1Departments of Pediatric Surgery, Sophia Children's Hospital, 2Cell Biology and Genetics, and 3Pathology, Erasmus Medical Centre Rotterdam, Rotterdam, The Netherlands
Submitted 23 April 2004 ; accepted in final form 15 September 2004
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ABSTRACT |
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mouse; bronchial and pulmonary system
Lung development is divided into distinctive stages with the earliest stages consisting of the embryonic stage, embryonic day (E) 910 in mice and 46 wk in humans, and the pseudoglandular stage, E1016 in mice and 616 wk in humans. Models of pulmonary vascular morphogenesis at these early stages are derived from morphological data. Based on vascular casts and electron microscopy of murine lungs, deMello et al. (5) suggested that two processes are involved in the formation of the pulmonary vessels: the central vessels are formed by angiogenesis, defined as branching of new vessels from preexisting ones, and peripheral vessels by vasculogenesis, defined as development of blood lakes in the mesenchyme. A connection between the central and peripheral vascular lumen would be established through a lytic process around E13/14, and pulmonary circulation would start. A comparative analysis of serial sections of human embryos suggested that the same processes would also occur in humans (4). In addition, they concluded that pulmonary arteries and veins were dissociated in their timing and pattern of branching, since "distal veins are present throughout the mesenchyme and establish a central luminal connection with the main pulmonary vein before an airway or artery is present at the same level" (4).
Although it is generally accepted that the distal vasculature arises by vasculogenesis, recent morphological studies have questioned the basic mechanism of formation of the proximal pulmonary vessels. Using heterozygous mice with a targeted insertion of the bacterial lacZ gene into the flk-1 locus, Schachtner et al. (30) showed that the proximal and distal vascular structures were already connected at gestational age 10.5. However, only the proximal portion of the pulmonary artery contained a lumen (30). They also demonstrated that lung vessel development occurred at all stages and directly corresponded to overall lung growth. In another study, Hall et al. (11) used lungs from human embryos to stain serial sections with endothelial-specific antibodies and showed continuity of circulation between the heart and the distal lung vascular plexus from 38 days of gestation onward. They concluded that the intrapulmonary arteries originate from a continuous expansion and coalescence of the primary capillary plexus that would form by vasculogenesis during the pseudoglandular stage. In addition, they showed that the same mechanism takes place to form the pulmonary veins (10). In contrast to the definition of deMello and coworkers (4, 5), they defined vasculogenesis as differentiation of angioblasts from mesoderm to form primitive blood vessels, without the formation of hematopoietic lakes.
In spite of the lack of consensus of how the lung vasculature develops, many molecular players involved in blood vessel formation are already identified. Epithelial cells from the lung bud are suggested to induce the expansion of the capillary plexus through vascular growth factors (14). Three different growth factor systems have been described to act via endothelial cell-specific tyrosine kinases: VEGFs, angiopoietins and ephrins (37). VEGF is required for vasculogenesis and angiogenesis, and VEGF isoforms are expressed in lung epithelial (14, 18) and mesenchymal cells (8, 9). Furthermore, lung endothelial cells express Flk-1, the receptor for VEGF-A (8, 30), and in vitro experiments showed that VEGF has a potential role in lung vascular morphogenesis (14, 38). Tie2 and its ligand Ang-1 play a role in the regulation of angiogenesis (37). Gene disruption of either Tie2 or Ang-1 in mice leads to the formation of an abnormal vascular network with immature vessels that lack proper organization (28, 34). It is likely that these factors are involved in the stabilization of the network rather than its initial formation. Colen et al. (3) demonstrated expression of Ang-1 and Ang-2 in the mouse lung from E9.5 onward.
Because two opposing models on lung vessel development exist, we have performed an ontogenic morphological study of the lung vasculature in relation to the airways in mice ranging from E9.5, when the lung starts to become morphologically discernible, until the midphase of the pseudoglandular stage at E13.5. We performed analysis of fetal lungs from transgenic mice expressing the bacterial lacZ gene under the control of the Tie2 promoter (Tie2-LacZ) as well as serial section analysis of normal lungs using antibodies against platelet endothelial cell adhesion molecule (PECAM)-1, Friend leukemia integration site 1 (Fli-1), and -smooth muscle (SM) actin to specifically identify endothelial cells and blood vessels. Mouse embryos were processed to keep the blood circulation intact, thereby maintaining the vascular tone and the integrity of the vasculature. Hence, individual vessels could be identified through serial section analysis, and their origin and connections could be described with accuracy. Furthermore, circulating cells, which are primitive erythrocytes formed by the blood islands in the yolk sac at the gestational age we investigated, are trapped in vessels during fixation, and this proves that these vessels are connected with the embryonic circulation. We report that even the earliest vessels formed in the lung are already connected with the heart vascular structures and thus are part of the embryonic circulation. Because our findings are not consistent with the current models, we propose distal angiogenesis as a new model for lung vascular morphogenesis.
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MATERIALS AND METHODS |
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For paraffin embedding, the yolk sac and placenta were removed and the embryos were fixed in 4% PFA for 30 min (E9.5), 45 min (E10.5), 2 h (E11.5) at room temperature, or overnight (E12.5 and E13.5) at 4°C. After two PBS washes, the tissue was processed for paraffin embedding. Embryos were placed in coronal or transversal orientation, completely sectioned, and the 4-µm-thick sections were used for hematoxylin and eosin staining and immunohistochemistry with antibodies raised against PECAM-1 (rat monoclonal, MEC13.3, 1:50, BDPharMingen) and Fli-1 (rabbit polyclonal, C-19, 1:1,000, Santa Cruz Biotechnology) as endothelial cell markers. PECAM-1 is a 130-kDa transmembrane glycoprotein expressed on the surface of endothelial cells (6), and Fli-1 is a 50-kDa ETS domain-containing transcription factor expressed in hemangioblasts, angioblasts, and (early) endothelial cells (27). We also used an antibody against -SM actin (mouse monoclonal, 1A4, 1:400, NeoMarkers) to assess the muscularization of the vascular and airway walls. Before incubation with the primary antibody, the sections were dewaxed and endogenous peroxidase was blocked by incubation in 3% hydrogen peroxidase in methanol for 20 min. Antigen unmasking was performed with trypsin treatment (1.25 mg/ml for 5 min at room temperature) for PECAM-1 and
-SM actin and with microwave treatment in 10 mM citric acid buffer (pH adjusted to 6.0, 9 min at 450 W) for Fli-1. Sections were blocked with 5% BSA in PBS for 30 min and incubated with primary antibody diluted in 5% BSA in PBS overnight at 4°C. As secondary antibody, we used rabbit anti-rat IgG-peroxidase (Dako) for PECAM-1, goat anti-rabbit IgG-peroxidase (Dako) for Fli-1, and goat anti-mouse IgG-peroxidase (Dako) for
-SM actin, all diluted 1:100 in 5% BSA in PBS for 2 h at room temperature. Antibody binding was detected by 3,3'-diaminobenzidine, and slides were counterstained with hematoxylin.
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RESULTS |
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The primitive gut originates after the completion of gastrulation when a crescent layer of endodermal cells starts folding to form a tube. The foregut, which is the anterior part of this gut-like structure, gives rise to a number of organs, like the thyroid glands and lungs. Immediately caudal to the fourth pharyngeal arch, the first morphological sign of the lung starts to become visible at E9.5 when a cluster of cells buds from the ventral site of the foregut and invades the surrounding splanchnic mesenchyme. Subsequently, this lung bud grows and splits into the prospective left and right lobes, running alongside the future esophagus. The dorsal mesocardium, or heart stalk, connects the atrial myocardial wall with the splanchnic mesenchyme ventral to the foregut (36). We found that already at E9.510, the lung vasculature is part of the splanchnic plexus surrounding the developing esophagus and airways (Fig. 1, A and F). The maintenance of the blood circulation while fixing the embryos made it easy to define and trace the vessels throughout the serial sections, because they appeared as an open structure and contained circulating primitive erythrocytes. The afferent and efferent components form a Tie2-, PECAM-1-, and Fli-1-positive plexus of capillaries that surrounds the proximal foregut (Fig. 1, AC) and is continuous with the developing aortic arches and dorsal aorta (Fig. 1A). A capillary network of Tie2 (Fig. 1A)-, PECAM-1 (Fig. 1B)-, and Fli-1 (Fig. 1C)-positive endothelial cells surrounds the two airway buds and is in close contact with the epithelial cells of the airway (Fig. 1, D and E). Primitive erythrocytes, exclusively produced by the blood islands of the yolk sac, are frequently observed within the main vessels, the dorsal aorta and heart structures (Fig. 1C), and the capillaries, indicating that even the smallest vessels are connected to the embryonic circulation (Fig. 1, D and E). The venous confluence of this network runs through the dorsal mesocardium (Fig. 1C) and forms an invagination at the entrance of the atrium, described as the pulmonary pit (36).
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At E11.5, lung asymmetry has become obvious: the left lung has one branch, whereas the right lung has four main branches, the primordia of the cranial, the middle, the caudal, and the accessory lobe (Fig. 1J). The proximal afferent vessels can now be clearly identified as two vascular tubes that run alongside the trachea, the right and left pulmonary arteries (Fig. 1, J and L). Proximally, the right pulmonary artery is still connected to the right sixth aortic arch but has been lateralized toward the left (Fig. 1J, inset). The left sixth aortic arch, which will form the ductus arteriosus, is more obvious than the right sixth aortic arch, which eventually degenerates (Fig. 1J, inset). The vessels that surround the lung buds are positive for Tie2, PECAM-1, and Fli-1 (Fig. 1, J, K, and M) and are filled with primitive erythrocytes. The efferent vessels form a vascular tube, the common pulmonary vein (Fig. 1, J and K). SM cells start to enfold the proximal parts of the arteries, airways (Fig. 1, N and O), and, to a lesser extent, veins (data not shown). However, the growing part of the distal airways and surrounding vessels are not yet muscularized.
At E12.5, the esophagus and trachea are only attached by mesothelial linings (Fig. 2A, inset). The lung has a single left lobe and four clearly distinguishable right lobes (Fig. 2A). Each lobe of the lung has undergone further branching, which occurs in an axial fashion, increasing length and number of generations from the periphery to the center. The airway terminal buds are completely surrounded by a polygonal, irregular capillary plexus (Fig. 2, A and inset), which contains a lumen filled with primitive erythrocytes (Fig. 2B), indicating a direct and closed connection with the embryonic circulation. The endothelial cells of these vessels form a Tie2 (Fig. 2A)-, PECAM-1 (Fig. 2, C and D)-, and Fli-1 (Fig. 2E)-positive network close to the epithelial cells of the terminal bud. The proximal airways and vessels as well as the trachea and dorsal aorta are clearly -SM actin positive, indicative for a maturation process. In contrast, the immature distal airways and vasculature are not yet muscularized (Fig. 2F).
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DISCUSSION |
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Already at the first morphological sign of lung development, the vasculature consists of Tie2-, PECAM-1-, and Fli-1-positive endothelial cells that are part of a capillary network in the splanchnic mesoderm (Fig. 1, A, F, and G). The proximal and distal structures from this plexus are connected to each other and are continuous with the heart vascular structures (Fig. 1, C, H, and I). The presence of yolk sac-derived primitive erythrocytes within the vascular lumen proves that there is already blood circulation in the lung and connection with the embryonic circulation from the earliest point of lung development (Fig. 1, D, E, and G). Remodeling of this plexus forms the main trunks of the proximal vessels, which is especially notable in the case of the pulmonary artery that changes from a plexus alongside the trachea at E9.5 into two muscularized vascular tubes at E11.5 (Fig. 1, A, F, and J). Serial sections demonstrated that both the proximal and distal vessels of the afferent and efferent pulmonary vasculature were positive for both Fli-1 and PECAM-1 from E9.5 onward. The endothelial cells of the network of capillaries that surrounds the growing epithelial buds, the effective component, were also positive for both antigens during early lung development.
We clearly demonstrate for the first time that from the earliest morphological sign of lung development, a vascular network exists that is in contact with the embryonic circulation. Through the preparation of vascular casts, deMello et al. (5) investigated the development of the pulmonary vasculature in mouse and concluded that there was no connection between proximal and distal structures of the mouse lung before E13.5. This technique, although very valuable and informative, has considerable limitations when studying vascular networks that consist mainly of capillaries and very small vessels. Schachtner et al. (30) used heterozygous Flk-1-LacZ knockin mice to study the lung vasculature, starting their whole mount analysis at E11.5, 2 days after the initiation of lung development. They concluded that only the proximal part of the growing pulmonary artery contained a lumen through analysis of E10.5 mouse embryo sections (30). Our data are partly in line with the work of Hall et al. (10), who showed continuity from the proximal and distal structures of the lung using three-dimensional reconstruction of immunostained 34-day human lung. Hall et al. identified PECAM-1-positive cells in vessel walls, some of which had a narrow lumen (10, 11). We used a tissue processing procedure that keeps the blood circulation intact and prevents the collapsing of the vessels. It is clear that the lumen of the proximal arteries is narrower than the lumen of the proximal veins, due to differences in pressure and muscularization. This may explain why deMello et al. (4) observed that early veins were diffusely present throughout the mesenchyme, establishing a central luminal connection to the main pulmonary vein before airways or arteries were present at the same level, thus leading to the conclusion that veins and arteries are dissociated in their timing and pattern of branching. Schachtner et al. (30) named these venous drainages "lacunae," which are very clear at E13.5. However, our analysis of serial sections revealed that these structures are, in fact, the pulmonary veins. Based on hematoxylin and eosin-stained sections, deMello et al. described the presence of "hematopoietic lakes" in E10 mouse lung (5) and 33-day human lung (4), but analysis of serial sections of embryonic lungs fixed with intact circulation did not reveal structures that could resemble these hematopoietic lakes. We conclude that the description of hematopoietic lakes in the lung is based on morphology, and these lakes most likely are collapsed vessels containing trapped primitive erythrocytes.
Vasculogenesis and hematopoiesis are intimately associated extraembryonically, and this led to the description of the existence of a common precursor cell, the hemangioblast (26). Poole and Coffin (24) used QH-1 antibodies as a label for angioblasts to study the major vessel primordia in quail chick chimeras. They defined vasculogenesis as the in situ formation of vessels from the aggregation of angioblasts into a cord that later acquires a lumen and angiogenesis as the formation of new vessels by sprouting of capillaries from existing ones. Later studies concluded that vasculogenesis gives rise to the heart and the first primitive vascular plexus, whereas angiogenesis is responsible for the remodeling and expansion of this primitive plexus (23). A vasculogenic study in early mouse embryos identified the angioblast as a mesodermal Tal-1+/Flk-1+/PECAM-1 cell (6). The presence of angioblasts in the lung has been described morphologically (4, 5) and as isolated PECAM-1-positive (10, 11) or Flk-1-positive (30) endothelial cells. Flk-1 is expressed by undifferentiated endothelial cells and angioblasts, but Schachtner et al. (30) showed that LacZ expression driven by the Flk-1 promoter overlaps with PECAM-1 expression in the lung. DeMello et al. (4, 5) suggested that the lung itself produces blood cells in hematopoietic lakes as part of a vasculogenic process. However, during our analysis of serial sections of the different embryonic stages of lung development, we have never encountered blood cells other than primitive and definitive erythrocytes. If the lung produced blood cells, it would be conceivable that intermediate hematopoietic precursor cells are observed in the sections, even if their frequency is very low. Furthermore, Medvinsky et al. (20) previously performed colony-forming unit spleen assays on several embryonic tissues and demonstrated that the embryonic lung does not contain hematopoietic progenitors; therefore, the lung is not recognized as an organ with the capacity to produce hematopoietic cells. Hematopoiesis has been described extraembryonically in the hematopoietic islands of the yolk sac as well as intraembryonically in the trunk intermediate cell mass and later in the aorta-gonad-mesonephros, liver, spleen, and bone marrow (7).
We hypothesize that the vasculature grows primarily by expansion of existing vessels, but we cannot exclude at this moment the possibility that a minor population of putative angioblast-like cells also contributes to the growth of the capillary network. Therefore, we have performed immunostaining with Flk-1 to investigate the existence of an angioblast-like cell in the lung mesenchyme that may contribute to the expanding vascular network. Positive Flk-1 staining was identified in the endothelial cells of the vessels in a pattern that resembled our presented Tie2 staining (data not shown), confirming the pattern of X-gal stainings reported by Schachtner et al. (30) with the Flk-1-LacZ knockin mice. However, we were unable to localize isolated Flk-1-positive cells that were not part of the vasculature that could serve as a putative angioblast. In addition, we have performed CD34 immunostainings, and the results support our data shown in this paper (data not shown). In contrast, Han et al. (12) describe CD34+ solitary cells, or groups forming a single layer, in the lung mesenchyme of 4-wk-old human embryonic lungs. Maeda et al. (18) also described endothelial cells in distal lung mesenchyme that are separated from the proximal vessels and that may be derived from mesenchymal cells. Although we formally cannot exclude that there may be scattered CD34+ cells in the mesenchyme not linked to the vasculature, we believe that these apparent single cells are part of a network that can only be revealed through careful analysis of serial sections.
Although we were unable to identify individual Flk-1+ or CD34+ cells, we cannot evocatively exclude the existence of angioblast-like cells in the lung mesenchyme. However, we reason that the contribution of putative intrapulmonary angioblast-like cells to the developing vasculature would be minimal. It remains interesting to speculate how the vasculature can expand so rapidly to keep up with the branching airways. Given the enormous expansion of the network required to match the growth of the airways in a short time, it is hard to understand that a small number of angioblasts would have a major contribution to the growth of the vascular network. It could be possible that circulating cells contribute to the expanding network of vessels, since the endothelial cell divisions and stretching may not fully explain the speed of growth (17). Although these authors refer to the process whereby circulating endothelial cell progenitors incorporate and differentiate into existing blood vessels as vasculogenesis, we would suggest calling this process angiogenesis since it is the expansion of an preexisting vessel.
Our observations of the gradual muscularization of the proximal vessels as part of their maturation process confirms the work described by Hall et al. (10, 11) in humans. They showed that veins acquired -SM actin at 56 days, whereas arteries did at 38 days. In mice, we first detected
-SM actin-positive cells in the vascular wall of arteries and veins at gestational age E11.5 (Fig. 1, N and O). Two muscularized pulmonary arteries run alongside the trachea at E11.5, as also shown by a confocal microscopic study in mouse lungs (35). Two recent papers (12, 18) used double immunolabeling for PECAM-1 and
-SM actin in human fetal lungs with the conclusion that there are two populations of endothelial cells in the lung vascular system. One population of vascular plexi independent of
-SM actin-positive adjacent cells and another population of vascular plexi were juxtaposed to
-SM actin-positive cells. Both groups imply that vasculogenesis would rely on the endothelial cells that are independent from the
-SM actin, whereas angiogenesis would be based on the
-SM actin-dependent endothelial cells. In addition, the authors concluded that both plexi communicate at the midpseudoglandular stage. According to Maeda et al. (18), the intrapulmonary arteries make contact with a preexisting capillary network that develops independently. This would support the findings of deMello et al. (4, 5), but as we already discussed, our data do not support this hypothesis. We have clearly shown that already at the onset of lung development there is a clear vascular network surrounding the growing lung buds, and our data support the idea that angiogenesis is the major process by which the lung vasculature grows and expands. In addition, our results demonstrate that the vessels forming in the distal mesenchyme are in contact with those in the proximal mesenchyme. Our analysis of serial sections revealed that there was just one vascular network and that proximal vessels undergo muscularization as part of the wall maturation as they accommodate the increasing blood flow and pressure. Muscularization is part of the gradual maturation process of airways and vessels. We have shown that the proximal structures in the lung are
-SM actin positive before the immature distal structures, which confirms the findings by Hall et al. (10, 11).
Distal angiogenesis as a new concept for lung vascular morphogenesis. Pulmonary vascular morphogenesis has been described to occur either by a combination of central angiogenesis and vasculogenesis with the formation of hematopoietic lakes (Fig. 3A) (4, 5, 33) or just by vasculogenesis with formation of new vessels from endothelial precursor cells (Fig. 3A) (10, 11, 15). However, our data do not support the existence of central angiogenesis or distal vasculogenesis in lung development. Therefore, we propose "distal angiogenesis" as a model for pulmonary vascular morphogenesis (Fig. 3, A and B). Distal angiogenesis is the formation of new capillaries from preexisting ones at the periphery of the lung. On the basis of extensive and detailed morphological observations, we define the concept of the "tip zone" as the distal part of the branching airway that lacks the layer of SM cells. It is wrapped by a polygonal meshwork of capillaries, the effective component, that expands by distal angiogenesis as the lung bud grows, finally leading to the alveolar capillary plexus. We hypothesize that epithelial-endothelial interactions are decisive, inducing angiogenesis at the tip zone, which ensures the coordinate expansion of the vascular network as the branching proceeds (Fig. 3B). Newly formed vessels remodel dynamically, as they form part of the afferent or efferent component. This vascular remodeling implies that some vessels will grow and fuse with neighboring vessels, whereas others will remain small or degenerate (Fig. 3B).
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In summary, we performed a detailed ontogenic morphological analysis of the pulmonary vasculature from the earliest embryonic stage onward. The present study describes the development of the different vascular components (arteries, capillaries, and veins) of the lung in relation with the developing airways. We conclude that the vasculature is part of the embryonic circulation from the moment the lung starts to develop. This implies that the presence of blood vessels could be more important for the development of the lung than previously anticipated. The endothelial cells of the splanchnic mesoderm may be involved in the prepatterning of the presumptive lung region, like it has been shown for liver and pancreas development where endothelial cells are involved in the induction of these organs (16). Our observations led us to propose distal angiogenesis as a new concept for lung vascular development. We defined the concept of the tip zone, where the epithelial-endothelial interactions are crucial to determine the expansion of the lung vascular network. Based on our observations, we propose that angiogenesis already starts at the embryonic phase of lung development and is the major blood vessel-forming process.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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