in cardiac septation, it is a relatively late process, being initialized around stage H/H28 and being basically completed around stage H/H38. To unravel the molecular mechanisms that are responsible for the induction and regulation of myocardialization, an in vitro culture system in which myocardialization could be mimicked and manipulated was developed. Using this in vitro myocardialization assay it was observed that under the standard culture conditions (i) whole OFT explants from stage H/H20 and younger did not spontaneously myocardialize the collagen matrix, (ii) explants from stage H/H21 and older spontaneously formed extensive myocardial networks, (iii) the myocardium of the OFT could be induced to myocardialize and was therefore "myocardialization-competent" at all stages tested (H/H16-30), (iv) myocardialization was induced by factors produced by, most likely, the nonmyocardial component of the outflow tract, (v) at none of the embryonic stages analyzed was ventricular myocardium myocardialization-competent, and finally, (vi) ventricular myocardium did not produce factors capable of supporting myocardialization.     

Bone marrow-independent adventitial macrophage progenitor cells contribute to angiogenesis

Pathological angiogenesis promotes tumor growth, metastasis, and atherosclerotic plaque rupture. Macrophages are key players in these processes. However, whether these macrophages differentiate from bone marrow-derived monocytes or from local vascular wall-resident stem and progenitor cells (VW-SCs) is an unresolved issue of angiogenesis. To answer this question, we analyzed vascular sprouting and alterations in aortic cell populations in mouse aortic ring assays (ARA). ARA culture leads to the generation of large numbers of macrophages, especially within the aortic adventitia. Using immunohistochemical fate-mapping and genetic in vivo-labeling approaches we show that 60% of these macrophages differentiate from bone marrow-independent Ly6c⁺/Sca-1⁺ adventitial progenitor cells. Analysis of the NCX^−/− mouse model that genetically lacks embryonic circulation and yolk sac perfusion indicates that at least some of those progenitor cells arise yolk sac-independent. Macrophages represent the main source of VEGF in ARA that vice versa promotes the generation of additional macrophages thereby creating a pro-angiogenetic feedforward loop. Additionally, macrophage-derived VEGF activates CD34⁺ progenitor cells within the adventitial vasculogenic zone to differentiate into CD31⁺ endothelial cells. Consequently, depletion of macrophages and VEGFR2 antagonism drastically reduce vascular sprouting activity in ARA. In summary, we show that angiogenic activation induces differentiation of macrophages from bone marrow-derived as well as from bone marrow-independent VW-SCs. The latter ones are at least partially yolk sac-independent, too. Those VW-SC-derived macrophages critically contribute to angiogenesis, making them an attractive target to interfere with pathological angiogenesis in cancer and atherosclerosis as well as with regenerative angiogenesis in ischemic cardiovascular disorders.Subject terms: Stem-cell differentiation, Stem-cell niche, Adult stem cells, Cell signalling     

The anatomical components of the cardiac outflow tract of chondrichthyans and actinopterygians

The outflow tract of the fish heart is the segment interposed between the ventricle and the ventral aorta. It holds the valves that prevent blood backflow from the gill vasculature to the ventricle. The anatomical composition, histological structure and evolutionary changes in the fish cardiac outflow tract have been under discussion for nearly two centuries and are still subject to debate. This paper offers a brief historical review of the main conceptions about the cardiac outflow tract components of chondrichthyans (cartilaginous fish) and actinopterygians (ray‐finned fish) which have been put forward since the beginning of the nineteenth century up to the current day. We focus on the evolutionary origin of the outflow tract components and the changes to which they have been subject in the major extant groups of chondrichthyans and actinopterygians. In addition, an attempt is made to infer the primitive anatomical design of the heart of the gnathostomes (jawed vertebrates). Finally, several areas of further investigation are suggested. Recent work on fish heart morphology has shown that the cardiac outflow tract of chondrichthyans does not consist exclusively of the myocardial conus arteriosus as classically thought. A conus arteriosus and a bulbus arteriosus, devoid of myocardium and mainly composed of elastin and smooth muscle, are usually present in cartilaginous and ray‐finned fish. This is consistent with the suggestion that both components coexisted from the onset of the gnathostome radiation. There is evidence that the conus arteriosus appeared in the agnathans. By contrast, the evolutionary origin of the bulbus is still unclear. It is almost certain that in all fish, both the conus and bulbus develop from the embryonic second heart field. We suggest herein that the primitive anatomical heart of the jawed vertebrates consisted of a sinus venosus containing the pacemaker tissue, an atrium possessing trabeculated myocardium, an atrioventricular region with compact myocardium which supported the atrioventricular valves, a ventricle composed of mixed myocardium, and an outflow tract consisting of a conus arteriosus, with compact myocardium in its wall and valves at its luminal side, and a non‐myocardial bulbus arteriosus that connected the conus with the ventral aorta. Chondrichthyans have retained this basic anatomical design of the heart. In actinopterygians, the heart has been subject to notable changes during evolution. Among them, the following two should be highlighted: (i) a decrease in size of the conus in combination with a remarkable development of the bulbus, especially in teleosts; and (ii) loss of the myocardial compact layer of the ventricle in many teleost species.     

N-cadherin is required for neural crest remodeling of the cardiac outflow tract

Cardiac neural crest cells undergo extensive cell rearrangements during the formation of the aorticopulmonary septum in the outflow tract. However, the morphogenetic mechanisms involved in this fundamental process remain poorly understood. To determine the function of the Ca2+-dependent cell adhesion molecule, N-cadherin, in murine neural crest, we applied the Cre/loxP system and created mouse embryos genetically mosaic for N-cadherin. Specifically, deletion of N-cadherin in neural crest cells led to embryonic lethality with distinct cardiovascular defects. Neural crest cell migration and homing to the cardiac outflow tract niche were unaffected by loss of N-cadherin. However, N-cadherin-deficient neural crest cells were unable to undergo the normal morphogenetic changes associated with outflow tract remodeling, resulting in persistent truncus arteriosus in the majority of mutant embryos. Other mutant embryos initiated aorticopulmonary septum formation; however, the neural crest cells were unable to elongate and align properly along the midline and remained rounded with limited contact with their neighbors. Interestingly, rotation of the outflow tract was incomplete in these mutants suggesting that alignment of the channels is dependent on N-cadherin-generated cytoskeletal forces. A second cardiac phenotype was observed where loss of N-cadherin in the epicardium led to disruption of heterotypic cell interactions between the epicardium and myocardium resulting in a thinned ventricular myocardium. Thus, we conclude that in addition to its role in myocardial cell adhesion, N-cadherin is required for neural crest cell rearrangements critical for patterning of the cardiac outflow tract and in the maintenance of epicardial-myocardial cell interactions.     

Biochemistry and biology: Heart-to-heart to investigate cardiac progenitor cells

Background

Cardiac regenerative medicine is a rapidly evolving field, with promising future developments for effective personalized treatments. Several stem/progenitor cells are candidates for cardiac cell therapy, and emerging evidence suggests how multiple metabolic and biochemical pathways strictly regulate their fate and renewal.

Scope of review

In this review, we will explore a selection of areas of common interest for biology and biochemistry concerning stem/progenitor cells, and in particular cardiac progenitor cells. Numerous regulatory mechanisms have been identified that link stem cell signaling and functions to the modulation of metabolic pathways, and vice versa. Pharmacological treatments and culture requirements may be exploited to modulate stem cell pluripotency and self-renewal, possibly boosting their regenerative potential for cell therapy.

Major conclusions

Mitochondria and their many related metabolites and messengers, such as oxygen, ROS, calcium and glucose, have a crucial role in regulating stem cell fate and the balance of their functions, together with many metabolic enzymes. Furthermore, protein biochemistry and proteomics can provide precious clues on the definition of different progenitor cell populations, their physiology and their autocrine/paracrine regulatory/signaling networks.

General significance

Interdisciplinary approaches between biology and biochemistry can provide productive insights on stem/progenitor cells, allowing the development of novel strategies and protocols for effective cardiac cell therapy clinical translation. This article is part of a Special Issue entitled Biochemistry of Stem Cells.     

他汀类药物对外周血内皮祖细胞的影响

本文旨在探讨他汀类药物氟伐他汀对外周血内皮祖细胞(endothelial progenitor cells,EPCs)数量和功能的影响.用密度梯度离心从外周血获取单个核细胞,将其接种在人纤维连接蛋白(human fibronectin)包被的培养板中,培养7 d后,收集贴壁细胞,加入不同浓度氟伐他汀(分别为0.01、0.1、1、10μmol/L)和辛伐他汀(1 μmol/L),培养一定的时间(6、12、24、48 h).用激光共聚焦显微镜鉴定FITC-UEA-I和DiI-acLDL双染色阳性细胞为正在分化的EPCs,用流式细胞仪检测其表面标志进一步鉴定EPCs,在倒置荧光显微镜下计数.采用MTT比色法、改良的Boyden小室、粘附能力测定实验和体外血管生成试剂盒观察EPCs的增殖能力、迁移能力、粘附能力和体外血管生成能力.结果显示,氟伐他汀可显著增加外周血EPCs的数量,并且EPCs数量随氟伐他汀浓度增加及作用时间延长而增加,1 μmol/L浓度氟伐他汀作用24h对EPCs的数量影响最为显著(较对照组增加15倍,P＜0.05).在动物实验中,喂养氟伐他汀3周后,大鼠的EPCs也较对照组增加2倍(P＜0.05),进一步支持了体外实验的结果.氟伐他汀和辛伐他汀也显著改善外周血EPCs的粘附能力、迁移能力、增殖能力和体外血管生成的能力,相同浓度的氟伐他汀和辛伐他汀(1 μmol/L)对EPCs数量和功能的影响并无显著差异.上述观察结果提示他汀类药物可增加EPCs的数量,改善EPCs功能.     

Targeted deletion of Hand2 in cardiac neural crest-derived cells influences cardiac gene expression and outflow tract development

The basic helix-loop-helix DNA binding protein Hand2 has critical functions in cardiac development both in neural crest-derived and mesoderm-derived structures. Targeted deletion of Hand2 in the neural crest has allowed us to genetically dissect Hand2-dependent defects specifically in outflow tract and cardiac cushion independent of Hand2 functions in mesoderm-derived structures. Targeted deletion of Hand2 in the neural crest results in misalignment of the aortic arch arteries and outflow tract, contributing to development of double outlet right ventricle (DORV) and ventricular septal defects (VSD). These neural crest-derived developmental anomalies are associated with altered expression of Hand2-target genes we have identified by gene profiling. A number of Hand2 direct target genes have been identified using ChIP and ChIP-on-chip analyses. We have identified and validated a number of genes related to cell migration, proliferation/cell cycle and intracellular signaling whose expression is affected by Hand2 deletion in the neural crest and which are associated with development of VSD and DORV. Our data suggest that Hand2 is a multifunctional DNA binding protein affecting expression of target genes associated with a number of functional interactions in neural crest-derived cells required for proper patterning of the outflow tract, generation of the appropriate number of neural crest-derived cells for elongation of the conotruncus and cardiac cushion organization. Our genetic model has made it possible to investigate the molecular genetics of neural crest contributions to outflow tract morphogenesis and cell differentiation.     

Bone marrow-derived endothelial progenitor cells contribute to the angiogenic switch in tumor growth and metastatic progression

Emerging evidence indicates that bone marrow (BM)-derived endothelial progenitor cells (EPCs) contribute to angiogenesis-mediated growth of certain tumors in mice and human. EPCs regulate the angiogenic switch via paracrine secretion of proangiogenic growth factors and by direct luminal incorporation into sprouting nascent vessels. While the contributions of EPCs to neovessel formation in spontaneous and transplanted tumors and to the metastatic transition have been reported to be relatively low, remarkably, specific EPC ablation in vivo has resulted in severe angiogenesis inhibition and impaired primary and metastatic tumor growth. The existence of a BM reservoir of EPCs, and the selective involvement of EPCs in neovascularization, have attracted considerable interest because these cells represent novel target for therapeutic intervention. In addition, EPCs are also being used as pharmacodynamic surrogate markers for monitoring cancer progression, as well as for optimizing efficacy of anti-angiogenic therapies in the clinic. This review will focus primarily on recent advances and emerging concepts in the field of EPC biology and discuss ongoing debates involving the role of EPCs in tumor neovascularization. For detailed information on the in vitro characterization of EPCs contribution to non-tumor pathologies, the reader is directed towards several excellent reviews and publications [F. Bertolini, Y. Shaked, P. Mancuso and R.S. Kerbel, Nat. Rev., Cancer 6 (2006) 835–845. [1]] [J.M. Hill, T. Finkel and A.A. Quyyumi, Vox Sang. 87 Suppl 2 (2004) 31–37. [2]] [A.Y. Khakoo and T. Finkel, Annu. Rev. Med. 56 (2005) 79–101. [3]] [H.G. Kopp, C.A. Ramos and S. Rafii, Curr. Opin. Hematol. 13 (2006) 175–181. [4]; K.K. Hirschi, D.A. Ingram and M.C. Yoder, Arterioscler. Thromb. Vasc. Biol. 28 (2008) 1584–1595. [5]; F. Timmermans, J. Plum, M.C. Yoder, D.A. Ingram, B. Vandekerckhove and J. Case, J. Cell. Mol. Med. 13 (2009) 87–102. [6]] and reviews by Bertolini, Voest and Yoder in this issue.     

Lymphatic endothelial progenitor cells contribute to de novo lymphangiogenesis in human renal transplants

De novo lymphangiogenesis influences the course of different human diseases as diverse as chronic renal transplant rejection and tumor metastasis. The cellular mechanisms of lymphangiogenesis in human diseases are currently unknown, and could involve division of local preexisting endothelial cells or incorporation of circulating progenitors. We analyzed renal tissues of individuals with gender-mismatched transplants who had transplant rejection and high rates of overall lymphatic endothelial proliferation as well as massive chronic inflammation. Donor-derived cells were detected by in situ hybridization of the Y chromosome. We compared these tissues with biopsies of essentially normal skin and intestine, and two rare carcinomas with low rates of lymphatic endothelial proliferation that were derived from individuals with gender-mismatched bone marrow transplants. Here, we provide evidence for the participation of recipient-derived lymphatic progenitor cells in renal transplants. In contrast, lymphatic vessels of normal tissues and those around post-transplant carcinomas did not incorporate donor-derived progenitors. This indicates a stepwise mechanism of inflammation-associated de novo lymphangiogenesis, implying that potential lymphatic progenitor cells derive from the circulation, transmigrate through the connective tissue stroma, presumably in the form of macrophages, and finally incorporate into the growing lymphatic vessel.     

Fgf15 is required for proper morphogenesis of the mouse cardiac outflow tract

Evidence in animal models indicates that signaling networks functioning in the developing pharyngeal arches regulate stereotyped processes critical for proper development of the aortic arch and cardiac outflow tract. Here, we describe the phenotype of mice lacking fibroblast growth factor 15 (Fgf15), which encodes a secreted signaling molecule expressed within the developing pharyngeal arches. Homozygous Fgf15 mutants present heart defects consistent with malalignment of the aorta and pulmonary trunk. These defects correlate with early morphological defects of the outflow tract due to aberrant behavior of the cardiac neural crest. We demonstrate that Fgf15 expression within the pharyngeal arches is unaltered by a loss of Tbx1, a key regulator of pharyngeal arch development implicated in DiGeorge syndrome. In addition, Fgf15 and Tbx1 do not interact genetically, suggesting that Fgf15 operates through a pathway independent of Tbx1. These studies reveal a novel role of Fgf15 during development of the cardiac outflow tract.     

BMP receptor IA is required in mammalian neural crest cells for development of the cardiac outflow tract and ventricular myocardium

The neural crest is a multipotent, migratory cell population arising from the border of the neural and surface ectoderm. In mouse, the initial migratory neural crest cells occur at the five-somite stage. Bone morphogenetic proteins (BMPs), particularly BMP2 and BMP4, have been implicated as regulators of neural crest cell induction, maintenance, migration, differentiation and survival. Mouse has three known BMP2/4 type I receptors, of which Bmpr1a is expressed in the neural tube sufficiently early to be involved in neural crest development from the outset; however, earlier roles in other domains obscure its requirement in the neural crest. We have ablated Bmpr1a specifically in the neural crest, beginning at the five-somite stage. We find that most aspects of neural crest development occur normally; suggesting that BMPRIA is unnecessary for many aspects of early neural crest biology. However, mutant embryos display a shortened cardiac outflow tract with defective septation, a process known to require neural crest cells and to be essential for perinatal viability. Surprisingly, these embryos die in mid-gestation from acute heart failure, with reduced proliferation of ventricular myocardium. The myocardial defect may involve reduced BMP signaling in a novel, minor population of neural crest derivatives in the epicardium, a known source of ventricular myocardial proliferation signals. These results demonstrate that BMP2/4 signaling in mammalian neural crest derivatives is essential for outflow tract development and may regulate a crucial proliferation signal for the ventricular myocardium.     

Role of myocardial hypoxia in the remodeling of the embryonic avian cardiac outflow tract

The embryonic cardiac outflow myocardium originates from a secondary heart-forming field to connect the developing ventricles with the aortic sac. The outflow tract (OFT) subsequently undergoes complex remodeling in the transition of the embryo to a dual circulation. In avians, elimination of OFT cardiomyocytes by apoptosis (stages 25-32) precedes coronary vasculogenesis and is necessary for the shortening of the OFT and the posterior rotation of the aorta. We hypothesized that regional myocardial hypoxia triggers OFT remodeling. We used immunohistochemical detection of the nitroimidazole EF5, administered by intravascular infusion in ovo, as an indicator of relative tissue oxygen concentrations. EF5 binding was increased in the OFT myocardium relative to other myocardium during these stages (25-32) of OFT remodeling. The intensity of EF5 binding paralleled the prevalence of apoptosis in the OFT myocardium, which are first detected at stage 25, maximal at stage 30, and diminished by stage 32. Evidence of coincident hypoxia-dependent responses included the expression of the vascular endothelial growth factor (VEGF) receptor 2 by the OFT myocardium, the predominant expression of VEGF122 (diffusible) isoform in the OFT, and the recruitment of QH1-positive pro-endothelial cells to the OFT and vasculogenesis. Exposure of embryos to hyperoxia (95% O(2)/5% CO(2)) during this developmental window reduced the prevalence of cardiomyocyte apoptosis and attenuated the shortening and rotation of the OFT, resulting in double-outlet right ventricle morphology, similar to that observed when apoptosis is directly inhibited. These results suggest that regional myocardial hypoxia triggers cardiomyocyte apoptosis and remodeling of the OFT in the transition to a dual circulation, and that VEGF autocrine/paracrine signaling may regulate these processes.     

Evolutionary and developmental origins of the cardiac neural crest: Building a divided outflow tract

The cardiac neural crest cells (CNCCs) have played an important role in the evolution and development of the vertebrate cardiovascular system: from reinforcement of the developing aortic arch arteries early in vertebrate evolution, to later orchestration of aortic arch artery remodeling into the great arteries of the heart, and finally outflow tract septation in amniotes. A critical element necessary for the evolutionary advent of outflow tract septation was the co‐evolution of the cardiac neural crest cells with the second heart field. This review highlights the major transitions in vertebrate circulatory evolution, explores the evolutionary developmental origins of the CNCCs from the third stream cranial neural crest, and explores candidate signaling pathways in CNCC and outflow tract evolution drawn from our knowledge of DiGeorge Syndrome. Birth Defects Research (Part C) 102:309–323, 2014. © 2014 Wiley Periodicals, Inc.