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Development of Human Embryonic and Fetal Dermal Vasculature
Development of Human Embryonic and Fetal Dermal Vasculature Carole L. Johnson, Ph.D. and Karen A. Holbrook, Ph.D Department of Biological Structure (CL),KAH) and Department of Medicine (Dermatology) Seattle, Washington, U.S.A.
(KAH), University of Washington,
This report summarizes recent advances in the understanding of the structure and organization of the microvasculature in developing human skin. Previous observations suggested that the skin contains no blood vessels as late as eight weeks estimated gestational age (EGA). Computer reconstructions, in conjunction with light and transmission electron micros copy (TEM), however, demonstrated that specimens as young as 35 - 45 d show a level of vascular complexity pre viously unknown. The computer reconstructions showed that the vasculature was organized in one or two planes paral lel to the epidermis. A simple, single plane was evident in specimens 40 - 50 d EGA, whereas specimens 50 - 75 d EGA showed two planes. Fewer vessels were continuous through out the tissue sample in the younger specimens compared with the older specimens. Superior views highlighted the continuities and connections of vessels. The younger speci-
mens showed more discontinuous segments of vessels when compared with the network established in the older speci mens. In the earliest specimens examined morphologically (35 - 40 d), simple, capillarylike vessels were morphologi cally identifiable in presumptive dermis. The samples studied by TEM revealed detailed structure of the vessel wall includ ing extreme attenuations and projections, plasmalemmal ves icles, and junctions similar to adult endothelial cells. Little or no basal lamina surrounded the vessel. The basal lamina first appeared in the form of amorphous deposits and eventually thickened and became continuous. By the end of the first trimester, the basal lamina still lacked the organization of adult cutaneous arterial and venous segments. These findings suggest that the major vascular organization of the dermis is defined in the first trimester of development.] Invest Dermatol
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cells that are already committed and differentiated. Different stim uli may promote the formation of vessels by these two mechanisms. In vitro studies have shown that capillary endothelial cells re spond variably to extracellular matrix components [5,6]. Interstitial collagens stimulated proliferation and migration, whereas basement membrane collagens promoted differentiation and aggregation into tubelike structures. Angiogenesis in vivo and in vitro can be induced by a large number of so-called angiogenic factors [7], that may be circulating factors produced by the tissue [8]. What mechanism or mechanisms do human dermal vessels use to establish their extensive network? How and when are these mecha nisms turned on or oft'? How influential are the matrix components? If angiogenic factors are present, what cells produce them? Are the vessels derived from already existing vasculature in muscle or are embryonic dermal mesenchymal cells capable of differentiating into endothelial cells in situ? There are no answers to these questions, but as a starting point, we can explore the environment of the embry onic and fetal dermis in which these vessels organize or into which they grow.
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ontroversy has existed for years over the precise ori gin of the vascular system. In 1900 His [1] proposed the angioblast theory, postulating that cells from the yolk sac differentiated into specialized cells, termed angioblasts. He believed that these cells had the p0tential to further differentiate into either red blood cells or endothelial cells. In opposition to the angioblast theory was the local origin theory, proposed in 1906 by Rlickett and Mollier [2]. They sug gested that mesenchyme anywhere in the body can transform into vascular tissue. Today, the local origin theory is more in favor [3]. Coffin and Poole [4] stated that embryonic vascularization is a programmed process in development and proposed two mecha nisms for vessel formation. One mechanism is by in situ localization and assembly of migrating, presumptive endothelial cells into a vascular cord that subsequently enlarges and forms a lumen as a definite blood vessel. The second mechanism is by sprouting of existing vessels. The first mechanism credits the mesenchymal cells with a certain plasticity that fits the local origin theory, whereas the second implies an origin of endothelial cells from other endothelial
REVIEW OF EMBRYONIC AND FETAL DERMAL DEVELOPMENT
This study was funded by PHS grants HD 17664 and AR 21557. Reprint requests to: Karen A. Holbrook,Department ofBiologica1 Struc ture SM-20, School of Medicine, University of Washington, Seattle,WA 98195 Abbreviations: AP: a1ka1ine phosphatase CAM: chorioallantoic membrane EC: endothelial cell EGA: estimated gestational age TEM: transmission electton microscopy
0022-202X/89/S03.50
During the early part of the first trimester (5 - 7 wks) the dermis is predominately cellular. These mesenchymal cells are separated by an extracellular matrix of fine, fibrillar components in a gelatinous matrix. The presumptive dermis is very homogeneous with vir tually no distinguishable deep border [9]. By 8-9 wks, the dermis, although still cellular, displays an in creased amount of collagen in the extracellular matrix. The dermis is delineated from the subcutaneous tissue by a deep border that
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reflects differences in the organization of the dermal matrix and the fibers of the subcutaneous tissue in the plane of the skin. By 1012 wks, a cellular to fibrous transition period is defined. Collagen fibrils increase in diameter, and greater numbers of fibrils associate into fiber bundles [10]. Papillary and reticular dermal regions are distinguishable by 14 wks [11]. The papillary region has a higher density of cells and finer collagen compared with the deeper tissue. In addition to changes in cell density and extracellular matrix, the dermis is in vaded by the downward growth (invagination) of developing epi dermal appendages. Concurrently, structural changes (i.e., bone and muscle formation) underlying the dermis tend to promote regional variations in cutaneous thickness. Collagen types I, III, and V are evenly distributed throughout the dermal and subdermal connective tissue regardless of fetal age [11]. The relative amounts are type I collagen, 70%-75%; type III, 18%-21%; and type V, 6%-8%, in contrast to adult skin where type I is 85%-900/0; type III, 8%-11%; and type V, 2%-4%. Hyal uronic acid and dermatan sulfate are the major glycosaminoglycan components [12]. Hyaluronic acid is highest in the embryonic stage, although overall the glycosaminoglycan content of fetal skin is generally higher than in the adult human skin. Thus, embryonic vascular development proceeds within the con fines of a watery, cellular tissue which has few distinguishable land marks. Fetal vasculature, on the other hand, is elaborated in a much more fibrous and eventually more organized tissue with intruding or molding structures at both its superficial and deep borders. Con sidering the changes in the surrounding tissue, what techniques have been used to obtain information about the developing vascula ture? METHODS A number of methods have contributed pieces to the developmental
story of cutaneous vasculature. Some have provided data from ani mal models, but have not necessarily been successful for obtaining
data on human skin vasculature.
Histochemical Staining
Staining with alkaline phosphatase
(AP) was one of the first methods used for studying vasculature in
fetal skin [13]. Tissue from 45 embryos and fetuses ranging in age from 4-32 wks was stained for AP activity. Thick frozen sections of skin from the scalp, forehead, face, chest, abdomen, neck, legs, palms, soles, fingers, and toes were fixed in neutral formalin and stained by a Gomori cobalt technique and azo-coupling dye to re veal AP. Most of the mesenchymal cells and fibroblasts in early fetal skin had some enzymatic activity. Later, fibroblasts, histiocytes, and mast cells showed variable intensities of AP activity. When blood vessels first appeared, they consisted of a tube of endothelial cells that were very high in AP. As soon as the mesen chymal cells surrounding the endothelial tubes differentiate into the muscle fibers and connective tissue of the tunicae, the endothelial cells gradually lose their reactivity for AP. Only the capillaries re main positive. Although AP is a widely accepted marker for endo thelial cells, its functional significance is uncertain. AP may play a role in the metabolism of carbohydrate, protein, fat, and in mediat ing the transport of phosphate ions through membranes [13].
Capillary Microscopy Although not used on fetal skin, capillary microscopy was used to study cutaneous circulation in 40 living infants ranging in age from 1 d to 17 wks [14]. By this technique observations can be made on the patterns of the microcirculation. The skin at birth showed a disorderly capillary network that was particularly prominent in skin creases. By the end of the first post natal week, the capillary network lost some of its haphazard appear ance and assumed a more orderly pattern. In the second week capil lary loops began to appear in dermal papillae as small superficial dilatations or buds. The authors stated that the number of observa tions were relatively small, and minor variations in vessel shape and number were relatively immense. Because of the difficulty in im mobilizing the infants, satisfactory photographic records were not obtained.
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Perfusion has been suggested as a method to study vessels in human fetuses; it has been used widely to infiltrate and form casts of vessels in embryonic and adult animals. Although largely successful, it is not without drawbacks [15]. Many parts of the body are not easily perfused and a collapsed or constricted blood vessel may not be filled. The perfusion technique may yield a spotty and unreliable picture of vascular patterns. It would be quite diffi cult to use to demonstrate blood vessels in very small pieces of skin, such as those available for human embryonic and fetal studies.
Perfusion
Antibodies that recognize matrix components of the vessel wall, the basement membrane and the endothelial cells have revealed differences in the developing vasculature from that of the neonate or adult. Tonnesen et al [16] immunostained samples of skin from adult, neonatal foreskin, and four second-trimester fetuses (14-18 wks gestation) to detect expression of fibronectin, laminin, and factor VIII-related antigen associated with the developing cuta neous microvasculature. Antilaminin staining of blood vessels was consistently bright and linear in fetal, neonatal, and adult skin. A reciprocal relationship was shown by intense fibronectin staining during human blood vessel development and prominent factor VIII-related antigen staining in mature blood vessels. The relation ship supports the hypothesis that fibronectin plays a role in human blood vessel morphogenesis, and that factor VIII-related antigen is a marker for endothelial cell differentiation. Another immunolabeling study investigated matrix proteins [11]. Concentrations of types III and V collagen around vessels and nerves as early as 5-6 wks EGA were observed and persisted throughout gestation.
Immunolabeling
In a review paper, Holbrook and Hoff suggested that the embryonic dermis appeared to be dis tinguished from subcutaneous tissue by a plexus of horizontally arranged vessels [91. Small capillaries from this plexus were sug gested to have invaded the dermis and to lie in close proximity to the epidermis. By the end of the first trimester, vessels as well as nerves extended throughout the dermis, reaching the dermal-epidermal junction. These were isolated observations integrated into a general story on skin development.
Light and Electron Microscopy
Computer Graphics Our laboratory has recently used computer graphics in conjunction with light microscopy to delineate the de velopmental patterns of early human vasculature [17,18]. Computer reconstructions are effective and efficient for visualizing the spacial organization of vessels in three dimensions. Two particular advan tages include the ability to assemble and reassemble the model for repetitive analysis of the whole of parts of the model and to analyze of data from the model from different angles. Some angles empha size particular data, for example the branching and/or connections are more apparent in the superior view. As our results will be dis cussed later in the text, a brief discussion of the method is given here. Tissue embedded in Epon was serially sectioned, stained and pho tographed. The negatives of the photographed sections were mag nified through a photographic enlarger and the contours of the vessels were traced onto paper. The profiles of the traced vessels were digitized using software developed at the University of Wash ington in the Department of Biological Structure. The information was transferred to a Vax 11/750 via Ethernet and contours of the blood vessels were visualized on a SiliconGraphics Iris 30-20 graphics monitor. THE OLD DEVELOPMENTAL STORY What kind of story has evolved from studies using these diverse methods? In 1983 Ryan [19], citing Serri et al, stated that the skin of 8-wk-old embryos lacks blood vessels. During the next 4 wks, an gioblastic transformation of mesenchymal cells was suggested by positive staining of cells for alkaline phosphatase. "The arterial and venous channels in the subcutaneous tissues fully communicate with central vessels and connect with the developing plexuses of the dermis during the 3rd and 4th month."
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During the fourth month a few vessels in the dermis begin to develop muscle and adventitial coats [20]. Until the perivasculature is established, budding and anastomosis are probably relatively unre stricted and occur wherever there is a stimulus to growth and orga nization. During the fifth month new vessel formation was consid ered to occur largely by budding from already formed vessels, but differentiation from primitive mesenchymal cells was still consid ered a possibility. THE NEW DEVELOPMENTAL STORY In contrast to the old developmental story, we have demonstrated that dermal vasculature exists much earlier than previously docu mented. Not only are the vessels morphologically identifiable, they are also organized in first a single plane, and then two planes parallel to the epidermis. By the end of the first trimester, both the patterns and morphology of vessels are suggestive of the adult vascular system.
Embryonic Stage (35 60 d)
Endothelial cells (Ee) of the vessel wall are morphologically identifiable in presumptive dermis of a 35 - 40-d embryo by light and transmission electron microscopy (Fig 1). The identification is based on characteristics embryonic EC share with adult EC: comparable shape and size, similar thickness in the wall structure (as narrow as 0.2-0.4 .urn), presence of plasma lemmal vesicles with their characteristic uniform diameter of 60 70 nm, and similar cell junctions, which frequently interconnect in intricate patterns. The membranes may be separated by a fairly uniform distance, but also appear to fuse at points along the bounda-
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ries. Simple tubelike vessels evolve from the joining of the endothe lial cells in the presumptive dermis, even in the earliest specimens examined (35 - 40 d). Although the lumenal sizes vary consider ably, the surfaces generally appear smooth. Blood cells are present in some of the vessels. Basal lamina around the vessels is absent. No computer reconstruction is available for the youngest specimens. The youngest specimen for which computer reconstruction of cutaneous vasculature is available is 40 - 45 d EGA. The reconstruc tion, corroborated with light micrographs, shows that the vessels lie in a single plane parallel to the epidermis and the dermal-subcutane ous interface (Fig 2). The superior view of the reconstruction illus trates apparent discontinuities in the digitized vessels, and in fact, no single vessel could be followed throughout the thickness (500.urn) of the sample. Only two vessels were continuous in sections 200 to 300.u thick, and one vessel appeared in 130 continuous sections. Examination of increasingly older specimens shows a gradually evolving complexity and density in the developing vasculature. Al though a single plane of vessels is demonstrated in the 45 - 50-d-old specimens, more vessels, in seemingly greater density are present than in the younger samples, as demonstrated by both light micro graphs and computer reconstructions (Fig 2). Reconstructions illus trate the branching and/or connections of the vessels, and a vascular network is apparent. The discontinuities are not nearly as marked as in the youngest specimen. The apparent discontinuities in the vessels in the younger speci mens pose interesting questions as to the mechanisms of vessel for mation in the developing skin. It may be, however, that the discon tinuities simply refl ect limitations of the method: technical difficulties in tracing nonlumenized vessels, resolving small sprouts,
Figure 1. Electron micrograph of youngest specimen studied. The superior border of the micrograph is the two-layered epidermis,below the presumptive dermis. Arrows point to two vessels,each containing a blood cell.
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Figure 2.
Computer reconstructions of embryonic vascular patterns,a cross sectional view (a) and a lateral view (b). The top specimen in both views is 40-45 d EGA, the middle is 45-50 d,and the bottom is 50-55 d. A comparison of the views from the youngest to the oldest specimen shows an increasing complexity from a very simple,single plane to two planes in close approximation with a greater density of vessels.
and finely filamentous projections, and identifying vessels which may be sectioned through the wall tangentially. It is also possible that the discontinuities may reflect in situ formation of vessels. As stated earlier, the dermis at 5-7 wk contains mainly mesenchymal cells. It is unknown if these cells are able to differentiate in situ into endothelial cells, or if they are able to differentiate in the earliest specimens, is there a specific time or change in the cells or their environment when the endothelial cells lose this ability. A component of the extracellular matrix may have an important role in directing endothelial cell migration. The loose structural matrix would be conducive to the growth of vascular endothelial cells by sprouting or in situ formation, because vascular sprouts appear to progress along the path of least resistance. The change in the environment from cellular to fibrous dermis may have impor tant consequences for vascular development. Concurrent with questions about early vascular development are questions about the influence of nerve growth patterns on vessels. It is not known what initiates migration of nerves into the skin or what influence this event has on angiogenesis. The migration of nerves into the skin appears to precede vessels. Although the two may follow a similar path, nerves seem to migrate in closer proxim ity to the epidermis than blood vessels. Once the nerves are distrib uted, does the epidermis produce or stimulate the production of an angiogenic factor that directs the vasculature closer to the dermal epidermal border? By 50 d EGA, two closely positioned planes of vessels are estab lished and within the planes there is heterogeneity in the diameter and structure of the vessel wall. The vessels appear to become seg mentally differentiated. The walls of the vessels are either thick and
substantial with the nuclei contained within the wall or thin and attenuated with nuclei that bulge into the lumen. Vessels with the thicker appearing walls consistently seemed to contain more blood cells. In addition to developing two planes of vasculature, the individ ual vessels are more continuous. The vessels develop into longer and longer tubes, as demonstrated in both the lateral and superior recon structions. In contrast the earlier vessels are in short segments, per haps indicative of the discontinuities observed. During the early embryonic stage, morphologically recognized subendothelial stroma appears. Ultrastructural examination of em bryonic tissue reveals little or no basal lamina around the endothelial tubes. With increasing age, EC-related matrix changes in appear ance from amorphous deposits to a thicker stromal layer that even tually becomes continuous around the endothelium. The structure of the basal lamina distinguishes between arteries and veins as well as describing perivasculature cells. In adults, as the arterial capillary is traced, the basement membrane material begins to develop lamellae within its previously homogeneous framework until a venous segment is reached in which the entire vascular wall is multilaminated [21]. Dense layers 250-1000 A thick alternate with less-dense zones. No comparable organization exists surrounding embryonic blood vessels, and although cells lie in close proximity to the vessels, none are enclosed within a basal lamina. In summary, simple capillarylike vessels first seen in the embry onic period evolve into vessels that are surrounded by matrix and perivasculature cells. Their organization appears to be nonrandom and assembles first into one, and then into two planes parallel to the epidermis.
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Early Fetal Vasculature (60-80 d) Early fetal vasculature is quite adultlike in architectural organization and morphology. The vessels are largely intact, continuous, and patent. Two planes of vasculature are well defined and parallel to the epidermis. The ves sels in the superficial plane demonstrate a honeycomb network that overlie larger vessels. In the computer reconstruction of a specimen aged 70-75 d, four major vessels were parallel to each other and to the epidermis and continuous throughout the 500 J.l of tissue exam ined. At this stage, there is greater heterogeneity in the vessels of the two planes. Those of the superior plane have seemingly smaller lumens, compared with those of the deeper plane (Fig 3). Other comparisons include: vessels with thinner appearing walls contain blood cells less frequently than those vessels with more substantial walls; more interconnections occur with the vessels containing blood cells; and few connections occur between the straighter, larger vessels and the branching, smaller vessels. Such differences prompt attempts to classify vessels as either arte rioles or venules. However, the most distinctive and reliable feature distinguishing adul� arterial segments from venous segments is the structure of the basal lamina. By the end of the first trimester, the thickness of the basal lamina has increased and in some cases, sur rounds a potential perivascular cell (Fig 4). However, similar to the embryonic vasculature, lack of organization precludes segmental differentiation. Second Trimester Light and electron microscopy demonstrate a continuing maturation of the vascular system in the dermis. By electron microscopy, Weibel Palade bodies, ultrastructural Ee markers, appear at around 100 d (Fig 5). Weibel Palade bodies serve as storage and/or processing vesicles for factor VIII-related antigen both in vitro and in vivo [22,23]. In Tonneson's study [16], factor VIII-related antigen staining of fetal vessels was granular, scant, and focal. Although the ultrastructural localization of the antigen in fetal Ee has not been done, its known existence in weibel Palade bodies would suggest that early second trimester Ee may have reached a level of maturation previously unrecognized.
Figure 3.
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Another aspect of maturation is the development of a more sub stantial basal lamina. Figure 5 demonstrates areas where the basal lamina may be developing as a multilaminated layer, indicative of venous segments. However, the structure is still quite simplistic compared with the adult, because the striking feature of adult der mal blood vessels is the thick vascular wall composed of basement membrane material, elastic fibers smooth muscle cells, or pericytes and variable amounts of individual collagen fibrils [21]. At 140 d EGA, small vessels in very close proximity to the epidermis show even more development with increasing thickness, numbers of peri vascular cells, and amounts of collagen. More information about the organization and morphology needs to be added to the unfolding story of fetal vasculature. New methods have potential to supply this information for both fetal and embryonic vasculature. MORE QUESTIONS, NEW METHODS We have shown the presence of vasculature in the earliest examined specimens (35-40 d) and its establishment in an organized plane. Do changes in morphology reflect functional capabilities or do functional demands initiate accommodating structures? What are the functional demands or metabolic requirements of embryonic and fetal skin? In Vivo Adapting methods specifically for fetal cells is very im portant considering that endothelial cells are a heterogeneous popu lation of cells. They display unique differentiated functions related to the requirements of the tissue. For example, the uptake of chemi cally modified low density lipoproteins in vivo is mediated by spe cific receptors on the endothelial cells of the liver, spleen, marrow, adrenal, and to a lesser extent, the ovary [24]. The uptake could reflect their requirements for cholesterol: for bile acid production in the liver, hormone production in the ovary, and adrenal and blood cell membrane formation in the spleen and bone marrow. What receptors do embryonic endothelial cells have and do they reflect specific functional requirements?
Light micrographs contrasting the differences in vessels. (a) View indicating the location of 6 vessels in the dermis. (b and c) High-power view of II. Vessell has a large,empty lumen,in contrast to vessels 2 and 3. Vessel 4 is filled with blood cells and has potential perivascular cells close to ablurninal surface, in contrast to vessels 4 and 5.
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ISS
70-75 days
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Figure 4. arrow).
Electron micrograph showing uneven thickness of subendothelial stroma
Human fetal skin is less available for in vivo experimentation than animal models. Other methods, however, can be adapted to acquire comparable information, for example, the chorioallantoic mem brane (CAM) system. Although cumbersome and semiquantitative by itself, the CAM method combined with appropriately designed computer reconstruction experiments can generate such morpho metric analysis as areas and volumes of vessels, number of branch points in vessels, distances between vessels and volume of tissue supplied by the vasculature. As we have established the normal pattern and morphology in vivo, embryonic skin grown on a CAM could be similarly compared and then growth factors and/or angio genic factors could be investigated to answer questions about poten tial factors in the early developing dermis. Our lab has examined the development of embryonic and fetal human skin grafted to nude mice. This method has proven to be very effective in supporting epidermal morphogenesis, but it has not been used to evaluate vascular development. The kidney capsule has been used as a common site of grafting because it offers a rich source of vessels to revascularize the graft. If the development of the vascu lar system in skin could be sustained, this method might promote studies on regulation of formation and/or differentiation. Cell Culture Methodologies for microvascular endothelial cell isolation and refinements in cell culture permit successful cultiva tion from various animal and human tissue [5,6,25,26,27]. How-
(arrows). Potential perivascular cell appears to be included in stroma (right
ever, culturing embryonic endothelial cells is not a routine proce dure. Microvascular cells from tissue as young as neonatal foreskin have been cultured and the synthesis of type IV collagen, laminin, fibronectin, and thrombospondin was compared with adult tissue [28]. The ultrastructure and the profile of secretory proteins depos ited in the subendothelial matrix by the two cell types were nearly identical. Would embryonic endothelial cells give the same results or is there a time point when the cells become capable of such synthesis? Because embryologic material appears to have greater plasticity than older adult tissue, interest exists regarding endothelial cell proliferation and migration in different gel matrices. Because em bryonic and fetal vascular cells develop an organized plane of vascu lature in a matrix high in hyaluronic acid, would the cells alter their organizational "program" in a different matrix? Specific Markers Information about differentiation has been fa cilitated by the use of specific markers, e.g., the family of keratins has provided markers for understanding epidermal differentiation. Factor VIII-related antigen has been used as marker for endothelial cells, but all vascular endothelial cells do not appear to produce factor VIII, and production has not been reliable in determining the origin or pathological conditions [14,29,30,31]. Lectins also have been used as markers, and [30,32,33] monoclonal antibodies have been developed for specific endothelial cell populations [32,34,35],
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100 da-ys
Figure 5.
Electron micrograph of vessel in second trimester specimen.(a) Basal lamina is significantly more developed (compare with Fig 4).Multilaminated areas are indicated (arrowhead). (b) High-power of a. Weibel-Palade bodies are present (arrows).
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but due to the heterogeneity among these cells, there is no assurance that a monoclonal antibody for a specific adult population would recognize a fetal population of endothelial cells. Having such markers available to immunostain the developing vasculature would help solve the puzzle of the apparent discontinuities seen in the computer reconstructions.
Molecular Biology
Molecular biology is providing a means to an even greater understanding of development and much information will be gained regarding vasculature. Using endothelial cells from cultured human umbilical vein, the process of forming tubular net works was found to result in decreased levels of the sis messenger RNA transcript and increased levels of the messenger RNktran script for fibronectin [36]. The situation was reversed on transition from the organized structure to a proliferative monolayer. What signals tell proliferating embryonic or fetal endothelial cells to dif ferentiate? In summary, more information about normal human developing dermal vasculature needs to be obtained to solve important unan swered questions. New or modified methods will increase knowl edge about normal development and provide a better understanding of developmental anomalies of the vasculature. Vascular defects are the most common congenital malformations in newborns. Heman giomas, although usually benign, are frequendy of cosmetic con cern and may deform related tissue. Some lesions are components of syndromes with such severe consequences as mental retardation, hemiplegia, and convulsions. It is unknown what factors affect the organization and segmental differentiation in utero but early estab lishment of the cutaneous vasculature indicates there is the potential for abnormalities to occur quite early. Future studies of the micro vasculature in human embryonic skin are expected to reveal when and what factors may influence both normal and abnormal develop ment. REFERENCES 1.
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32. Holden CA,Spaull J,Williams R,Spry CJ. Jones RR,Jones EW: The detection of endothelial cell antigens in cutaneous tissue using methacarn and periodate lysine paraformaldehyde fixation. J Im munol Methods 91:45-52.1986 33. Miettinen M,Holthofer H.Lehto V. Miettinen A, Virtanen I: Ulex europaeus I lectin as a marker for tumors derived from endothelial cells.Am J Clin PathoI79:32-36.1983
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Smith LT,Holbrook KA. Madri JA: Collagen types I,III, V in human embryonic and fetal skin. Am J Anat 175:507-521,1986
34. Hamburger AW, Reid YA, Pelle BA, Breth LA. Beg N, Ryan U: Isolation and characterization of monoclonal antibodies reactive with endothelial cells.Tissue 8£ Cell,17:451-459. 1985
12.
Varma RS,Varma R: Glycosaminoglycans and proteoglycans of skin. In, Glycosaminoglycans and Proteoglycans in Physiological Pro cesses of Body Systems.Basel,Karger pp 151-164,1982
35. Schlingeman RO,Dingjan GM.Emeis JJ,Blok J.Warnaar SO.Ruiter DJ: Monoclonal antibody PAL-E specific for endothelium.Lab In vest 52:71-76.1985
13. Serri F.Huber W, Montagna W: Studies of the skin of the fetus and the child. IV. Distribution of alkaline phosphatase in the skin of the fetus.Arch DermatoI87:234-245,1963
36. Jaye M, McConathy E, Drohan W, Tong B. Deuel T, Maciag T: Modulation of the sis gene transcript during endothelial cell differ entiation in vitro.Science 228:882-885,1985
(KAH), University of Washington,
This report summarizes recent advances in the understanding of the structure and organization of the microvasculature in developing human skin. Previous observations suggested that the skin contains no blood vessels as late as eight weeks estimated gestational age (EGA). Computer reconstructions, in conjunction with light and transmission electron micros copy (TEM), however, demonstrated that specimens as young as 35 - 45 d show a level of vascular complexity pre viously unknown. The computer reconstructions showed that the vasculature was organized in one or two planes paral lel to the epidermis. A simple, single plane was evident in specimens 40 - 50 d EGA, whereas specimens 50 - 75 d EGA showed two planes. Fewer vessels were continuous through out the tissue sample in the younger specimens compared with the older specimens. Superior views highlighted the continuities and connections of vessels. The younger speci-
mens showed more discontinuous segments of vessels when compared with the network established in the older speci mens. In the earliest specimens examined morphologically (35 - 40 d), simple, capillarylike vessels were morphologi cally identifiable in presumptive dermis. The samples studied by TEM revealed detailed structure of the vessel wall includ ing extreme attenuations and projections, plasmalemmal ves icles, and junctions similar to adult endothelial cells. Little or no basal lamina surrounded the vessel. The basal lamina first appeared in the form of amorphous deposits and eventually thickened and became continuous. By the end of the first trimester, the basal lamina still lacked the organization of adult cutaneous arterial and venous segments. These findings suggest that the major vascular organization of the dermis is defined in the first trimester of development.] Invest Dermatol
C
cells that are already committed and differentiated. Different stim uli may promote the formation of vessels by these two mechanisms. In vitro studies have shown that capillary endothelial cells re spond variably to extracellular matrix components [5,6]. Interstitial collagens stimulated proliferation and migration, whereas basement membrane collagens promoted differentiation and aggregation into tubelike structures. Angiogenesis in vivo and in vitro can be induced by a large number of so-called angiogenic factors [7], that may be circulating factors produced by the tissue [8]. What mechanism or mechanisms do human dermal vessels use to establish their extensive network? How and when are these mecha nisms turned on or oft'? How influential are the matrix components? If angiogenic factors are present, what cells produce them? Are the vessels derived from already existing vasculature in muscle or are embryonic dermal mesenchymal cells capable of differentiating into endothelial cells in situ? There are no answers to these questions, but as a starting point, we can explore the environment of the embry onic and fetal dermis in which these vessels organize or into which they grow.
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ontroversy has existed for years over the precise ori gin of the vascular system. In 1900 His [1] proposed the angioblast theory, postulating that cells from the yolk sac differentiated into specialized cells, termed angioblasts. He believed that these cells had the p0tential to further differentiate into either red blood cells or endothelial cells. In opposition to the angioblast theory was the local origin theory, proposed in 1906 by Rlickett and Mollier [2]. They sug gested that mesenchyme anywhere in the body can transform into vascular tissue. Today, the local origin theory is more in favor [3]. Coffin and Poole [4] stated that embryonic vascularization is a programmed process in development and proposed two mecha nisms for vessel formation. One mechanism is by in situ localization and assembly of migrating, presumptive endothelial cells into a vascular cord that subsequently enlarges and forms a lumen as a definite blood vessel. The second mechanism is by sprouting of existing vessels. The first mechanism credits the mesenchymal cells with a certain plasticity that fits the local origin theory, whereas the second implies an origin of endothelial cells from other endothelial
REVIEW OF EMBRYONIC AND FETAL DERMAL DEVELOPMENT
This study was funded by PHS grants HD 17664 and AR 21557. Reprint requests to: Karen A. Holbrook,Department ofBiologica1 Struc ture SM-20, School of Medicine, University of Washington, Seattle,WA 98195 Abbreviations: AP: a1ka1ine phosphatase CAM: chorioallantoic membrane EC: endothelial cell EGA: estimated gestational age TEM: transmission electton microscopy
0022-202X/89/S03.50
During the early part of the first trimester (5 - 7 wks) the dermis is predominately cellular. These mesenchymal cells are separated by an extracellular matrix of fine, fibrillar components in a gelatinous matrix. The presumptive dermis is very homogeneous with vir tually no distinguishable deep border [9]. By 8-9 wks, the dermis, although still cellular, displays an in creased amount of collagen in the extracellular matrix. The dermis is delineated from the subcutaneous tissue by a deep border that
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reflects differences in the organization of the dermal matrix and the fibers of the subcutaneous tissue in the plane of the skin. By 1012 wks, a cellular to fibrous transition period is defined. Collagen fibrils increase in diameter, and greater numbers of fibrils associate into fiber bundles [10]. Papillary and reticular dermal regions are distinguishable by 14 wks [11]. The papillary region has a higher density of cells and finer collagen compared with the deeper tissue. In addition to changes in cell density and extracellular matrix, the dermis is in vaded by the downward growth (invagination) of developing epi dermal appendages. Concurrently, structural changes (i.e., bone and muscle formation) underlying the dermis tend to promote regional variations in cutaneous thickness. Collagen types I, III, and V are evenly distributed throughout the dermal and subdermal connective tissue regardless of fetal age [11]. The relative amounts are type I collagen, 70%-75%; type III, 18%-21%; and type V, 6%-8%, in contrast to adult skin where type I is 85%-900/0; type III, 8%-11%; and type V, 2%-4%. Hyal uronic acid and dermatan sulfate are the major glycosaminoglycan components [12]. Hyaluronic acid is highest in the embryonic stage, although overall the glycosaminoglycan content of fetal skin is generally higher than in the adult human skin. Thus, embryonic vascular development proceeds within the con fines of a watery, cellular tissue which has few distinguishable land marks. Fetal vasculature, on the other hand, is elaborated in a much more fibrous and eventually more organized tissue with intruding or molding structures at both its superficial and deep borders. Con sidering the changes in the surrounding tissue, what techniques have been used to obtain information about the developing vascula ture? METHODS A number of methods have contributed pieces to the developmental
story of cutaneous vasculature. Some have provided data from ani mal models, but have not necessarily been successful for obtaining
data on human skin vasculature.
Histochemical Staining
Staining with alkaline phosphatase
(AP) was one of the first methods used for studying vasculature in
fetal skin [13]. Tissue from 45 embryos and fetuses ranging in age from 4-32 wks was stained for AP activity. Thick frozen sections of skin from the scalp, forehead, face, chest, abdomen, neck, legs, palms, soles, fingers, and toes were fixed in neutral formalin and stained by a Gomori cobalt technique and azo-coupling dye to re veal AP. Most of the mesenchymal cells and fibroblasts in early fetal skin had some enzymatic activity. Later, fibroblasts, histiocytes, and mast cells showed variable intensities of AP activity. When blood vessels first appeared, they consisted of a tube of endothelial cells that were very high in AP. As soon as the mesen chymal cells surrounding the endothelial tubes differentiate into the muscle fibers and connective tissue of the tunicae, the endothelial cells gradually lose their reactivity for AP. Only the capillaries re main positive. Although AP is a widely accepted marker for endo thelial cells, its functional significance is uncertain. AP may play a role in the metabolism of carbohydrate, protein, fat, and in mediat ing the transport of phosphate ions through membranes [13].
Capillary Microscopy Although not used on fetal skin, capillary microscopy was used to study cutaneous circulation in 40 living infants ranging in age from 1 d to 17 wks [14]. By this technique observations can be made on the patterns of the microcirculation. The skin at birth showed a disorderly capillary network that was particularly prominent in skin creases. By the end of the first post natal week, the capillary network lost some of its haphazard appear ance and assumed a more orderly pattern. In the second week capil lary loops began to appear in dermal papillae as small superficial dilatations or buds. The authors stated that the number of observa tions were relatively small, and minor variations in vessel shape and number were relatively immense. Because of the difficulty in im mobilizing the infants, satisfactory photographic records were not obtained.
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Perfusion has been suggested as a method to study vessels in human fetuses; it has been used widely to infiltrate and form casts of vessels in embryonic and adult animals. Although largely successful, it is not without drawbacks [15]. Many parts of the body are not easily perfused and a collapsed or constricted blood vessel may not be filled. The perfusion technique may yield a spotty and unreliable picture of vascular patterns. It would be quite diffi cult to use to demonstrate blood vessels in very small pieces of skin, such as those available for human embryonic and fetal studies.
Perfusion
Antibodies that recognize matrix components of the vessel wall, the basement membrane and the endothelial cells have revealed differences in the developing vasculature from that of the neonate or adult. Tonnesen et al [16] immunostained samples of skin from adult, neonatal foreskin, and four second-trimester fetuses (14-18 wks gestation) to detect expression of fibronectin, laminin, and factor VIII-related antigen associated with the developing cuta neous microvasculature. Antilaminin staining of blood vessels was consistently bright and linear in fetal, neonatal, and adult skin. A reciprocal relationship was shown by intense fibronectin staining during human blood vessel development and prominent factor VIII-related antigen staining in mature blood vessels. The relation ship supports the hypothesis that fibronectin plays a role in human blood vessel morphogenesis, and that factor VIII-related antigen is a marker for endothelial cell differentiation. Another immunolabeling study investigated matrix proteins [11]. Concentrations of types III and V collagen around vessels and nerves as early as 5-6 wks EGA were observed and persisted throughout gestation.
Immunolabeling
In a review paper, Holbrook and Hoff suggested that the embryonic dermis appeared to be dis tinguished from subcutaneous tissue by a plexus of horizontally arranged vessels [91. Small capillaries from this plexus were sug gested to have invaded the dermis and to lie in close proximity to the epidermis. By the end of the first trimester, vessels as well as nerves extended throughout the dermis, reaching the dermal-epidermal junction. These were isolated observations integrated into a general story on skin development.
Light and Electron Microscopy
Computer Graphics Our laboratory has recently used computer graphics in conjunction with light microscopy to delineate the de velopmental patterns of early human vasculature [17,18]. Computer reconstructions are effective and efficient for visualizing the spacial organization of vessels in three dimensions. Two particular advan tages include the ability to assemble and reassemble the model for repetitive analysis of the whole of parts of the model and to analyze of data from the model from different angles. Some angles empha size particular data, for example the branching and/or connections are more apparent in the superior view. As our results will be dis cussed later in the text, a brief discussion of the method is given here. Tissue embedded in Epon was serially sectioned, stained and pho tographed. The negatives of the photographed sections were mag nified through a photographic enlarger and the contours of the vessels were traced onto paper. The profiles of the traced vessels were digitized using software developed at the University of Wash ington in the Department of Biological Structure. The information was transferred to a Vax 11/750 via Ethernet and contours of the blood vessels were visualized on a SiliconGraphics Iris 30-20 graphics monitor. THE OLD DEVELOPMENTAL STORY What kind of story has evolved from studies using these diverse methods? In 1983 Ryan [19], citing Serri et al, stated that the skin of 8-wk-old embryos lacks blood vessels. During the next 4 wks, an gioblastic transformation of mesenchymal cells was suggested by positive staining of cells for alkaline phosphatase. "The arterial and venous channels in the subcutaneous tissues fully communicate with central vessels and connect with the developing plexuses of the dermis during the 3rd and 4th month."
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During the fourth month a few vessels in the dermis begin to develop muscle and adventitial coats [20]. Until the perivasculature is established, budding and anastomosis are probably relatively unre stricted and occur wherever there is a stimulus to growth and orga nization. During the fifth month new vessel formation was consid ered to occur largely by budding from already formed vessels, but differentiation from primitive mesenchymal cells was still consid ered a possibility. THE NEW DEVELOPMENTAL STORY In contrast to the old developmental story, we have demonstrated that dermal vasculature exists much earlier than previously docu mented. Not only are the vessels morphologically identifiable, they are also organized in first a single plane, and then two planes parallel to the epidermis. By the end of the first trimester, both the patterns and morphology of vessels are suggestive of the adult vascular system.
Embryonic Stage (35 60 d)
Endothelial cells (Ee) of the vessel wall are morphologically identifiable in presumptive dermis of a 35 - 40-d embryo by light and transmission electron microscopy (Fig 1). The identification is based on characteristics embryonic EC share with adult EC: comparable shape and size, similar thickness in the wall structure (as narrow as 0.2-0.4 .urn), presence of plasma lemmal vesicles with their characteristic uniform diameter of 60 70 nm, and similar cell junctions, which frequently interconnect in intricate patterns. The membranes may be separated by a fairly uniform distance, but also appear to fuse at points along the bounda-
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ries. Simple tubelike vessels evolve from the joining of the endothe lial cells in the presumptive dermis, even in the earliest specimens examined (35 - 40 d). Although the lumenal sizes vary consider ably, the surfaces generally appear smooth. Blood cells are present in some of the vessels. Basal lamina around the vessels is absent. No computer reconstruction is available for the youngest specimens. The youngest specimen for which computer reconstruction of cutaneous vasculature is available is 40 - 45 d EGA. The reconstruc tion, corroborated with light micrographs, shows that the vessels lie in a single plane parallel to the epidermis and the dermal-subcutane ous interface (Fig 2). The superior view of the reconstruction illus trates apparent discontinuities in the digitized vessels, and in fact, no single vessel could be followed throughout the thickness (500.urn) of the sample. Only two vessels were continuous in sections 200 to 300.u thick, and one vessel appeared in 130 continuous sections. Examination of increasingly older specimens shows a gradually evolving complexity and density in the developing vasculature. Al though a single plane of vessels is demonstrated in the 45 - 50-d-old specimens, more vessels, in seemingly greater density are present than in the younger samples, as demonstrated by both light micro graphs and computer reconstructions (Fig 2). Reconstructions illus trate the branching and/or connections of the vessels, and a vascular network is apparent. The discontinuities are not nearly as marked as in the youngest specimen. The apparent discontinuities in the vessels in the younger speci mens pose interesting questions as to the mechanisms of vessel for mation in the developing skin. It may be, however, that the discon tinuities simply refl ect limitations of the method: technical difficulties in tracing nonlumenized vessels, resolving small sprouts,
Figure 1. Electron micrograph of youngest specimen studied. The superior border of the micrograph is the two-layered epidermis,below the presumptive dermis. Arrows point to two vessels,each containing a blood cell.
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Figure 2.
Computer reconstructions of embryonic vascular patterns,a cross sectional view (a) and a lateral view (b). The top specimen in both views is 40-45 d EGA, the middle is 45-50 d,and the bottom is 50-55 d. A comparison of the views from the youngest to the oldest specimen shows an increasing complexity from a very simple,single plane to two planes in close approximation with a greater density of vessels.
and finely filamentous projections, and identifying vessels which may be sectioned through the wall tangentially. It is also possible that the discontinuities may reflect in situ formation of vessels. As stated earlier, the dermis at 5-7 wk contains mainly mesenchymal cells. It is unknown if these cells are able to differentiate in situ into endothelial cells, or if they are able to differentiate in the earliest specimens, is there a specific time or change in the cells or their environment when the endothelial cells lose this ability. A component of the extracellular matrix may have an important role in directing endothelial cell migration. The loose structural matrix would be conducive to the growth of vascular endothelial cells by sprouting or in situ formation, because vascular sprouts appear to progress along the path of least resistance. The change in the environment from cellular to fibrous dermis may have impor tant consequences for vascular development. Concurrent with questions about early vascular development are questions about the influence of nerve growth patterns on vessels. It is not known what initiates migration of nerves into the skin or what influence this event has on angiogenesis. The migration of nerves into the skin appears to precede vessels. Although the two may follow a similar path, nerves seem to migrate in closer proxim ity to the epidermis than blood vessels. Once the nerves are distrib uted, does the epidermis produce or stimulate the production of an angiogenic factor that directs the vasculature closer to the dermal epidermal border? By 50 d EGA, two closely positioned planes of vessels are estab lished and within the planes there is heterogeneity in the diameter and structure of the vessel wall. The vessels appear to become seg mentally differentiated. The walls of the vessels are either thick and
substantial with the nuclei contained within the wall or thin and attenuated with nuclei that bulge into the lumen. Vessels with the thicker appearing walls consistently seemed to contain more blood cells. In addition to developing two planes of vasculature, the individ ual vessels are more continuous. The vessels develop into longer and longer tubes, as demonstrated in both the lateral and superior recon structions. In contrast the earlier vessels are in short segments, per haps indicative of the discontinuities observed. During the early embryonic stage, morphologically recognized subendothelial stroma appears. Ultrastructural examination of em bryonic tissue reveals little or no basal lamina around the endothelial tubes. With increasing age, EC-related matrix changes in appear ance from amorphous deposits to a thicker stromal layer that even tually becomes continuous around the endothelium. The structure of the basal lamina distinguishes between arteries and veins as well as describing perivasculature cells. In adults, as the arterial capillary is traced, the basement membrane material begins to develop lamellae within its previously homogeneous framework until a venous segment is reached in which the entire vascular wall is multilaminated [21]. Dense layers 250-1000 A thick alternate with less-dense zones. No comparable organization exists surrounding embryonic blood vessels, and although cells lie in close proximity to the vessels, none are enclosed within a basal lamina. In summary, simple capillarylike vessels first seen in the embry onic period evolve into vessels that are surrounded by matrix and perivasculature cells. Their organization appears to be nonrandom and assembles first into one, and then into two planes parallel to the epidermis.
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Early Fetal Vasculature (60-80 d) Early fetal vasculature is quite adultlike in architectural organization and morphology. The vessels are largely intact, continuous, and patent. Two planes of vasculature are well defined and parallel to the epidermis. The ves sels in the superficial plane demonstrate a honeycomb network that overlie larger vessels. In the computer reconstruction of a specimen aged 70-75 d, four major vessels were parallel to each other and to the epidermis and continuous throughout the 500 J.l of tissue exam ined. At this stage, there is greater heterogeneity in the vessels of the two planes. Those of the superior plane have seemingly smaller lumens, compared with those of the deeper plane (Fig 3). Other comparisons include: vessels with thinner appearing walls contain blood cells less frequently than those vessels with more substantial walls; more interconnections occur with the vessels containing blood cells; and few connections occur between the straighter, larger vessels and the branching, smaller vessels. Such differences prompt attempts to classify vessels as either arte rioles or venules. However, the most distinctive and reliable feature distinguishing adul� arterial segments from venous segments is the structure of the basal lamina. By the end of the first trimester, the thickness of the basal lamina has increased and in some cases, sur rounds a potential perivascular cell (Fig 4). However, similar to the embryonic vasculature, lack of organization precludes segmental differentiation. Second Trimester Light and electron microscopy demonstrate a continuing maturation of the vascular system in the dermis. By electron microscopy, Weibel Palade bodies, ultrastructural Ee markers, appear at around 100 d (Fig 5). Weibel Palade bodies serve as storage and/or processing vesicles for factor VIII-related antigen both in vitro and in vivo [22,23]. In Tonneson's study [16], factor VIII-related antigen staining of fetal vessels was granular, scant, and focal. Although the ultrastructural localization of the antigen in fetal Ee has not been done, its known existence in weibel Palade bodies would suggest that early second trimester Ee may have reached a level of maturation previously unrecognized.
Figure 3.
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Another aspect of maturation is the development of a more sub stantial basal lamina. Figure 5 demonstrates areas where the basal lamina may be developing as a multilaminated layer, indicative of venous segments. However, the structure is still quite simplistic compared with the adult, because the striking feature of adult der mal blood vessels is the thick vascular wall composed of basement membrane material, elastic fibers smooth muscle cells, or pericytes and variable amounts of individual collagen fibrils [21]. At 140 d EGA, small vessels in very close proximity to the epidermis show even more development with increasing thickness, numbers of peri vascular cells, and amounts of collagen. More information about the organization and morphology needs to be added to the unfolding story of fetal vasculature. New methods have potential to supply this information for both fetal and embryonic vasculature. MORE QUESTIONS, NEW METHODS We have shown the presence of vasculature in the earliest examined specimens (35-40 d) and its establishment in an organized plane. Do changes in morphology reflect functional capabilities or do functional demands initiate accommodating structures? What are the functional demands or metabolic requirements of embryonic and fetal skin? In Vivo Adapting methods specifically for fetal cells is very im portant considering that endothelial cells are a heterogeneous popu lation of cells. They display unique differentiated functions related to the requirements of the tissue. For example, the uptake of chemi cally modified low density lipoproteins in vivo is mediated by spe cific receptors on the endothelial cells of the liver, spleen, marrow, adrenal, and to a lesser extent, the ovary [24]. The uptake could reflect their requirements for cholesterol: for bile acid production in the liver, hormone production in the ovary, and adrenal and blood cell membrane formation in the spleen and bone marrow. What receptors do embryonic endothelial cells have and do they reflect specific functional requirements?
Light micrographs contrasting the differences in vessels. (a) View indicating the location of 6 vessels in the dermis. (b and c) High-power view of II. Vessell has a large,empty lumen,in contrast to vessels 2 and 3. Vessel 4 is filled with blood cells and has potential perivascular cells close to ablurninal surface, in contrast to vessels 4 and 5.
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ISS
70-75 days
I
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.� / '
Figure 4. arrow).
Electron micrograph showing uneven thickness of subendothelial stroma
Human fetal skin is less available for in vivo experimentation than animal models. Other methods, however, can be adapted to acquire comparable information, for example, the chorioallantoic mem brane (CAM) system. Although cumbersome and semiquantitative by itself, the CAM method combined with appropriately designed computer reconstruction experiments can generate such morpho metric analysis as areas and volumes of vessels, number of branch points in vessels, distances between vessels and volume of tissue supplied by the vasculature. As we have established the normal pattern and morphology in vivo, embryonic skin grown on a CAM could be similarly compared and then growth factors and/or angio genic factors could be investigated to answer questions about poten tial factors in the early developing dermis. Our lab has examined the development of embryonic and fetal human skin grafted to nude mice. This method has proven to be very effective in supporting epidermal morphogenesis, but it has not been used to evaluate vascular development. The kidney capsule has been used as a common site of grafting because it offers a rich source of vessels to revascularize the graft. If the development of the vascu lar system in skin could be sustained, this method might promote studies on regulation of formation and/or differentiation. Cell Culture Methodologies for microvascular endothelial cell isolation and refinements in cell culture permit successful cultiva tion from various animal and human tissue [5,6,25,26,27]. How-
(arrows). Potential perivascular cell appears to be included in stroma (right
ever, culturing embryonic endothelial cells is not a routine proce dure. Microvascular cells from tissue as young as neonatal foreskin have been cultured and the synthesis of type IV collagen, laminin, fibronectin, and thrombospondin was compared with adult tissue [28]. The ultrastructure and the profile of secretory proteins depos ited in the subendothelial matrix by the two cell types were nearly identical. Would embryonic endothelial cells give the same results or is there a time point when the cells become capable of such synthesis? Because embryologic material appears to have greater plasticity than older adult tissue, interest exists regarding endothelial cell proliferation and migration in different gel matrices. Because em bryonic and fetal vascular cells develop an organized plane of vascu lature in a matrix high in hyaluronic acid, would the cells alter their organizational "program" in a different matrix? Specific Markers Information about differentiation has been fa cilitated by the use of specific markers, e.g., the family of keratins has provided markers for understanding epidermal differentiation. Factor VIII-related antigen has been used as marker for endothelial cells, but all vascular endothelial cells do not appear to produce factor VIII, and production has not been reliable in determining the origin or pathological conditions [14,29,30,31]. Lectins also have been used as markers, and [30,32,33] monoclonal antibodies have been developed for specific endothelial cell populations [32,34,35],
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100 da-ys
Figure 5.
Electron micrograph of vessel in second trimester specimen.(a) Basal lamina is significantly more developed (compare with Fig 4).Multilaminated areas are indicated (arrowhead). (b) High-power of a. Weibel-Palade bodies are present (arrows).
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but due to the heterogeneity among these cells, there is no assurance that a monoclonal antibody for a specific adult population would recognize a fetal population of endothelial cells. Having such markers available to immunostain the developing vasculature would help solve the puzzle of the apparent discontinuities seen in the computer reconstructions.
Molecular Biology
Molecular biology is providing a means to an even greater understanding of development and much information will be gained regarding vasculature. Using endothelial cells from cultured human umbilical vein, the process of forming tubular net works was found to result in decreased levels of the sis messenger RNA transcript and increased levels of the messenger RNktran script for fibronectin [36]. The situation was reversed on transition from the organized structure to a proliferative monolayer. What signals tell proliferating embryonic or fetal endothelial cells to dif ferentiate? In summary, more information about normal human developing dermal vasculature needs to be obtained to solve important unan swered questions. New or modified methods will increase knowl edge about normal development and provide a better understanding of developmental anomalies of the vasculature. Vascular defects are the most common congenital malformations in newborns. Heman giomas, although usually benign, are frequendy of cosmetic con cern and may deform related tissue. Some lesions are components of syndromes with such severe consequences as mental retardation, hemiplegia, and convulsions. It is unknown what factors affect the organization and segmental differentiation in utero but early estab lishment of the cutaneous vasculature indicates there is the potential for abnormalities to occur quite early. Future studies of the micro vasculature in human embryonic skin are expected to reveal when and what factors may influence both normal and abnormal develop ment. REFERENCES 1.
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3. Matthews JT: Embryologic development of the vascular system. In, Dean RH and O'Neill Jr JA (eds). Vascular Disorders of Childhood. Lea 8£ Febiger,Philadelphia,pp 1-20,1983 4. Coflin JD,Poole TJ: Embryonic vascular development: Immunohis tochemical identification of the origin and subsequent morphogen esis of the major vessel primordia in quail embryos.Development 102:735-748,1988 5. Madri JA,Pratt BM,Tucker AM: Phenotypic modulation of endothe lial cells by transforming growth factor-beta depends upon the com position and organization of the extracellular matrix. J Cell Bioi 106:1375-1384,1988 6. Kubota Y,Kleinman HK,Martin GR,Lawley TJ: Role oflaminin and basement membrane in the morphological differentiation of human endothelial cells into capillary-like structures. J Cell Bioi 107:1589-1598,1988 7.
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Ryan TJ: Structure,pattern and shape of blood vessels of the skin.In, Jarret A (ed). The Physiology and Pathophysiology of the Skin. Academic Press. New York,pp 577-651,1973
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22. Wagner DD, Olmsted JB, Marder VJ: Immunolocalization of von Willebrand protein in Weibel-Palade bodies of human endothelial cells.J Cell Bioi 95:355-360,1982 23. Warhol MJ,Sweet JM: The ultrastructural localization of von Wille brand factor in endothelial cells.Am J PathoI117:310-315,1984 24. Pitas RE. Boyles J. Mahley RW, Bissel OM: Uptake of chemically modified low density lipoproteins in vivo is mediated by specific endothelial cells.J Cell BioI100:103-117,1985 25.
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