Embryonic development of coronary vasculature in ratsCorrosion casting studies.код для вставкиСкачать
THE ANATOMICAL RECORD PART A 270A:109 –116 (2003) Embryonic Development of Coronary Vasculature in Rats: Corrosion Casting Studies ANNA RATAJSKA,1* BOGDAN CISZEK,2 AND AGNIESZKA SOWIŃSKA3 1 Department of Pathological Anatomy, Medical University of Warsaw, Warsaw, Poland 2 Department of Anatomy, Medical University of Warsaw, Warsaw, Poland 3 Department of Pathology, Children’s Memorial Health Institute, Warsaw, Poland ABSTRACT The aim of this study was to analyze the development of coronary vessels at different stages of embryonic life in rats using corrosion casts and scanning electron microscopy (SEM). We studied morphologic details of vessel maturation, expansion, and pattern formation from the stage of development when the coronary system forms patent connections with the aorta and the right atrium (embryonic day 16 (ED16)) to full-term fetus (ED21). The internal surface morphologies of the arterial and venous vessel walls were different and were dependent on the distance from the oriﬁce and the capillary system. They also depended on the maturation state of a given vessel. In various branches of the coronary system we demonstrated round, fusiform or polygonal, endothelial cell imprints. The capillary network was dense, however, at the early stages of development, it formed a thin layer over the myocardium. By ED21 capillaries assumed an orientation parallel to the long axes of the cardiac myocytes. During all stages of development, different forms of angiogenesis by intussusceptive growth were observed. Splitting of the vessel wall occurred in two or three points along the vessel, forming two- or three-link chains. Certain areas of vessels resembled doughnuts, from which several sister vessels originated. The coronary arteries were situated deep within the myocardial wall. The major coronary veins were mostly located on the surface of the capillary plexuses of the myocardial wall. In conclusion, this method of vessel casting enables the detection of angiogenesis by intussusceptive growth, and the visualization of a capillary’s position to the myocardial wall, thickness of the capillary plexuses, and the internal surface morphology of major vessels. Anat Rec Part A 270A:109 –116, 2003. © 2003 Wiley-Liss, Inc. Key words: coronary vessel; corrosion cast; fetal rat heart; angiogenesis; scanning electron microscope Vascularization of the rat myocardium starts relatively late during embryonic development, on embryonic day 13 (ED13) (Heinzberger, 1983). The formation of new blood vessels in the embryonic heart is accomplished by at least two processes: 1) vasculogenesis (Rongish et al., 1994; Risau, 1997), which includes in situ formation of cell clusters consisting of angioblasts and erythroblasts, which then differentiate into endothelial cells and erythrocytes, respectively; and 2) angiogenesis (Tomanek et al., 1996), i.e., the sprouting of new vessels from preexisting ones. Throughout embryonic life, both vasculogenesis and angiogenesis take part in the formation of coronary vessels. Endothelial cells differentiate (vasculogenesis) and then coalesce, forming a lumen. Subsequently, they arborize (angiogenesis) to ﬁnally form a continuous, patent system of tubes called primitive vessels, within which blood circulates (Risau, 1997). © 2003 WILEY-LISS, INC. Despite the growing number of publications in this ﬁeld, some of the morphologic details of coronary vessel angiogenesis are still undeﬁned. The heart is a pump that undergoes vascularization very late, considering the rat’s Grant sponsor: KBN; Grant number: 6P05A02520; Grant sponsor: Medical University of Warsaw. *Correspondence to: Anna Ratajska, Department of Pathological Anatomy, Medical University of Warsaw, Chałubińskiego 5, 02-004 Warsaw, Poland. Fax: ⫹48-22-629-98-92. E-mail: email@example.com Received 21 December 2001; Accepted 1 October 2002 DOI 10.1002/ar.a.10011 110 RATAJSKA ET AL. Fig. 1. a: ED16 heart, posterior surface with the atria removed. Surface vessels of the left ventricle were removed to demonstrate the trabecular system of this region of the heart. The thin capillary plexus covers the right ventricle. One vein runs along the atrioventricular sulcus and congregates vessels coming from the direction of the apex. b: Higher magniﬁcation of the coronary venous system of the right ventricle, with details of the capillary plexus. Round endothelial cell imprints can be seen on the surface of venules. Bar ⫽ 100 m. short embryonic period of life (between ED13 and ED21). Some forms of angiogenesis extend into early postnatal life (Tomanek, 1996). During the early period of embryonic development, the avascular heart is nourished from the lumen by blood circulating within the trabecular system (Ošt’ádal et al., 1975). Subsequently, the myocardial wall thickens, and trabeculae gradually become ﬂatter and wider (Ošt’ádal et al., 1975). On ED16 –17, coronary arteries form patent connections with the aorta (Bogers et al., 1988; Ratajska and Fiejka, 1999), and probably at about the same time (based on the studies in quail heart by Vrancken Peeters et al. (1997a) the venous system forms a connection with the right atrium. Corrosion casts of the coronary system can only be made after formation of patent coronary vessel connections with the heart’s chambers. The number of studies of heart vascularization has dramatically increased during the last decade due to the development of various morphological, immunohistochemical (Poelmann et al., 1993; Vrancken Peeters et al., 1997a, b, 1999), and India ink injection Fig. 2. ED16 heart. a: Conal part with the pulmonary trunk (pt) in the front and the aorta (a) behind on the left; a vessel plexus surrounding the truncus arteriosus and the conus forms a thin layer of capillaries. b: Higher magniﬁcation of the ascending aorta with the right coronary sinus (the oriﬁce of the right coronary artery marked with arrow); endothelial cell imprints within the sinus and the proximal aorta are polygonal. c: Higher magniﬁcation of the proximal part of the left coronary artery coursing from the aorta (which is hidden behind the pulmonary trunk), with longitudinally oriented endothelial cell imprints in the proximal part (arrow) and oval or round endothelial cell imprints at a certain distance from the oriﬁce. Bar ⫽ 100 m. CORROSION CAST OF CORONARY VESSELS 111 Fig. 3. ED16 heart. The anterior surface of the right ventricle with a connection of a capillary with the ventricular chamber marked with arrow (ﬁstula). Bar ⫽ 100 m. techniques (Waldo et al., 1990; Vrancken Peeters et al., 1997b), as well as retrovirus labeling methods (Mikawa and Fischman, 1992; Mikawa and Gourdie, 1996). However, little is known about the formation and distribution of coronary arteries, veins, and capillaries, and their three dimensional pattern during fetal heart development in rats. The corrosion cast technique has been successfully utilized in studies of abnormal patterning of vessels within the myocardium (Bockman et al., 1989; Sans-Coma et al., 1999). In the present study we applied this technique to extend our knowledge of the normal development of coronary vessels. We studied rat heart vascularization during the period of embryonic life between ED16 (i.e., the time when the coronary system makes patent connections with the aorta) and ED21 (full-term fetus). MATERIALS AND METHODS All procedures were performed according to the requirements of the Animal Care Ethics Committee of Poland, and in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals. Pregnant dams of the Wistar Albino Glasgow (WAG) strain were used for the experiments. The animals were kept in cages with standard laboratory food and water at libitum (12/12-hr light/dark cycles). Day 0 of pregnancy was estimated by the presence of spermatozoa in the early-morning vaginal smear. Dams were anesthetized with narcotan and chlorohydrate (100 mg/kg b.w., i.p.), and the fetuses were removed and additionally treated with chlorohydrate. The fetuses were then divided into six age groups: ED16 –ED21 (full term). At least ﬁve fetuses were used in each experimental group. Corrosion Cast Technique The fetuses were perfused via the umbilical artery with 3 ml of a prewarmed (37°C) heparinized saline (12.5 I.U./ ml), containing 3% dextran, M.W. 70,000 and 0.025% lidocain (Lignocain, Polfa, Poland) until a pure solution from the umbilical vein was obtained. Subsequently, perfusion ﬁxation was carried out with 2–3 ml of 0.08% glutaraldehyde/0.66% paraformaldehyde in 0.15 M cacody- Fig. 4. a: ED18 heart. Maturation of the venous system. A vein coursing horizontally (above) with its surface marked with round imprints of endothelial cell nuclei and transverse indentations. b: ED19 heart with “bypass vessels” (microvessels that bypass the main route of a major vein and coalesce with this vein again at a lower level). Bar ⫽ 100 m. late buffer, pH 7.4, 37°C. Finally, a mixture consisting of 8 ml Mercox威 CL-2R (Vilene Comp., Japan) and 2 ml methyl methacrylate (Fluka) containing 0.2 g MA initiator per 10 ml of the casting medium was injected (in a volume of 2 ml). Hearts were removed and placed in water overnight at 50°C to allow the resin to harden. Subsequently, the soft tissue of the myocardium was macerated with 10% KOH and rinsed with tap water (Lametschwandtner et al., 1990; Miodoński and Litwin, 1999). The resulting vascular casts were dried, mounted onto specimen stubs, coated with carbon and gold, and examined in a Jeol JSM-35C scanning electron microscope at 25kV. Histological Examination Some fetal hearts of 21-day-old rats were ﬁxed in 4% buffered formalin, dehydrated in a series of increasing alcohol concentrations, and embedded in parafﬁn. Serial sections were cut and stained with hematoxylin-eosin. Morphometry and Statistical Analysis Scanning electron microscopy (SEM) images were used for measurements of proximal diameters of both coronary 112 RATAJSKA ET AL. Fig. 5. Different forms of angiogenesis by intussusceptive growth demonstrated in embryonic hearts. a: ED18 heart: angiogenesis is visible as a chain of three consecutive splitting loops along the same capillary (arrow). b: ED19 heart: angiogenesis within a vein representing a doughnut-like structure (arrow); there are three or four vessels branching off in various directions. Bar ⫽ 100 m. arteries and distal branches of two major veins running on the posterior surface of the ventricles. The same respective vessels were measured at the earliest and the latest stages of development (ED16 and ED21). At least three measurements were taken of every vessel by the use of an image analysis program (Multiscan). The results were presented as the mean value ⫾ standard deviation. RESULTS In ED16 hearts the outer surface of the myocardial wall was covered with vascular plexuses, which appeared to be discontinuous over the whole myocardium. The trabecular system of the ventricles was visible under the thin capillary surface (Fig. 1a and b). Capillaries and primordial veins and arteries were distinguished. Morphologically, these vessels were very similar at this stage of development. However, the veins and arteries had larger diameters than the capillaries (44 m for veins, 36.6 ⫾ 2.9 m for arteries, and 15.4 ⫾ 3.84 for capillaries). Capillaries over the surface of the heart close to the apex were oriented haphazardly (not shown). The surface of veins was Fig. 6. a: The conus of an ED21 heart with capillaries running obliquely in the proximal part (upstream) and transversely in the distal part (downstream). b: Higher magniﬁcation of the distal part of the heart conus with capillaries arranged transversely; arrow points to the cranial direction; pt, pulmonary trunk. Bar ⫽ 100 m. marked with round endothelial cell imprints. The major veins were located on the posterior surface of the ventricles. The veins at this stage of heart development were short. Thus, the proximal end of these vessels was situated not far from the base of the heart. The course of some veins overlapped the atrioventricular sulcus. The conus of the heart was covered with a thin capillary plexus forming a wreath around this part of the heart (Fig. 2a). The vessel plexus in this area consisted of one layer of capillaries, under which the surface of the conotruncus was seen. The coronary sinuses were imprinted with polygonal nuclei of endothelial cells. Imprints of the same shape were also found in the wall of the aortic and pulmonary trunk roots (Fig. 2b). The proximal part of the coronary artery had fusiform endothelial cell imprints oriented longitudinally to the direction of the blood ﬂow, whereas at a certain distance from the coronary sinus the imprints on the coronary artery surface were round (Fig. 2c). In one case we observed a coronary vessel ﬁstula (a connection of a capillary with the ventricular chamber) (Fig. 3, arrow). On ED17, the thickness of the capillary plexus increased within the myocardial wall. The major veins were CORROSION CAST OF CORONARY VESSELS Fig. 7. a: ED19 heart. Parallel orientation of the capillary plexus on the posterior surface of the left ventricle. b: ED21 heart, right-posterior surface: superﬁcial capillaries run in a vertical orientation (long arrow); beneath. Capillaries run in a horizontal orientation (short arrow). Bar ⫽ 100 m. located on the posterior surface of the left and right ventricles, and their proximal ends were located closer to the apex of the heart compared with the respective veins of ED16 hearts (not shown). Both veins merged with their minor tributaries into the coronary sinus, or the right vein went directly into the right atrium, independently of the coronary sinus. Some veins were situated on the lateral and anterior surfaces of the outﬂow tract. Coronary arteries and their branches coursed deep within the myocardial wall, except for their very proximal portion, which ran subepicardially. Starting from ED17 and later during development, the capillaries within the myocardial wall tended to be oriented parallel to the long axes of the cardiac myocytes. ED18 and ED19 hearts were completely covered with dense capillary plexuses that ran within the thickened myocardial wall. Distal branches of the coronary arteries were completely covered with thick capillary plexuses. Venules and their minor tributaries could be distinguished by the presence of round or oval endothelial cell imprints on their surface, and by transversely oriented indentations (Fig. 4a; compare with the ED16 heart in Fig. 1a, in which the veins are devoid of indentations). Some 113 Fig. 8. a: ED20 heart viewed from the front; the pulmonary trunk has been removed. The aorta with the coronary sinuses and the proximal coronary arteries can be seen. b: Proximal part of the left coronary artery at the level of branching (close to the aortic sinus), with longitudinally oriented endothelial cell imprints. Bar ⫽ 100 m. veins on the posterior wall of the ventricle formed an unusual pattern with “bypassing” capillaries (Fig. 4b). In the latter case, these vessels were highly convoluted and formed structures, bypassing the main route of the great vein and then coalescing with the same vein again at a lower level. Intussusceptive angiogenesis was distinguishable on the surface of the ventricular wall at all stages (Fig. 5a and b). Various forms of intussusceptive angiogenesis were identiﬁed by the presence of small pits and holes, and capillaries splitting into two sister vessels (not shown). In some areas capillary splitting occurred in two or three adjacent places along the capillary wall, creating two- or three-link chain structures, respectively (Fig. 5a). In another case a fragment of a small vein formed a structure resembling a doughnut, from which several descendent vessels originated (Fig. 5b). On ED20 –21 the right ventricular conus was covered with a thick, dense capillary plexus, within which capillaries took on a speciﬁc orientation along cardiac myocytes traversing obliquely within the conal wall (Fig. 6a). Distally (upstream), at the origin of the pulmonary trunk (at 114 RATAJSKA ET AL. Fig. 9. ED19 heart with the atria removed. The right-posterior surface of the ventricles with major veins coursing superﬁcially. The veins on the heart conus are short, whereas the proximal ends of the veins running on the posterior part of the ventricles are close to the apex. Bar ⫽ 100 m. the arterio-myocardial border) the capillaries were oriented circularly (Fig. 6b). In the fetal myocardium at these stages of development, the capillary system imitated the orientation of cardiac myocytes. In certain parts of the myocardial wall, the capillary system formed strata with a different orientation of capillaries. In the posterior part of the left ventricle, capillaries were oriented parallel to the tributaries of the major vein, i.e., transversely to the long axis of the heart (Fig. 7a). The posterior wall of the right ventricular myocardium (close to the interventricular septum) contained vertically and horizontally oriented capillaries in different strata (Fig. 7b). Both coronary arteries and their branches (Fig. 8a and b) together with the venous system (Fig. 9) could be distinguished. The mean diameter of coronary arteries at their proximal courses was 90.9 ⫾ 13 m, whereas the mean diameter of veins measured at their distal courses was 81.3 ⫾ 9.7 m. The arteries and veins took an analogous position and course to the respective vessels in adult rat heart. Some veins coursing within the subepicardium of the proximal interventricular sulcus accompanied the superﬁcial branches of the left coronary artery (Fig. 10). The veins coursing within the conal and lateral parts of the heart were short: their proximal ends were situated in the middle of the ventricular length (Fig. 9). The veins of the right ventricular epicardium did not accompany the respective branches of the right coronary artery. The same was true for the veins running on the posterior surface of both ventricles (Fig. 10). On ED20 –21, the coronary artery sinuses within the aortic wall were entirely developed (Fig. 8a and b). Fusiform endothelial cell imprints within the proximal portion of the coronary artery were oriented longitudinally to the blood stream, whereas endothelial cell imprints within the sinuses of Valsalva did not have such a regular orientation, and assumed polygonal shapes. DISCUSSION The present work describes for the ﬁrst time the sequence of events taking place during embryonic coronary Fig. 10. ED21 heart, H&E staining. a: The proximal part of the left coronary artery (large arrowhead) branching off the aorta with an accompanying vein (small arrowhead); the vein courses subepicardially within the conal part of the heart. b: At the level of the artioventricular junction the subepicardially coursing veins (small arrowheads) accompany two branches of the left coronary artery, whereas two branches of the right coronary artery (large arrowhead) do not have their venous counterparts subepicardially; bar ⫽ 200 m. c: Major veins run subepicardially (small arrowheads) on the posterior surface of the ventricles, whereas the respective arteries (or their branches) course at a certain distance within the myocardial wall (large arrowheads). d: Higher magniﬁcation of the area shown in b, representing the anterior interventricular septum with four branches of the left coronary artery (two of them marked with large arrowheads) and accompanying veins (small arrowheads). Bar ⫽ 100 m. angiogenesis by means of the corrosion cast method. Since the ﬁrst steps of the angiogenic process involve interactions between cell clusters that do not form continuous channels within the myocardium (Bogers et al., 1989; Poelmann et al., 1993; Rongish et al., 1994), it is not possible to study these stages (vasculogenesis) by the corrosion cast technique. The corrosion cast technique can be very useful in demonstrating those angiogenic events that occur after the formation of a patent system of vessels continuous with the fetal circulation. Studies by van Groningen et al. (1991) have dealt with rat heart vascularization in the prenatal and early postnatal periods of life by corrosion cast and quantitative methods. We focused only on the embryonic period, and extended the study by adding some important data to this topic. First, we have described in detail for the ﬁrst time the coronary system at the very early stages of development (ED16), when coronary arteries ﬁrst form patent connections with the aorta. Second, we found that in ED16 hearts the coronary system consists of a very thin layer of capillary plexuses and larger vessels (primordial arteries and veins). The larger vessels on the posterior side of the heart have the surface morphology of veins with typical round endothelial cell imprints. As the process of vein maturation proceeds, additional marks on their internal surface can be seen in the form of transverse indentations. During later stages of development this system of vessels thickens as the myocardium enlarges, and the trabeculae gradually become ﬂatter. Third, we conﬁrmed previous observations from corrosion casts made on ED16 and CORROSION CAST OF CORONARY VESSELS ED21 (van Groningen et al., 1991) that angiogenesis is characterized by certain morphological signs, such as the formation of “splitting vessels” which divide into sister vessels. These events correspond to the time sequence of new vessel formation by intussusceptive growth (Burton and Palmer, 1989; Burri and Tarek, 1990; Patan et al., 1993; Miodoński et al., 1998). In addition we demonstrated the existence of other forms of angiogenesis by intussusceptive growth, during which new vessels start to develop from veins, creating structures similar to doughnuts. In the case of “bypass” vessels, the newly formed vessels could originate either from a large vein that gives off capillaries, or from a single capillary that further splits into two vessels (one remaining a capillary, and the other differentiating into a large vein). We have also shown with this method that coronary vessel maturation and differentiation involves certain changes on their internal surface by characteristic nuclear imprints of endothelial cells. These imprints occur ﬁrst when coronary circulation becomes patent and connects with the whole-body circulation. The shapes of these imprints may depend on the velocity of blood ﬂow within the vessel, and may be related to the vessel shape. For example, within the sinuses of the aorta and the pulmonary trunk (with wall shapes similar to hemispheres) the imprints are polygonal; in the ascending aorta and the proximal part of the coronary arteries (cylinder-like shapes) the imprints are fusiform, with the long axes oriented longitudinally to the direction of the blood ﬂow. Interestingly, in very young fetuses the endothelial cell imprints are oval on the surface of coronary arteries at a certain distance from the oriﬁce, as in veins (low blood ﬂow velocity). The surface of postarterial capillaries within the capillary plexuses is smooth. Our present study indicates that the early system (ED16) of venules tends to be localized close to the atrioventricular sulcus, leaving the rest of the myocardial wall void of larger coronary vessels. The venules (precursors of major veins) are very short at this stage of development. The same tendency was demonstrated in our previous study with regard to the arterial system: the length of the coronary artery is small compared with ventricular length on ED16 –17, and increases rapidly at later stages of development (Ratajska et al., 2000). Other studies of quail hearts (Vrancken Peeters et al., 1997b), in which the India ink injection technique was used, also suggest the existence of very short coronary arteries and veins just after the formation of patent connections with the aortic lumen. These studies indicate that the expansion of the venous and arterial systems during embryonic life proceeds toward the heart apex. The capillary plexus of the myocardial wall is very thin at this stage and appears to be discontinuous. The demonstration of capillaries throughout the myocardium with other techniques (Ratajska and Fiejka, 1999) may indicate a lack of patency between the respective groups of capillary plexuses at these early stages. During later stages of development (starting from ED18) the diameter of both coronary arteries and veins increases markedly. The maturation of veins during all stages of development is demonstrated by a change of their internal surface morphology: in ED16 hearts endothelial cell imprints are weak, and later (ED18 –21) the imprints on the internal surface of veins are more prominent and accompanied by transverse indentations. The shape of aortic sinuses changes during development (com- 115 pare Figs. 2b and 8a). There are also some differences among species. Compared with chicken embryonic hearts at the same developmental stage (Aikawa and Kawano, 1982), in which the aortic sinuses are oval and ﬂat in shape, in the rat the sinuses are round and bulgy. Since corrosion casting samples are very fragile, we were unable to use this technique to analyze the surface morphology of deeply located coronary arteries, branches, and vessels covered by dense capillaries. Thus, to localize the arterial system we performed histological analysis of serial sections of ED21 hearts. The arteries and their branches are located deep within the myocardial wall. Interestingly, the veins in most areas of the heart usually do not accompany the respective arteries within the rat myocardium, with the exception of veins running on the left surface of the outﬂow tract, which are situated close to the respective arteries (namely, the superior branches of the left coronary artery). Major veins have a tendency to run on the posterior surface of the ventricles and on the lateral surfaces of the outﬂow tract. The veins of the outﬂow tract are short. By the end of fetal life, the pattern of major vessel distribution reaches a ﬁnal shape and position equivalent to those seen in the adult rat heart (Jons and Olson, 1954; Dbalý et al., 1968; Beighley et al., 1997). The course and branching system of the major vessels, however, appear to depend on the rat strain. The morphological pattern of the capillary plexuses at the beginning of their formation is irregular, except for the conal part of the heart, where they become oriented circularly around the conus in the early stages of development. At later developmental stages the capillaries are usually oriented parallel to the long axes of the myocytes. Thus, during the earlier stages of development, which are characterized by immature shapes of cardiac cells, myocardial capillaries also show an irregular orientation. In this study we presented one example of a connection between the coronary system and the ventricular chamber. We suspect that these connections (ﬁstulas) are rare during normal heart development, because in our other studies (performed by serial section analysis) we found few of them (data not published). Interestingly, at the bases of the major vessels extending from the heart (the pulmonary trunk and the aorta), the capillary system is oriented in a very regular way and tends to encircle the roots of the great vessels. This pattern of distribution may reﬂect the direction of cardiac myocytes in this region of the heart. ACKNOWLEDGMENTS The authors are thankful to Prof. B. Woźniewicz, the head of the Department of Pediatric Pathology, Children’s Memorial Health Institute in Warsaw, for access to the scanning electron microscope. We are also grateful to Prof. A. Miodoński, Collegium Medicum of the Jagiellonian University of Kraków, for his expert suggestions and discussion. 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