Book - Contributions to Embryology Carnegie Institution No.65
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Direct Growth of Veins by Sprouting
By Florence R. Sabin Anatomical Laboratory, Johns Hopkins Medical School. With one plate (1922).
In the chapter on the development of the vascular system in Keibel and Mall's Manual of Human Embryology, published in 1911 and 1912, Evans gave an analysis of the progress of embryology in connection with this system up to that time. By his own work he then demonstrated in a series of beautiful studies that the method of injection, as applied to the embryo, had made possible a great advance in the phase of the subject concerned with the spread of vessels over the body. In the introduction he said :
- "The two fundamental questions involved in the development of the vascular system are (1) What is the origin of the blood-vessels in the body of the embryo? (2) What is the primitive form of the vessels in any area, and the manner of change from this to that of the adult? These two aspects of the subject thus concern themselves with the problem of the cellular antecedents of the endothelium, on the one hand, and with the principles governing the architecture of the vascular system, on the other. To the former problem it is still impossible to give any decisive answer, but to the latter I trust the reader will see that a flood of new light has come."
It is now possible, I think, to give a definite answer to the first question; we know just how blood-vessels begin, and it is therefore possible to show that this knowledge of the fundamental genesis of the vascular system calls for certain extensions and modifications of the prevailing views on the second question.
A more careful examination of the old problem of angiogenesis, opened up by the early embryologists, Wolff, Pander, von Baer, and others, in their studies on blood-islands, has shown that blood-vessels begin by the differentiation of a new type of cell, the angioblast of His or vasoformative cell of Ranvier. The final proof that vessels are formed intracellularly was not obtained until the methods of tissue-culture permitted the process to be actually watched in a living specimen. The angioblast has certain characteristics. When it divides it forms syncytial masses, which have two essential properties: (1) the power of liquefying in the center, with the formation of plasma and vesicles; (2) the power of sprouting, by which these groups of cells join similar groups, forming vessels or plexuses. Both of these processes are necessary for the formation of the vascular system. It has thus become clear that the most fundamental concept, in connection with the vascular system, is that its essential tissue, endothelium, differentiates from mesenchyme. This means that the processes by which vessels form are essentially different from the processes by which the great tissue spaces (such as the arachnoidal spaces and periotic spaces) form. Weed (1917) has followed the development of the spaces of the arachnoid, Streeter (1917) the periotic spaces, and Shields, in a paper now in preparation, the tendon-sheaths; in all of these structures the formation or differentiation of a mesothelial lining is the last stage in the process, while in the formation of the blood-vessels the differentiation of the lining-cell, endothelium, is the first stage in the process.
Having determined that the primary point about the vascular system is that it starts by the differentiation of a new cell, which increases by division and by sprouting, it is of first importance to determine whether the differentiation of new angioblasts is limited in time or whether it continues throughout life, either generally or in certain specific places. This question was tested by restudying the regeneration of blood-vessels, after intestinal anastomoses made' in adult dogs, in conjunction with Dr. Halsted and Dr. Holman (1920), who performed the operations. No evidence could be found of a differentiation of new angioblasts; rather, the vascular system was restored by an active division of preexisting endothelium of small arteries, veins, and capillaries, involving a return of this endothelium to its embryonic angioblastic condition. Thus in these studies the new vessels, when first formed, were connected with the old, but showed a lumen as irregular as the lumen of embryonic vessels during their transformation from solid angioblastic masses. It thus seems likely that we must look for a phase in embryonic or fetal development when the differentiation of new angioblasts ceases, all subsequent new growth of vessels being accounted for by the division of preexisting endothelium. Thus the complete story of the development of the vascular system must take into consideration how far each vessel arises by the differentiation of angioblasts and how far by division and sprouting, and when, for each organ or area of the body, the differentiation of new vasoformative cells ceases.
As far as we have gone in this study, it has been found that throughout the first 7 days of incubation in the chick there is a differentiation of new angioblasts to be made out in the area pellucida. This differentiation of new angioblasts is extremely extensive during the whole of the second day; from the third day on it becomes relatively greatly diminished ; but almost any blastoderm up to the seventh day of incubation, which is as far as the process has yet been followed, will show one or two small vesicles unconnected with the main plexus. It is an interesting point that the solid masses of angioblasts are much rarer than the vesicles, only one or two masses of angioblasts having been found in about 80 specimens, while most of the specimens show one or two vesicles. The reason for this is that the liquefaction takes place in a short time, only one or two hours being required to transform a solid mass into a hollow vesicle, while it takes a long time for the vesicles to join the main plexus. One only rarely sees the process during the time of observation of a single specimen, representing on an average 5 hours. This difference in duration in the two processes explains why the isolated vesicles are so much more common in sections than the solid masses of angioblasts.
Concerning the primary vessels of the embryo, it was first noted that a large part of the dorsal aorta of the chick could be seen in the living blastoderm to differentiate in situ from angioblasts. In this volume is a study of the origin of the pulmonary vessels in the chick, by Buell. He has demonstrated that the period in which the vessels begin, i. e., on the second day of incubation, is a stage in which the vessels are represented by a mass of solid angioblasts. These angioblasts first appear as a solid mass of cells connected with the wall of the sinus venosus and are readily distinguishable by their structure from undifferentiated mesenchyme. Buell was unable to find any clumps of angioblasts unconnected with the main mass, so he had no evidence of a direct differentiation of these cells from mesenchyme; rather, they seem to come directly from the wall of the sinus venosus; but he had abundant evidence that the period of origin of the pulmonary vessels falls well within the angioblastic stage of the vascular system. This mass of angioblasts forms at a stage when the lung-bud lies directly dorsal to the sinus venosus. The cells spread over the surface of the gut, making a plexus which connects with the dorsal aorta, the ventral aorta, and both cardinal veins. By the liquefaction of their cytoplasm the plexus of angioblasts becomes a plexus of vessels. The pulmonary veins form in the angioblasts that are directly connected with the sinus venosus, while the arteries form in the more dorsal loops of the post-branchial plexus, the formation of the pulmonary artery slightly preceding the completion of the pulmonary arch. Thus the fundamental morphology of the vascular system of the lung in the chick is established.
This volume also contains a study by Miss Finley of another phase of this problem. She has studied the invasion of the subcutaneous tissue of the head of the human embryo by the vascular system. In the head there are two primary vascular plexuses: One in the meninges, the forerunner of the vessels of the central nervous system, the meninges and the skull, which begins very early; the other the subcutaneous plexus, which develops late. Its late appearance makes this subcutaneous plexus a favorable place to study the problem of the differentiation of angioblasts in a late embryonic or early fetal stage. Miss Finley has found evidence of a progressive differentiation of angioblasts in front of an invading zone of vessels. There are four zones, beginning at the periphery: (1) An avascular area, with undifferentiated mesenchyme. (2) A zone in which the vascular system consists of a massive plexus of cells. This vascular plexus, interestingly enough, consists very largely of masses of red cells, with a somewhat incomplete endothelial border, so that the observations have a very important bearing on the method of origin of the red blood-cells in the mammal. The process is clearly an intermediate one between the condition found in the chick, where the red cells arise within vessels, and a process of a diffuse origin of red cells which would subsequently have to migrate into vessels. These observations will be of especial value in the restudy of mammalian bone-marrow, where the question of the relation of the origin of red cells to endothelium has not been satisfactorily cleared up. Along the edge of this angioblastic zone are a very few isolated masses or chains of angioblasts. Miss Finley has studied the tissue*, first in place and then in total preparations, stripped from the head of the embryo, so that she is sure of the very small number of such isolated clumps. (3) The third zone, which is formed from the second, consists of capillaries, some of which are empty, while some contain red cells. This zone probably does not have any circulation. (4) The fourth zone, leading to the neck, has definite vessels in which one can make out a pattern that may persist. Thus she has demonstrated an advancing zone in the angioblastic phase, definitely related to the formation of red cells, in human embryos about 30 mm. long, corresponding to the end of the second month of pregnancy.
It thus becomes clear that in the study of the development of the vascular system as a whole there are three great stages: First, a primary stage before the circulation begins, when there is a differentiation of angioblasts and the formation of a very primitive vascular system, including the heart, aorta, and primary veins; second, a long stage of invasion of the entire body by the vascular system, a process accomplished by both a progressive differentiation of new vessels and the continued division and growth of the vessels already formed; and third, the final stage, in which new growth or repair of the system is from preexisting endothelium.
An exceedingly valuable analysis of these recent modifications on the subject of the development of the vascular system was given by Streeter in 1918, in a study on the developmental alterations in the vascular system of the brain of the human embryo. He divided the development of the vessels of the brain into five successive periods: First, a stage of differentiation of primordial endothelial blood-containing channels, in which there are neither arteries nor veins and in which it is practically impossible to make out a vascular pattern that is even a forerunner of the pattern of the adult. This is the more strictly angioblastic phase. Second, a stage characterized by the formation of certain primitive arteries and veins and a capillary bed, through which blood circulates; the pattern is related to the existing functional needs of the tissues and yet is not to be interpreted too closely with reference to the adult pattern. Third and fourth, stages involved in the adaptation of the vascular pattern to changes in the general region, and later to changes in the specific developing organ, the vessels always conforming to alterations in structure and to the immediate functional requirements of the organ. Fifth, a period of the final histological differentiation of the ultimate, permanent arteries and veins. It is clear that the entire vascular system must be restudied with some such outline.
These new concepts, in connection with the blood- vascular system as a whole, apply with equal force to the subject of the lymphatic system. It has, I think, become clear that the fundamental concept that the lymphatic system is a part of the blood-vascular system, subject to the same laws of development, has been strengthened rather than weakened by these new studies; that is to say, all the observations that have gradually accumulated in connection with the development of the lymphatic system fall into line with the idea that the lymphatics also differentiate from angioblasts and develop as do the veins. In 1911 Huntington discussed the development of the lymphatic system from the standpoint of the two processes of differentiation and growth and has throughout believed that the Meyer-Lewis primordia â€” that is, the isolated vesicles shown by Lewis (1906) to characterize the pathway of developing lymphatic vessels arise locally. That these isolated vesicles of Lewis do arise locally in the origin of the main lymphatic trunks is undoubtedly true, since the time of their development corresponds with periods during which blood-vessels themselves have been proved to be increasing by a differentiation of angioblasts in loco. Their method of origin, however, has proved to be the most important point. In connection with the origin of bloodvessels it has been proved that these isolated vesicles of Lewis arise by a liquefaction of the center of a solid mass of cells, so that they form, not secondary to a collection of fluid in mesenchymal spaces, but by a transformation of mesenchyme cells into angioblasts which then produce both the fluid and the endothelial boundary.
It is interesting to note that all of the facts brought forward by Kampmeier (1922), in his recent restudy of the origin of the lymphatic system in amphibia, are virtually an account of the origin of the lymphatic system by the differentiation of angioblasts, their transformation into vessels, and their uniting to make lymphatic plexuses. When the subject is restudied, it will be found, I am sure, that the same sequence of events can be demonstrated in any of the zones in which lymphatics are differentiating; that is to say, the fundamental principles of the origin of the entire vascular system, including lymphatics, are known. It is, of course, clear that we are as far as ever from analyzing the cause of this differentiation and are stating merely a sequence of events, that the cell precedes the formation of the fluid of the blood or of the lymph rather than that fluid collects and causes a flattening out of cells to line a space. If the third hypothesis of Thoma, namely, that in the spread of vessels into organs it is, in the last analysis, the organs themselves that determine vessels, proves to be the most fundamental law in connection with the growth of the vascular system, certain factors in the environment of developing vessels are not beyond the range of experimentation. Indeed, such studies have already been started by Stockard (1915) and, if carried farther, might throw great light on the extent to which the vascular system is determined by its environment.
In the early studies of the spread of vessels over the embryo, as developed by the method of injection, there grew up the theory that the growth of vessels is wholly within the capillary bed. This was a natural deduction from the fact that during the stages in which vessels are spreading over the embryo the wall of the vessel is almost everywhere limited to a lining of endothelium, so that the idea was correlated with the fact that the entire vascular system started on the basis of the structure of the capillary. In fact, the aorta begins as a vessel with a lining of endothelium only and remains without either muscle or adventitia for a long time after the circulation has begun. Indeed, the heart is the only part of the vascular system in which the musculature begins to differentiate at the same time the endothelial lining is itself forming from angioblasts. It appears, then, that in the spreading of the vascular system the capillary plexus precedes the artery and vein. There are, however, exceptions to the general rule that each vessel comes from a preliminary plexus, since the aorta itself, certainly in a part of its course, forms from chains of angioblasts rather than from any very complicated plexus.
In the present paper are presented certain observations concerning the growth of veins, which have a bearing on these fundamental relations. In the study of the vessels in the area vasculosa of the living chick it has been found possible to make preparations of the area pellucida throughout the period of incubation. The embryo itself can be kept attached only through the early part of the fourth day, because it then becomes too heavy to remain against the cover-slip in the reversed position of the hanging drop preparation, and as it sags away from the over-slip it -drags the membranes with it. The area pellucida, however, with a rim of the opaca, can be mounted ; and although the circulation stops when the embryo is cut away, the cells continue to divide for a short period, so that certain processes can be watched. In such a preparation it was first noted that the granulocytes which develop outside the vessels could wander into the veins, even after a considerable thickness of the adventitia had developed, with just as great ease as they enter the capillaries; that is to say, the adventitia is no barrier whatever to the wandering of the leucocytes. It was then found that the same was true -with regard to sprouting. Sprouts put out from the walls of a vein could push their way between the adventitial cells as easily as through the looser tissue that surrounds a capillary.
Plate 1 shows examples of such sprouting from veins of the area pellucida in a chick of the fourth day of incubation which was grown for two hours on a cover-slip. In figure A is a long sprout consisting of endothelial cells, for the most part solid, which were growing out from the side of a large vein. It is clear that at the base of the sprout the adventitia is represented by two cells, one on each side, that are growing out with the endothelium; that is to say, the vessel is growing as a vein, not as a capillary that is to be transformed later into a vein. Toward the end of the outer endothelial cell is a tiny vesicle, which I think is the beginning of the lumen-forming process. It seems difficult to accept the idea that the lumen of a vessel may develop within the cytoplasm of a single cell, but the process has now been so frequently observed that there is no escape from the fact.
In figure B is another long sprout from a smaller vein, which shows even more clearly that sprouts grow as veins, for the adventitial cells have wandered even farther along the growing sprout. In this case the lumen of the vein has opened widely into the base of the sprout. On the margin of the main vein there is a considerable heaping up of adventitial cells and several are also seen along the new sprout. The last adventitial nucleus is on the upper side and is the third nucleus from the tip. The branch of the sprout which passes upward has already joined another vein not shown in the drawing. In the new growth of veins one often finds rather large blunt swellings on the side of vessels, like the zone at the base of the sprout in this figure. Such a swelling represents a proliferation of endothelium from which a sprout will eventually form a connection with a neighboring vessel. The beginning of this process is shown in figure C, where a group of three endothelial nuclei is to be seen at the base of a short endothelial sprout. This is also a vein, as can be seen from the adventitial nucleus at the right of the base of the sprout.
Thus from the living specimens is established the fact that not only do the preliminary angioblasts make plexuses by the process of sprouting, but that the resulting capillaries and the veins likewise have this property. The importance of the point concerns (1) the story of how the vessels of each organ develop originally and (2) how to visualize the processes of repair of vessels after injury. If veins can regenerate as veins, it means that we have a much more rational accounting for the rapidity with which vessels are repaired in wound-healing. In the case of the healing of the vessels in intestinal anastomosis, we know that vessels from one of the apposed surfaces of the intestine can be injected from the other surface on the fourth day after the operation. If veins can grow as veins, the reestablishment of the circulation can doubtless be more rapid than by a process of the pre- liminary development of a capillary bed out of which the larger vessels must subsequently form.
Along with the processes of growth in these living specimens, it is possible also to follow the important subject of the destruction of vessels. In the area vasculosa there are regions in which one finds an extensive plexus of capillaries followed a short time later by a stage in which the same area has only one or two large vessels. A most interesting place to follow such a change is in the origin of the main vein, which develops to accompany the primary stem of the omphalo-mesenteric artery. Such a transition must involve a destruction of vessels and one should be able to follow this process in a living specimen. Figure D is taken from the same blastoderm as the other figures, but shows veins which were disappearing rather than growing at the time the specimen was fixed. All of the other figures were near together in a growing zone, while this figure is taken farther along the course of the same veins, where branches were degenerating. The main large vein at the right of the figure is normal. From this vein are two branches in which both the endothelial and the adventitial cells are to be seen in a stage of advanced degeneration. The cells are full of vacuoles and granular detritus and lead over to another smaller vein on the left side. The specimen shows clearly that the first stage in the degeneration of a vessel is a preliminary collapse of the endothelium which obliterates the lumen of the vessel. The evidence for this is a solid core of endothelium in a structure that was a vein. This is probably an important step in preventing hemorrhage during the degeneration of vessels. In this specimen the next stage is the death of the cells, both endothelial and adventitial. It seems to me possible that in some cases there may be a retraction of the endothelial sprouts, after the collapsing of the lumen, instead of actual death of the cells, making the process the reverse of the sprouting which characterises the growth of vessels. If this takes place, it should be possible to find it in a living blastoderm, but so far it has not been observed. As a matter of fact, the methods of destruction of vessels in a growing zone are second in interest only to the methods of spreading of vessels, so often are the vessels formed and re-formed before the final pattern is reached.
It seems to me clear that the work of the past twenty years on the development of the vascular system has established its fundamental genesis and has given us the broad outlines on which the story of the spread of the vascular system over the body has become a feasible problem. Instead of lessening the interest in the problem, as one for which we can now see a conclusion, the whole subject has rather been opened up to a new experimental attack by which we may hope to analyze more deeply some of the factors in development that control and modify the system.
Buell, C. E., 1922. Origin of the pulmonary vessels in the chick. Contributions to Embryology (this volume) .
1 "inlet, E. B., 1922. The development of the subcutaneous vascular plexus in the head of the human embryo. Contributions to Embryology (this volume).
Holman, E., 1920. End-to-end anastomosis of the intestine by presection sutures. An experimental study. Johns Hopkins Hosp. Bull., vol. 31, p. 300.
Huntington, G. S., 1911. The anatomy and development of the systemic lymphatic vessels in the domestic cat. Memoirs of the Wistar Institute of Anatomy and Biology, Philadelphia, No. 1.
Kampmeieh, O. F., 1922. The development of the anterior lymphatics and lymph hearts in anuran embryos. Amer. Jour. Anat., vol. 30, p. 61.
Lewis, F. T., 1906. The development of the lymphatic system in rabbits. Amer. Jour. Anat., vol. 5, p. 95.
Sabin FR. Studies on the origin of blood-vessels and of red blood-corpuscles as seen in the living blastoderm of chicks during the second day of incubation. (1920) Contrib. Embryol., Carnegie Inst. Wash. No. 9 36: 213-262.
Stockakd, C. R., 1915. The origin of blood and vascular endothelium in embryos without a circulation of the blood and in the normal embryo. Amer. Jour. Anat., vol. 18, p. 227.
Streeter, G. L., 1917. The development of the scala tympani, scali vestibuli, and perioticular cistern in the human embryo. Amer. Jour. Anat., vol. 21, p. 299.
, 1918. The developmental alteration in the vascular system of the brain of the human embryo. Contributions to Embryology, vol. 8, Carnegie Inst. Wash. Pub. No. 271.
Weed, L. H., 1917. The development of the cerebro-spinal spaces in pig and in man. Contributions to Embryology, vol. 5, Carnegie Inst. Wash. Pub. No. 225.
Description of Plate
Fig. A. Endothelial sprout from wall of median anterior vein of the area pellueida of the yolk-sac of a chick (No. 312) on the fourth day of incubation. The specimen was grown on a cover-slip for 2 hours in Locke-Lewis solution and then fixed in Bouin's solution, stained in hematoxylin and eounterstained in eosin and orange G. X 525.
Fig. B. Branched endothelial sprout from the wall of a smaller vein from the same specimen. The reticular structure of the red blood-cells is an artefact due to the fixation. X 525.
Fig. C. Small sprout from a vein, taken from the same specimen, showing a heaping up of endothelial nuclei at its base. X 525.
Fig. D. View of degenerating veins along the course of the same vein as figure 1, but closer to the embryo. The large vein at the right is normal. The specimen shows the preliminary collapsing of the endothelium as evidenced by the solid core of endothelium, followed by the death of both endothelial and adventitial cells. X 525.
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