Plasticity of endothelial cells during arterial-venous differentiation in the avian embryo

Remodeling of the primary vascular system of the embryo into arteries and veins has long been thought to depend largely on the influence of hemodynamic forces. This view was recently challenged by the discovery of several molecules specifically expressed by arterial or venous endothelial cells. We here analysed the expression of neuropilin-1 and TIE2, two transmembrane receptors known to play a role in vascular development. In birds, neuropilin-1 was expressed by arterial endothelium and wall cells, but absent from veins. TIE2 was strongly expressed in embryonic veins, but only weakly transcribed in most arteries. To examine whether endothelial cells are committed to an arterial or venous fate once they express these specific receptors, we constructed quail-chick chimeras. The dorsal aorta, carotid artery and the cardinal and jugular veins were isolated together with the vessel wall from quail embryos between embryonic day 2 to 15 and grafted into the coelom of chick hosts. Until embryonic day 7, all grafts yielded endothelial cells that colonized both host arteries and veins. After embryonic day 7, endothelial plasticity was progressively lost and from embryonic day 11 grafts of arteries yielded endothelial cells that colonized only chick arteries and rarely reached the host veins, while grafts of jugular veins colonized mainly host veins. When isolated from the vessel wall, quail aortic endothelial cells from embryonic day 11 embryos were able to colonize both host arteries and veins. Our results show that despite the expression of arterial or venous markers the endothelium remains plastic with regard to arterial-venous differentiation until late in embryonic development and point to a role for the vessel wall in endothelial plasticity and vessel identity.     

Coronary artery embryogenesis in cardiac defects induced by retinoic acid in mice

BACKGROUND: Although normal coronary artery embryogenesis is well described in the literature, little is known about the development of coronary vessels in abnormal hearts. METHODS: We used an animal model of retinoic acid (RA)-evoked outflow tract malformations (e.g., double outlet right ventricle [DORV], transposition of the great arteries [TGA], and common truncus arteriosus [CTA]) to study the embryogenesis of coronary arteries using endothelial cell markers (anti-PECAM-1 antibodies and Griffonia simplicifolia I (GSI) lectin). These markers were applied to serial sections of staged mouse hearts to demonstrate the location of coronary artery primordia. RESULTS: In malformations with a dextropositioned aorta, the shape of the peritruncal plexus, from which the coronary arteries develop, differed from that of control hearts. This difference in the shape of the early capillary plexus in the control and RA-treated hearts depends on the position of the aorta relative to the pulmonary trunk. In both normal and RA-treated hearts, there are several capillary penetrations to each aortic sinus facing the pulmonary trunk, but eventually only 1 coronary artery establishes patency with 1 aortic sinus. CONCLUSIONS: The abnormal location of the vessel primordia induces defective courses of coronary arteries; creates fistulas, a single coronary artery, and dilated vessel lumens; and leaves certain areas of the heart devoid of coronary artery branches. RA-evoked heart malformations may be a useful model for elucidating abnormal patterns of coronary artery development and may shed some light on the angiogenesis of coronary artery formation.     

Normal stages of cardiac organogenesis in the mouse: I. Development of the external shape of the heart

Normal development of the mouse embryonic heart was studied at the organ level using microdissection and scanning electron microscopy (SEM). Altogether 225 embryos, sampled at 8-hour intervals between 11ed (ed = embryonic day; day of vaginal plug = 1ed) and 15ed were collected. Their hearts were fixed by high flow-low pressure perfusion, microdissected, and observed in SEM. Standardized frontal, right profile, and left profile SEM micrographs were obtained and analyzed. The main purpose of this study was to create a series of normal stages of mouse cardiac development as a reference for ongoing studies in experimental cardiac teratology (e.g., in fetal mouse trisomies). Comparisons with chick, human, and dog embryonic hearts, prepared using the same technique, show that the mouse embryonic heart is characterized by a relatively deep interventricular sulcus. The absence of a conoventricular sulcus in the mouse results in poor definition of the boundary between the conus and the right ventricle. The external separation of the aorta and the pulmonary artery is evident from 13ed onward. The respective positions of the great arteries (aorta dextroposterior, pulmonary artery sinistroanterior) does not change until the end of cardiac organogenesis (15ed in the mouse).     

Peritruncal Coronary Endothelial Cells Contribute to Proximal Coronary Artery Stems and Their Aortic Orifices in the Mouse Heart

Avian embryo experiments proved an ingrowth model for the coronary artery connections with the aorta. However, whether a similar mechanism applies to the mammalian heart still remains unclear. Here we analyzed how the main coronary arteries and their orifices form during murine heart development. Apelin (Apln) is expressed in coronary vascular endothelial cells including peritruncal endothelial cells. By immunostaining, however, we did not find Apln expression in endothelial cells of the aorta during the period of coronary vessel development (E10.5 to E15.5). As a result of this unique expression difference, Apln^CreERT2/+ genetically labels nascent coronary vessels forming on the heart, but not the aorta endothelium when pulse activated by tamoxifen injection at E10.5. This allowed us to define the temporal contribution of these distinct endothelial cell populations to formation of the murine coronary artery orifice. We found that the peritruncal endothelial cells were recruited to form the coronary artery orifices. These cells penetrate the wall of aorta and take up residence in the aortic sinus of valsalva. In conclusion, main coronary arteries and their orifices form through the recruitment and vascular remodeling of peritruncal endothelial cells in mammalian heart.     

Expression of alphaVbeta3 integrin in the chick embryo aortic endothelium

The integrin chain alphaV, expressed in association with beta3, by cells of the megakaryocytic/thrombocytic and endothelial lineages is thought to play an important role in angiogenesis. alphaVbeta3 expression by endothelial cells is not constitutive but induced by various stimuli in avian and human models. Here the developmental pattern of alphaVbeta3 expression was analysed in the chick embryo by immunocytochemistry, using a specific monoclonal antibody. On day 2 of development alphaVbeta3 expression was restricted to rare cells in the blood stream, in the embryo proper and in the yolk sac blood islands. AlphaVbeta3 expression by endothelial cells became detectable on day 3 and was restricted to the dorsal aorta. Interestingly it was absent from the intra-aortic hemopoietic clusters (E3.5) which, as we have showed previously, express the alphaIIbbeta3 integrin and display progenitor potentialities. However the endothelium underlying intra-embryonic hemopoietic clusters expressed this integrin. In contrast E6-7 para-aortic hemopoietic foci contained numerous alphaVbeta3 positive cells. Both alphaVbeta3 and alphaIIbbeta3 were expressed in these latter hemopoietic sites, while alphaVbeta3 was still selectively expressed by the aortic endothelium until E6. Thereafter, at E7 the pulmonary artery also expressed it. Since alphaIIbbeta3 is expressed by avian and murine multilineage hemopoietic progenitors, we then studied the hemopoietic potentialities of alphaVbeta3/alphaIIbeta3 double positive cells from embryonic bone marrow differentiating in vitro in erythro-myeloid conditions. Thrombocytic, erythroid and myeloid progenitor potentialities were found within the cell population expressing both beta3 integrins.     

Avian coronary endothelium is a mosaic of sinus venosus‐ and ventricle‐derived endothelial cells in a region‐specific manner

The origin of coronary endothelial cells (ECs) has been investigated in avian species, and the results showed that the coronary ECs originate from the proepicardial organ (PEO) and developing epicardium. Genetic approaches in mouse models showed that the major source of coronary ECs is the sinus venosus endothelium or ventricular endocardium. To clarify and reconcile the differences between avian and mouse species, we examined the source of coronary ECs in avian embryonic hearts. Using an enhanced green fluorescent protein‐Tol2 system and fluorescent dye labeling, four types of quail‐chick chimeras were made and quail‐specific endothelial marker (QH1) immunohistochemistry was performed. The developing PEO consisted of at least two cellular populations in origin, one was sinus venosus endothelium‐derived inner cells and the other was surface mesothelium‐derived cells. The majority of ECs in the coronary stems, ventricular free wall, and dorsal ventricular septum originated from the sinus venosus endothelium. The ventricular endocardium contributed mainly to the septal artery and a few cells to the coronary stems. Surface mesothelial cells of the PEO differentiated mainly into a smooth muscle phenotype, but a few differentiated into ECs. In avian species, the coronary endothelium had a heterogeneous origin in a region‐specific manner, and the sources of ECs were basically the same as those observed in mice.     

Ablation of the secondary heart field leads to tetralogy of Fallot and pulmonary atresia

Recent studies in chick and mouse embryos have identified a previously unrecognized secondary heart field (SHF), located in the ventral midline splanchnic mesenchyme, which provides additional myocardial cells to the outflow tract as the heart tube lengthens during cardiac looping. In order to further delineate the contribution of this secondary myocardium to outflow development, we labeled the right SHF of Hamburger-Hamilton (HH) stage 14 chick embryos via microinjection of DiI/rhodamine and followed the fluorescently labeled cells over a 96-h time period. These experiments confirmed the movement of the SHF into the outflow and its spiraling migration distally, with the right side of the SHF contributing to the left side of the outflow. In contrast, when the right SHF was labeled at HH18, the fluorescence was limited to the caudal wall of the lengthening aortic sac. We then injected a combination of DiI and neutral red dye, and ablated the SHF in HH14 or 18 chick embryos. Embryos were allowed to develop until day 9, and harvested for assessment of outflow alignment. Of the embryos ablated at HH14, 76% demonstrated cardiac defects including overriding aorta and pulmonary atresia, while none of the sham-operated controls were affected. In addition, the more severely affected embryos demonstrated coronary artery anomalies. The embryos ablated at HH18 also manifested coronary artery anomalies but maintained normal outflow alignment. Therefore, the myocardium added to the outflow by the SHF at earlier stages is required for the elongation and appropriate alignment of the outflow tract. However, at later stages, the SHF contributes to the smooth muscle component of the outflow vessels above the pulmonary and aortic valves which is important for the development of the coronary artery stems. This work suggests a role for the SHF in a subset of congenital heart defects that have overriding aorta and coronary artery anomalies, such as tetralogy of Fallot and double outlet right ventricle.     

Earlier accumulation of calcium, phosphorus, and magnesium in the coronary artery in comparison with the ascending aorta, aortic valve, and mitral valve

To explore reasons for a high accumulation of Ca and P occurring in the coronary artery of Thai with aging, the authors investigated age-related changes of elements in the coronary artery, ascending aorta near the heart, and cardiac valves in single individuals, and the relationships in the elements between the coronary artery and either the ascending aorta or cardiac valves. After an ordinary dissection by medical students at Chiang Mai University was finished, the anterior descending arteries of the left coronary artery, ascending aortas, mitral valves, and aortic valves were resected from the subjects. The subjects consisted of 17 men and 9 women, ranging in age from 46 to 76 yr. The element content was analyzed by inductively coupled plasma-atomic emission spectrometry. The average content of Ca and P was the highest in the coronary artery and decreased in the order aortic valve, ascending aorta, and mitral valve. The Ca, P, and Mg content increased in the coronary artery in the fifties and in the ascending aorta, aortic valve, and mitral valve in the sixties. It should be noted that the accumulation of Ca, P, and Mg occurred earlier in the coronary artery than in the ascending aorta, aortic valve, and mitral valve. It was found that with respect to the Ca, P, Mg, and Na contents, the coronary artery correlated well with both the aortic valve and ascending aorta, especially with the aortic valve, but it did not correlate with the mitral valves. This finding suggests that the accumulation of Ca, P, Mg, and Na occurs in the coronary artery together with the aortic valve and ascending aorta, but not together with the mitral valve. Because regarding the accumulation of Ca, P, and Mg, the ascending aorta and aortic valve are preceded by the coronary artery, it is unlikely that the accumulation of Ca, P, and Mg spreads from the ascending aorta or aortic valve to the coronary artery.     

Coronary artery anomalies and aortic valve morphology in the Syrian hamster

In the Syrian hamster, anomalies in the origin of the left coronary artery are significantly associated with the bicuspid condition of the aortic valve. In this species, bicuspid aortic valves are expressions of a trait, the variation of which takes the form of a phenotypic continuum, ranging from a tricuspid aortic valve with no commissural fusion to a bicuspid aortic valve with the aortic sinuses located in ventrodorsal orientation and devoid of any raphe. The intermediate stages of the continuum are represented by tricuspid aortic valves with a more or less extensive fusion of the ventral commissure and bicuspid aortic valves with a more or less developed raphe located in the ventral aortic sinus. The present study was designed to decide whether there is a gap between tricuspid and bicuspid aortic valves regarding the incidence of coronary artery anomalies, or whether this incidence varies according to the different tricuspid and bicuspid morphotypes of the continuum. The study was carried out in Syrian hamsters belonging to a single inbred family with a high incidence of tricuspid aortic valves with fusion of the ventral commissure, bicuspid aortic valves, and anomalies in the origin of the left coronary artery, i.e. single right coronary artery ostium in aorta, anomalous origin of the left coronary artery from the pulmonary artery, and anomalous origin of the left coronary artery from the dorsal aortic sinus. The specimens were examined by means of a stereomicroscope and, in several cases, scanning electron microscopy was also used. The relationships between anomalous coronary artery patterns and aortic valve morphologies were tested using a logistic regression model. The results obtained indicate that there is no discontinuity between tricuspid and bicuspid aortic valves regarding the incidence of coronary artery anomalies. The probability of occurrence of anomalous coronary artery patterns increases continuously according to the deviation degree of the aortic valve from its normal (tricuspid) design. The present findings suggest that in the Syrian hamster, the morphogenetic mechanisms involved in the formation of congenital anomalous aortic valves and anomalies in the origin of the left coronary artery, respectively, are strongly related from an aetiological viewpoint.     

The calcium channel agonist, Bay K-8644, antagonizes effects of diacetyl monoxime on cardiac tissues

Effects of a novel slow channel activator, Bay K-8644 (Bay K), were studied on slow action potential (APs) in young and old embryonic chick hearts, and on its antagonism of the effects of diacetyl monoxime (DAM). The slow APs of young hearts are mediated by slow Na+ channels, whereas those of old hearts are mediated by slow Ca2+ channels. In slow APs of old (13-18 days old) embryonic chick hearts superfused with a high (22 mM) K+ solution, Bay K (10-6 M) gradually increased the amplitude, maximum rate of rise (Vmax), and duration of the slow APs. The actions of Bay K persisted for a long time (greater than 30 min) after washout of the drug. DAM (10 mM) depressed the Vmax, duration and amplitude of the slow APs. Some of the changes in slow AP parameters produced by DAM, e.g., Vmax decrease, were antagonized by the addition of Bay K (10(-6) M). In 3-day-old embryonic chick hearts. Bay K potentiated the slow APs and DAM depressed them; Bay K antagonized these effects of DAM. Thus, the actions of Bay K and DAM are likely to be produced, respectively, via the activation and depression of slow Ca2+ channels in old embryonic chick hearts. In addition, the drugs seem to influence slow Na+ channels found in young embryonic chick hearts.     

The Electrophysiological Organization of the Embryonic Chick Heart

Both intracellular and surface electrodes were employed to record electrical activity from embryonic chick hearts between the ages of 3 and 20 days. Cells from the sinus venosus, sinoatrial (SA) valves, atrium, atrioventricular (AV) ring, and ventricle were localized and characterized on the basis of shape, amplitude, rise time, and duration of transmembrane potentials. The differences in transmembrane potentials from these various regions provided the basis for a hypothesis concerned with the distribution of pacemaker potentiality and one related to the origin of the His-Purkinje system. Action potentials recorded along the entire embryonic AV ring were comparable to those of the adult rabbit AV nodal cells in both configuration and sequence of activation and were thus categorized into three functional regions (AN, N, NH). Histological sections of 7 and 14 day hearts demonstrated muscular continuity between the right atrium and ventricle across the muscular AV valve.     

Embryonic vascular development: immunohistochemical identification of the origin and subsequent morphogenesis of the major vessel primordia in quail embryos

The development of the embryonic vasculature is examined here using a monoclonal antibody, QH-1, capable of labelling the presumptive endothelial cells of Japanese quail embryos. Antibody labelling is first seen within the embryo proper at the 1-somite stage. Scattered labelling of single cells appears ventral to the somites and at the lateral edges of the anterior intestinal portal. The dorsal aorta soon forms a continuous cord at the ventrolateral edge of the somites and continues into the head to fuse with the ventral aorta forming the first aortic arch by the 6-somite stage. The rudiments of the endocardium fuse at the midline above the anterior intestinal portal by the 3-somite stage and the ventral aorta extends craniad. Intersomitic arteries begin to sprout off of the dorsal aorta at the 7-somite stage. The posterior cardinal vein forms from single cells which segregate from somatic mesoderm at the 7-somite stage to form a loose plexus which moves mediad and wraps around the developing Wolffian duct in later stages. These studies suggest two modes of origin of embryonic blood vessels. The dorsal aortae and cardinal veins apparently arise in situ by the local segregation of presumptive endothelial cells from the mesoderm. The intersomitic arteries, vertebral arteries and cephalic vasculature arise by sprouts from these early vessel rudiments. There also seems to be some cell migration in the morphogenesis of endocardium, ventral aorta and aortic arches. The extent of presumptive endothelial migration in these cases, however, needs to be clarified by microsurgical intervention.     

Tracing the progeny of the aortic hemangioblast in the avian embryo

A population of hematopoietic progenitors becomes committed within the embryo proper in the floor of the aorta (P-Sp/AGM in the mouse). In birds, this first aspect of intraembryonic hematopoiesis is prominent during embryonic day 3 (E3) as endothelium-associated "intra-aortic clusters." Between E6 and E8, diffuse hematopoiesis then occurs as "para-aortic foci" located in the dorsal mesentery ventral to the aorta. These foci are not associated with endothelium. Whether these two hematopoietic cell populations arise from distinct or common progenitors is not known. We could recently trace back the origin of intra-aortic clusters in the avian embryo by labeling aortic endothelial cells (EC) in vivo with acetylated low-density lipoproteins. This approach established the derivation of early intraembryonic hemopoietic cells from the endothelium, but did not indicate how long during ontogeny such a relationship may exist, since the progeny of EC labeled at E2 could be traced for 1-2 days at most. Here we report that, when E2 aortic ECs were infected prior to the formation of intra-aortic clusters with a nonreplicative LacZ-bearing retroviral vector, numerous cells were labeled in the para-aortic foci at E6. In contrast, when the retroviral vector was inoculated at E4 rather than E2, that is, after the disappearance of intra-aortic clusters, no cells in the para-aortic foci were labeled. Taken together, our results demonstrate that ECs from the aortic floor seed the two aspects of aorta-associated hemopoiesis and that these ECs with hemangioblastic potential are present only transiently in the aorta.     

Expression of the Ca2+ mobilizing muscarinic system in the chick embryo correlates with morphogenesis

We describe muscarinic receptors and intracellular Ca2+ mobilization after cholinergic stimulation in cell suspensions prepared from chick embryos between day 2 (stage 12/13) and day 13 (stage 40) of development. Cell suspensions are prepared from whole chick embryos and from embryonic hearts, heads or brains, limb buds, and trunks. Muscarinic receptors are measured using [3H]quinuclidinylbenzilate as specific ligand. Intracellular Ca2+ mobilization is determined by changes of chlorotetracycline fluorescence. (1) Considerable amounts of muscarinic receptors are found in all parts of the embryo and at all stages tested. (2) The intracellular Ca2+ response after stimulation by muscarinic agonist shows a peak at day 3-4 (stage 23). (3) The pharmacological profile of the Ca2+ response remains constant during embryonic development and differs from the profiles of most adult systems. (4) The 'embryonic muscarinic system' is uniformly expressed in cells from neural and non-neural tissues. It appears and disappears independently of innervation.     

Blood-forming potential of vascular endothelium in the human embryo

Hematopoietic cells arise first in the third week of human ontogeny inside yolk sac developing blood vessels, then, one week later and independently, from the wall of the embryonic aorta and vitelline artery. To address the suggested derivation of emerging hematopoietic stem cells from the vessel endothelium, endothelial cells have been sorted by flow cytometry from the yolk sac and aorta and cultured in the presence of stromal cells that support human multilineage hematopoiesis. Embryonic endothelial cells were most accurately selected on CD34 or CD31 surface expression and absence of CD45, which guaranteed the absence of contaminating hematopoietic cells. Yet, rigorously selected endothelial cells yielded a progeny of myelo-lymphoid cells in culture. The frequency of hemogenic endothelial cells in the yolk sac and aorta reflected the actual blood-forming activity of these tissues, as a function of developmental age. Even less expected, a subset of endothelial cells sorted similarly from the embryonic liver and fetal bone marrow also exhibited blood-forming potential. These results suggest that a part at least of emerging hematopoietic cells in the human embryo and fetus originate in vascular walls.     

Multiple chick tropoelastin mRNAs

Several overlapping chick tropoelastin cDNAs were isolated from a lambda gt11 cDNA library constructed from whole 10 day chick embryo total RNA. Comparison of the nucleotide sequence of the 2.3 kb tropoelastin cDNA to the sequences published by Bressan et al. (1) and Tokimitsu et al. (2) revealed the presence of two inserts (72 and 30 base pairs) in the cDNA derived from embryonic tissue. Northern blot analysis of 14 day embryonic aortae RNA with tropoelastin cDNA clones showed hybridization to a 3.5 kb mRNA. However, S1 nuclease protection experiments performed on RNA extracted from the same tissue showed that at least two if not more tropoelastin mRNAs exist and that the proportion of each varies in the ages examined. These results provide an origin and substantiate the differential expression of the multiple tropoelastin polypeptides found in developing chick aortic tissue.     

Glucose Utilization by Chick Embryo Heart Homogenates

Homogenates of early chick embryos and homogenates of early chick embryonic hearts utilized the phosphogluconate pathway of glucose catabolism to a greater extent, relative to the glycolytic-Krebs cycle pathway, than did homogenates of hearts from older chick embryos or adult chicks. An abrupt drop in the relative participation by the phosphogluconate pathway in embryo heart homogenates occurs at about 5 to 7 days of incubation. Heart homogenates from adult chicks catabolize glucose almost entirely by the glycolytic-Krebs cycle pathway, with negligible participation by the phosphogluconate pathway.     

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.     

Development of atherosclerotic lesions in cholesterol-loaded rabbits.

To examine both of the target vessels and the optimal time of their endothelial denudation to study vascular restenosis after balloon injury in cholesterol-loaded rabbits, we made 36 atherosclerotic rabbits by feeding a hypercholesterol diet, and histologically examined the onset time and the development of atherosclerosis. Atheromatous changes were observed first after the 5th week in the thoracic aorta from the start of the diet, and then extended to the abdominal aorta, coronary artery with time. The atherosclerotic lesions in the thoracic aorta and the proximal portion of the coronary artery showed high-grade concentric intimal thickening with luminal stenosis. The abdominal aortic lesion mildly progressed. In the renal, carotid and femoral arteries, in contrast, slight atheroscleromatous changes developed during the diet period. These results suggest that the thoracic and abdominal aortas and the coronary artery would be suitable as target vessels to study vascular restenosis after balloon injury, and the endothelial denudation of these vessels should be performed between the 8th and 15th week in this diet protocol for an accurate analysis.