Diminished growth of atrioventricular cushion tissue in stage 24 retinoic acid-treated chicken embryos.

Stage 34 chicken hearts have shown a spectrum of looping disturbances, changed hemodynamics, and changed growth of both right ventricular myocardium and atrioventricular cushion tissue after retinoic acid treatment. To obtain more information about the onset of the malformations we studied stage 24, the stage between the previously studied stage 34 and the moment of treatment. Sixteen stage 24 chicken embryos were examined after treatment with 1 microg all-trans retinoic acid at stage 15 and compared with 6 sham operated embryos. Morphological examination was supported by graphic reconstructions. Absolute volumes of atrial, atrioventricular, and ventricular myocardia were measured by a point counting method. The absolute volumes of the endocardial cushions were measured as well. Fifteen (15/16) retinoic acid-treated hearts did not show marked malformations as far as could be detected with our current macroscopic and microscopic techniques. One (1/16) retinoic acid-treated heart showed an abnormal tubular C-shape with a less bended inner curvature and with an abnormal horizontally oriented atrioventricular canal. The dorsal cushion tissue of this atrioventricular canal was discontinuous with the dorsal mesocardium and covered the malpositioned myocardial border between the atrium and the atrioventricular canal. The volume measurements did show a difference between retinoic acid treatment and sham operations. The retinoic acid-treated hearts showed a significant volume decrease of the atrioventricular cushions. No significant differences were found in the volumes of the ventricular myocardium compared to the sham operated embryos. We hypothesize that, between stages 15 and 24, retinoic acid directly affects the myocardial wall and the cushion tissue formation. In the present material this has resulted in decreased atrioventricular cushion growth, in changed hemodynamics, and in a severe looping disturbance of one embryo. We further hypothesize that, between stages 24 and 34, the malformations with minor looping disturbances will become apparent. Thus, development beyond stage 24 would result in the spectrum of looping disturbances as has been found at stage 34. These latter morphological malformations would lead to increasing hemodynamic changes, resulting in changes in growth as a secondary effect

Acutely altered hemodynamics following venous obstruction in the early chick embryo

In the venous clip model specific cardiac malformations are induced in the chick embryo by obstructing the right lateral vitelline vein with a microclip. Clipping alters venous return and intracardiac laminar blood flow patterns, with secondary effects on the mechanical load of the embryonic myocardium. We investigated the instantaneous effects of clipping the right lateral vitelline vein on hemodynamics in the stage-17 chick embryo. 32 chick embryos HH 17 were subdivided into venous clipped (N=16) and matched control embryos (N=16). Dorsal aortic blood flow velocity was measured with a 20 MHz pulsed Doppler meter. A time series of eight successive measurements per embryo was made starting just before clipping and ending 5h after clipping. Heart rate, peak systolic velocity, time-averaged velocity, peak blood flow, mean blood flow, peak acceleration and stroke volume were determined. All hemodynamic parameters decreased acutely after venous clipping and only three out of seven parameters (heart rate, time-averaged velocity and mean blood flow) showed a recovery to baseline values during the 5h study period. We conclude that the experimental alteration of venous return has major acute effects on hemodynamics in the chick embryo. These effects may be responsible for the observed cardiac malformations after clipping

Anatomical and sonographic correlation of the fetal ductus arteriosus in first and second trimester pregnancy

Ultrasonic visualization of the ductus arteriosus in first and second trimester pregnancies was compared with postmortem preparations. Twenty human fetal postmortem specimens from 8 to 19 weeks menstrual age were examined, 11 with microscopic reconstruction, nine with macroscopic dissection. The angle between ductus arteriosus and aortic isthmus (upstream) and ductus arteriosus and descending aorta (downstream) was determined. In 52 normally developing fetuses between 14 and 27 weeks, the angle between the ductus arteriosus and the thoracic spine as visualized in real-time ultrasound was determined. In a further 19 normally developing fetuses between 14 and 25 weeks, ductal blood flow was visualized by colour velocity imaging (CVI). In anatomical preparations, the upstream angle was always less than 90° and the downstream angle was always 80° or more. These angles were unrelated to menstrual age. In both real-time and CVI ultrasound, the angle between ductus arteriosus and thoracic spine remained at approximately 90°. CVI showed highest blood flow velocities at the point of ductal insertion into the aorta. When performing Doppler ultrasound examinations in the fetal ductus arteriosus, no menstrual age dependent angle adjustment appears to be necessary

Ventricular diastolic filling characteristics in stage-24 chick embryos after extra-embryonic venous obstruction

Alteration of extra-embryonic venous blood flow in stage-17 chick embryos results in well-defined cardiovascular malformations. We hypothesize that the decreased dorsal aortic blood volume flow observed after venous obstruction results in altered ventricular diastolic function in stage-24 chick embryos. A microclip was placed at the right lateral vitelline vein in a stage-17 (52-64 h of incubation) chick embryo. At stage 24 (4.5 days of incubation), we measured simultaneously dorsal aortic and atrioventricular blood flow velocities with a 20-MHz pulsed-Doppler velocity meter. The fraction of passive and active filling was integrated and multiplied by dorsal aortic blood flow to obtain the relative passive and active ventricular filling volumes. Data were summarized as means +/- S.E.M. and analyzed by t-test. At similar cycle lengths ranging from 557 ms to 635 ms (P>0.60), dorsal aortic blood flow and stroke volume measured in the dorsal aorta were similar in stage-24 clipped and normal embryos. Passive filling volume (0.07+/-0.01 mm(3)) was decreased, and active filling volume (0.40+/-0.02 mm(3)) was increased in the clipped embryo when compared with the normal embryo (0.15+/-0.01 mm(3), 0.30+/-0.01 mm(3), respectively) (P<0.003). In the clipped embryos, the passive/active ratio was decreased compared with that in normal embryos (P<0.001). Ventricular filling components changed after partially obstructing the extra-embryonic venous circulation. These results suggest that material properties of the embryonic ventricle are modified after temporarily reduced hemodynamic load

Ablation of various regions within the avian vagal neural crest has differential effects on ganglion formation in the fore‐, mid‐ and hindgut

The vagal neural crest adjacent to the first seven somites gives rise to both ganglionic and ectomesenchymal derivatives. Ganglionic derivatives are the neurons and supportive cells of the enteric nervous system (ENS), cardiac, and dorsal root ganglia. Ectomesenchymal derivatives are cells in the cardiac outflow tract and the mesenchymal components of thymus and parathyroids. Ectomesenchymal derivatives are formed by a segment of the vagal neural crest, from the level of the otic vesicle down to the caudal boundary of the third somite, called the cardiac neural crest. We performed neural crest ablations to study regional differences within the avian vagal neural crest with regard to the formation of the ENS. Ablation of the entire vagal neural crest from the mid‐otic vesicle down to the seventh somite plus the nodose placode resulted in the absence of ganglia in the midgut (jejunum and ileum) and hindgut (colon). The foregut (esophagus, proventriculus, gizzard, and duodenum) was normally innervated. After ablation of the vagal neural crest adjacent to somites 3–5, ganglia were absent in the hindgut. Ablations of vagal neural crest not including this segment had no effect on the formation of the ENS. We surmise that the innervation of the hindgut in vivo depends specifically on the neural crest adjacent to somites 3–5, whereas innervation of the midgut can be accomplished by all segments within the vagal neural crest. The foregut can also be innervated by a source outside the vagal neural crest. To study intrinsic differences between various vagal neural crest segments regarding ENS formation, we performed chorioallantoic membrane cocultures of segments of quail vagal neural anlage and E4 chicken hindgut. We found that all vagal neural crest segments were able to give rise to enteric ganglia in the hindgut. When the neural crest of somites 6 and 7 was included in the segment, we also found melanocytes in the hindgut, suggesting that this segment is more related to trunk neural crest. Furthermore, we found that the vagal neural anlage from older embryos (>18 somites) showed an increased potential to form enteric ganglia. This suggests that vagal neural crest cells that have been in prolonged contact with the neural tube in vivo, because of either late emigration or delayed migration, have an increased probability to form enteric ganglia

Histopathology of aortic complications in bicuspid aortic valve versus Marfan syndrome: relevance for therapy?

Patients with bicuspid aortic valve (BAV) and patients with Marfan syndrome (MFS) are more prone to develop aortic dilation and dissection compared to persons with a tricuspid aortic valve (TAV). To elucidate potential common and distinct pathways of clinical relevance, we compared the histopathological substrates of aortopathy. Ascending aortic wall biopsies were divided in five groups: BAV (n = 36) and TAV (n = 23) without and with dilation and non-dilated MFS (n = 8). General histologic features, apoptosis, the expr

Common arterial trunk and ventricular non-compaction in Lrp2 knockout mice indicate a crucial role of LRP2 in cardiac development

Lipoprotein-related receptor protein 2 (LRP2) is important for development of the embryonic neural crest and brain in both mice and humans. Although a role in cardiovascular development can be expected, the hearts of Lrp2 knockout (KO) mice have not yet been investigated. We studied the cardiovascular development of Lrp2 KO mice between embryonic day 10.5 (E10.5) and E15.5, applying morphometry and immunohistochemistry, using antibodies against Tfap2α (neural crest cells), Nkx2.5 (second heart field), WT1 (epicardium derived cells), tropomyosin (myocardium) and LRP2. The Lrp2 KO mice display a range of severe cardiovascular abnormalities, including aortic arch anomalies, common arterial trunk (persistent truncus arteriosus) with coronary artery anomalies, ventricular septal defects, overriding of the tricuspid valve and marked thinning of the ventricular myocardium. Both the neural crest cells and second heart field, which are essential for the lengthening and growth of the right ventricular outflow tract, are abnormally positioned in the Lrp2 KO. T hi s explains the absence of the aorto-pulmonary septum, which leads to common arterial trunk and ventricular septal defects. Severe blebbing of the epicardial cells covering the ventricles is seen. Epithelial-mesenchymal transition does occur; however, there are fewer WT1-positive epicardium-derived cells in the ventricular wall as compared to normal, coinciding with the myocardial thinning and deep intertrabecular spaces. LRP2 plays a crucial role in cardiovascular development in mice. This corroborates findings of cardiac anomalies in humans with LRP2 mutations. Future studies should reveal the underlying signaling mechanisms in which LRP2 is involved during cardiogenesis

Epicardium-derived cells are important for correct development of the Purkinje fibers in the avian heart

During embryonic development, the proepicardial organ (PEO) grows out over the heart surface to form the epicardium. Following epithelial-mesenchymal transformation, epicardium-derived cells (EPDCs) migrate into the heart and contribute to the developing coronary arteries, to the valves, and to the myocardium. The peripheral Purkinje fiber network develops from differentiating cardiomyocytes in the ventricular myocardium. Intrigued by the close spatial relationship between the final destinations of migrating EPDCs and Purkinje fiber differentiation in the avian heart, that is, surrounding the coronary arteries and at subendocardial sites, we investigated whether inhibition of epicardial outgrowth would disturb cardiomyocyte differentiation into Purkinje fibers. To this end, epicardial development was inhibited mechanically with a membrane, or genetically, by suppressing epicardial epithelial-to-mesenchymal transformation with antisense retroviral vectors affecting Ets transcription factor levels (n = 4, HH39-41). In both epicardial inhibition models, we evaluated Purkinje fiber development by EAP-300 immunohistochemistry and found that restraints on EPDC development resulted in morphologically aberrant differentiation of Purkinje fibers. Purkinje fiber hypoplasia was observed both periarterially and at subendocardial positions. Furthermore, the cells were morphologically abnormal and not aligned in orderly Purkinje fibers. We conclude that EPDCs are instrumental in Purkinje fiber differentiation, and we hypothesize that they coo

Coding of coronary arterial origin and branching in congenital heart disease: The modified Leiden Convention

Objectives: Variations in coronary anatomy are common and may relate to the position of the coronary ostium relative to the aortic sinus, the angle of coronary take-off, or the course of the coronary arterial branches. Several classification systems have been proposed. However, they all lack a simple rationale that is applicable irrespective of the relative position of the great arteries, as well as in bicuspid aortic valves. We present a modification of a relatively simple system introduced in the early 1980s, designated the “Leiden Convention.” Methods: The first step of the Leiden Convention is that the clinician takes position in the nonfacing sinus of the aorta looking toward the pulmonary orifice. The right-hand facing sinus is sinus 1, and the left-hand facing sinus is sinus 2. The coronary branches arising from sinus 1 are annotated proceeding in a counterclockwise fashion toward sinus 2. “Usual” (normal) coronary anatomy would be 1R-2LCx. Given their clinical relevance, single sinus coronary arteries are discussed separately. Results: This system was originally designed and highly applicable in hearts with an altered great artery relationship, such as in the var