Marco C. DeRuiter

Neural Crest Cell Contribution to the Developing Circulatory System Implications for Vascular Morphology

In this study, the distribution patterns of neural crest (NC) cells (NCCs) in the developing vascular system of the chick were thoroughly studied and examined for a correlation with smooth muscle cell differentiation and vascular morphogenesis. For this purpose, we performed long-term lineage tracing using quail-chick chimera techniques and premigratory NCC infection with a replication-incompetent retrovirus containing the LacZ reporter gene in combination with immunohistochemistry. Results indicate that NCC deposition around endothelial tubes is influenced by anteroposterior positional information from the pharyngeal arterial system. NCCs were shown to be among the first cells to differentiate into primary smooth muscle cells of the arch arteries. At later stages, NCCs eventually differentiated into adventitial fibroblasts and smooth muscle cells and nonmuscular cells of the media and intima. NCCs were distributed in the aortic arch and pulmonary arch arteries and in the brachiocephalic and carotid arteries. The coronary and pulmonary arteries and the descending aorta, however, remained devoid of NCCs. A new finding was that the media of part of the anterior cardinal veins was also determined to be NC-derived. NC-derived elastic arteries differed from non-NC elastic vessels in their cellular constitution and elastic fiber organization, and the NC appeared not to be involved in designating a muscular or elastic artery. Boundaries between NC-infested areas and mesodermal vessel structures were mostly very sharp and tended to coincide with marked changes in vascular morphology, with the exception of an intriguing area in the aortic and pulmonary trunks.

Smooth Muscle Cell Origin and Its Relation to Heterogeneity in Development and Disease

Smooth muscle cells (SMC) of the vascular system form an intriguing population of cells that are relevant for maintaining vascular tone and function. They also play a key role in pathological processes in the vessel wall. If we focus on the development of intimal thickening in the latter function, it is clear that even this pathological subset presents itself in various forms. That is, arteriosclerosis after hypertension,1 atherosclerosis,2 and restenosis after percutaneous transluminal coronary angioplasty or coronary artery bypass grafting surgery3 have features in common as well as characteristics selective for each disease. Relevant to an understanding of the above processes is the basic question of whether we are dealing either with a SMC heterogeneity in origin or with a spatiotemporal heterogeneity in expression of differentiation markers. To add to this complexity there is an increasing evidence that already committed and differentiated cells can transdifferentiate into another cell type. In studying SMC heterogeneity, a combination of these factors is likely. It has been shown by several research groups that SMC heterogeneity exists within the vessel wall, varying from the adult rat4 5 to the human fetal population.6 These data are mainly based on in vitro cell culture studies. A different approach is to study the intact vascular wall and expression of differentiation markers.7 8 9 10 11 This approach shows a change in gene expression patterns with normal maturation and with development of intimal thickening of the vessel wall. During development of intimal thickening in various settings, including physiological circumstances,11 thickening experimentally induced by a perivascular cuff,12 and atherosclerosis in humans,13 reexpression of fetal genes11 has been observed as well as altered migration and proliferation patterns as compared with normal. The most recent addition to characteristics in development of intimal …

Unilateral Vitelline Vein Ligation Alters Intracardiac Blood Flow Patterns and Morphogenesis in the Chick Embryo

To study the role of blood flow in normal and abnormal heart development, an embryonic chicken model was developed. The effect of altered venous inflow on normal intracardiac blood flow patterns was studied by visualization of blood flow with India ink. At stage 17, India ink was injected into a capillary or small venule within a specific yolk sac region. After determination of the normal intracardiac flow pattern, the right lateral vitelline vein was ligated, and the new intracardiac flow pattern was studied. Ligation resulted in disturbance of normal intracardiac flow patterns, which was most obvious in the conotruncus. The long-term effect of these abnormal intracardiac flow patterns on the development of the heart and pharyngeal arch arteries was investigated by permanent ligation in ovo with a microclip at stage 17 and subsequent evaluation at stages 34, 37, and 45. These experiments revealed anomalies of the vascular system in 58 of the 91 ligated embryos studied. We observed intracardiac malformations consisting of subaortic ventricular septal defects (n = 52), semilunar valve anomalies (n = 19), atrioventricular anomalies (n = 7), and pharyngeal arch artery malformations (n = 32). It is concluded that abnormal intracardiac blood flow, resulting from hampered venous inflow, may result in serious intracardiac and pharyngeal arch artery malformations comparable to defects observed in embryonic chicken models subjected to neural crest ablation, cervical flexure experiments, and excessive retinoic acid treatment.

Basics of cardiac development for the understanding of congenital heart malformations.

Cardiovascular development has become a crucial element of transgene technology in that many transgenic and knockout mice unexpectedly present with a cardiac phenotype, which often turns out to be embryolethal. This demonstrates that formation of the heart and the connecting vessels is essential for the functioning vertebrate organism. The embryonic mesoderm is the source of both the cardiogenic plate, giving rise to the future myocardium as well as the endocardium that will line the system on the inner side. Genetic cascades are unravelled that direct dextral looping and subsequent secondary looping and wedging of the outflow tract of the primitive heart tube. This tube consists of a number of transitional zones and intervening primitive cardiac chambers. After septation and valve formation, the mature two atria and two ventricles still contain elements of the primitive chambers as well as transitional zones. An essential additional element is the contribution of extracardiac cell populations like neural crest cells and epicardium-derived cells. Whereas the neural crest cell is of specific importance for outflow tract septation and formation of the pharyngeal arch arteries, the epicardium-derived cells are essential for proper maturation of the myocardium and coronary vascular formation. Inductive signals, sometimes linked to apoptosis, of the extracardiac cells are thought to be instructive for differentiation of the conduction system. In summary, cardiovascular development is a complex interplay of many cell–cell and cell–matrix interactions. Study of both (transgenic) animal models and human pathology is unravelling the mechanisms underlying congenital cardiac anomalies.

Avoiding long-term disturbance to bladder and sexual function in pelvic surgery, particularly with rectal cancer.

Urinary and sexual dysfunction are common problems after rectal cancer surgery, and the likely cause is damage to the pelvic autonomic nerves during surgery. In recent years, attention has been focused on preserving the autonomic nerves through a technique which is usually combined with total mesorectal excision or radical pelvic lymphadenectomy. The autonomic nerves consist of the paired sympathetic hypogastric nerve, sacral splanchnic nerves, and the pelvic autonomic nerve plexus. We will demonstrate the anatomy of the pelvic autonomic nerves and the relation of these nerves to the mesorectal fascial planes, and review the medical literature on sexual and urinary dysfunction after rectal cancer surgery with and without autonomic nerve preservation.

A nerve‐sparing radical hysterectomy: guidelines and feasibility in Western patients

Surgical damage to the pelvic autonomic nerves during radical hysterectomy is thought to be responsible for considerable morbidity, i.e., impaired bladder function, defecation problems, and sexual dysfunction. Previous anatomical studies and detailed study of surgical techniques in various Japanese oncology centers demonstrated that the anatomy of the pelvic autonomic nerve plexus permits a systematic surgical approach to preserve these nerves during radical hysterectomy without compromising radicality. We introduced elements of the Japanese nerve-preserving techniques and carried out a feasibility study in ten consecutive Dutch patients. The technique involved three steps: first, the identification and preservation of the hypogastric nerve in a loose tissue sheath underneath the ureter and lateral to the sacro-uterine ligaments; second, the inferior hypogastric plexus in the parametrium is lateralized and avoided during parametrial transsection; third, the most distal part of the inferior hypogastric plexus is preserved during the dissection of the posterior part of the vesico-uterine ligament. The clinical study showed that the procedure is feasible and safe, except possibly when used with very obese patients and patients with broad, bulky tumors. Surgical preservation of the pelvic autonomic nerves in radical hysterectomy deserves consideration in the quest to improve both cure and quality of life in cervical cancer patients.

Development of the Cardiac Conduction Tissue in Human Embryos Using HNK-1 Antigen Expression Possible Relevance for Understanding of Abnormal Atrial Automaticity

BACKGROUND Abnormal atrial automaticity in young patients with structurally normal hearts is often located around the pulmonary veins and in sinus venosus-related parts of the right atrium. We hypothesize that these ectopic pacemaker sites correspond to areas of embryonic myocardium with an early phenotypic differentiation, as indicated by differences in antigen expression during normal cardiac development. METHODS AND RESULTS In human embryos ranging in age from 42 to 54 days of gestation, the development of the cardiac conduction system was studied with the use of HNK-1 immunohistochemistry. HNK-1 stains the developing atrioventricular conduction system, ie, the bundle branches, His bundle, right atrioventricular ring, and retroaortic ring. In addition, the myocardium around the common pulmonary vein showed transient HNK-1 antigen expression. In the right atrium, 3 HNK-1-positive connections were demonstrated between the sinoatrial node and the right atrioventricular ring. An anterior tract through the septum spurium connects the sinoatrial node with the anterior right atrioventricular ring, and 2 posterior tracts connect the sinoatrial node with the posterior right atrioventricular ring through the right venous valve (future crista terminalis) and sinus septum, encircling the coronary sinus. The medioposterior part of the right atrioventricular ring connected to the His bundle and the medioanterior part form 2 node-like structures. CONCLUSIONS In patients with abnormal atrial automaticity, the distribution of left and right atrial pacemaker foci correspond to areas of the embryonic myocardium that temporarily express the HNK-1 antigen.

Origin, Fate, and Function of Epicardium-Derived Cells (EPDCs) in Normal and Abnormal Cardiac Development

During heart development, cells of the primary and secondary heart field give rise to the myocardial component of the heart. The neural crest and epicardium provide the heart with a considerable amount of nonmyocardial cells that are indispensable for correct heart development. During the past 2 decades, the importance of epicardium-derived cells (EPDCs) in heart formation became increasingly clear. The epicardium is embryologically formed by the outgrowth of proepicardial cells over the naked heart tube. Following epithelial-mesenchymal transformation, EPDCs form the subepicardial mesenchyme and subsequently migrate into the myocardium, and differentiate into smooth muscle cells and fibroblasts. They contribute to the media of the coronary arteries, to the atrioventricular valves, and the fibrous heart skeleton. Furthermore, they are important for the myocardial architecture of the ventricular walls and for the induction of Purkinje fiber formation. Whereas the exact signaling cascades in EPDC migration and function still need to be elucidated, recent research has revealed several factors that are involved in EPDC migration and specialization, and in the cross-talk between EPDCs and other cells during heart development. Among these factors are the Ets transcription factors Ets-1 and Ets-2. New data obtained with lentiviral antisense constructs targeting Ets-1 and Ets-2 specifically in the epicardium indicate that both factors are independently involved in the migratory behavior of EPDCs. Ets-2 seems to be especially important for the migration of EPDCs into the myocardial wall, and to subendocardial positions in the atrioventricular cushions and the trabeculae. With respect to the clinical importance of correct EPDC development, the relation with coronary arteriogenesis has been noted well before. In this review, we also propose a role for EPDCs in cardiac looping, and emphasize their contribution to the development of the valves and myocardial architecture. Lastly, we focus on the congenital heart anomalies that might be caused primarily by an epicardial developmental defect.

Extraembryonic venous obstructions lead to cardiovascular malformations and can be embryolethal

OBJECTIVE To expand our knowledge concerning the effect of placental blood flow on human heart development, we used an embryonic chicken model in which extraembryonic blood flow was manipulated. METHODS First, one of the three major vitelline veins was ligated, while blood flow was visualized with Indian ink. In this way, we could study the effect of different ligation positions on intracardiac flow patterns. Secondly, these vitelline veins were ligated permanently with a microclip until cardiac septation was completed, thereafter, the hearts were morphologically evaluated. In this way, we could study the impact of the ligation position on the severity and frequency of heart malformations. On combining the results, we were able to study the effect of different intracardiac flow patterns on heart development. RESULTS Although ligation of each vein resulted in different intracardiac flow patterns, long-term ligation resulted in similar cardiovascular malformations in survivors. These consisted mainly of ventricular septum defects (VSDs), semilunar valve anomalies, and pharyngeal arch artery malformations. There was no significant difference (p > 0.05) between the ligation position and the incidence of cardiovascular malformations. However, the percentage mortality after clipping the left lateral vitelline vein was significantly higher (p < 0.05) than after ligation of either the right lateral or posterior vitelline vein. CONCLUSIONS Early extraembryonic venous obstruction leads to altered flow patterns, which probably result in shear stress changes. In postseptation stages, these result in a spectrum of cardiovascular malformations irrespective of the ligation position. A diminished incidence of VSDs in the oldest stage was attributed to delayed closure of the interventricular foramen.

Preservation of Left Ventricular Function and Attenuation of Remodeling After Transplantation of Human Epicardium-Derived Cells Into the Infarcted Mouse Heart

Background— Proper development of compact myocardium, coronary vessels, and Purkinje fibers depends on the presence of epicardium-derived cells (EPDCs) in embryonic myocardium. We hypothesized that adult human EPDCs might partly reactivate their embryonic program when transplanted into ischemic myocardium and improve cardiac performance after myocardial infarction. Methods and Results— EPDCs were isolated from human adult atrial tissue. Myocardial infarction was created in immunodeficient mice, followed by intramyocardial injection of 4×105 enhanced green fluorescent protein–labeled EPDCs (2-week survival, n=22; 6-week survival, n=15) or culture medium (n=24 and n=18, respectively). Left ventricular function was assessed with a 9.4T animal MRI unit. Ejection fraction was similar between groups on day 2 but was significantly higher in the EPDC-injected group at 2 weeks (short term), as well as after long-term survival at 6 weeks. End-systolic and end-diastolic volumes were significantly smaller in the EPDC-injected group than in the medium-injected group at all ages evaluated. At 2 weeks, vascularization was significantly increased in the EPDC-treated group, as was wall thickness, a development that might be explained by augmented DNA-damage repair activity in the infarcted area. Immunohistochemical analysis showed massive engraftment of injected EPDCs at 2 weeks, with expression of α-smooth muscle actin, von Willebrand factor, sarcoplasmic reticulum Ca2+-ATPase, and voltage-gated sodium channel (α-subunit; SCN5a). EPDCs were negative for cardiomyocyte markers. At 6-weeks survival, wall thickness was still increased, but only a few EPDCs could be detected. Conclusions— After transplantation into ischemic myocardium, adult human EPDCs preserve cardiac function and attenuate ventricular remodeling. Autologous human EPDCs are promising candidates for clinical application in infarcted hearts.