Recent studies on human embryonic and fetal lungs show that the pulmonary arteries form by vasculogenesis. Little is known of the early development of the pulmonary veins. Using immunohistochemical techniques and serial reconstruction, we studied 18 fetal and neonatal lungs. Sections were stained with antibodies specific for endothelium (CD31, von Willebrand factor) and smooth muscle ( α and γ smooth muscle actin, smooth muscle myosin, calponin, caldesmon, and desmin) and antibodies specific for the matrix glycoprotein tenascin, the receptor protein tyrosine kinase EphB4, and its ligand ephrinB2. Kiel University–raised antibody number 67 (Ki67) expression allowed qualitative assessment of cell replication. By 34 d gestation, there was continuity between the aortic sac, pulmonary arteries, capillaries, pulmonary veins, and atrium. The pulmonary veins formed by vasculogenesis in the mesenchyme surrounding the terminal buds during the pseudoglandular period and probably by angiogenesis in the canalicular and alveolar stages. EphB4 and ephrinB2 did not distinguish between presumptive venous and arterial endothelium as they do in mouse. All venous smooth muscle cells derived directly from the mesenchyme, gradually acquiring smooth muscle specific proteins from 56 d gestation. Thus, both pulmonary arteries and veins arise by vasculogenesis, but the origins of their smooth muscle cells and their cytoskeletal protein content are different.
Little is known about the embryonic and fetal development of human pulmonary veins. Historically, the pulmonary veins have received less attention than the pulmonary arteries, being regarded as passive conduits, whereas the arteries regulate pulmonary vascular resistance. However, recent studies have shown that despite being thin walled, isolated pulmonary veins show a greater response to both contractile and relaxant agonist stimulation and greater release of nitric oxide than pulmonary arteries (1). It is now apparent that the pulmonary veins form an active segment of the pulmonary circulation.
In a recent study of human embryos, serial reconstruction showed that the pulmonary arteries form by vasculogenesis from the spanchnopleural mesenchyme (2). Distinguishing presumptive arterial and venous endothelium from each other in the mesenchyme can be problematic. However, in mouse embryos, endothelial cells destined to become systemic venous cells expressed the tyrosine kinase receptor EphB4, whereas its cognate ligand, ephrinB2, was expressed on predestined systemic arterial endothelial tubes (3-5). Thus, in mice, venous and arterial endothelial cells are molecularly distinct from the earliest stages of development (5). The Eph receptor tyrosine kinase family and their ephrin ligands have been shown in early vertebrate development to be responsible for many boundary and guidance processes (6). In particular, EphB4 and ephrinB2 are essential for the development of the cardiovascular system in mouse embryos. During the development of pulmonary arteries, the airways appear to act as a template. However, in the mature lung, the veins lie at some distance from airway and artery, in the connective tissue septa, suggesting that the airways may have less of a direct influence on the control of vein development in utero.
In the present study we hypothesized that the endothelial expression of EphB4 and ephrinB2 in the lung mesenchyme of the human embryo would distinguish between presumptive pulmonary venous and pulmonary arterial endothelium. Furthermore, we considered that the difference in spatial relationship between veins with airways and arteries with airways would be reflected in the origin and composition of the venous smooth muscle. We used immunohistochemical techniques to study serial sections of human embryonic and fetal lungs. Reconstruction of the serial sections showed that in human embryos the pulmonary veins, like the pulmonary arteries, are originally derived by vasculogenesis from the mesenchyme surrounding the terminal airways. Reconstruction also confirmed the presence of the postnatal anatomic relationship of arteries, veins, and capillaries from embryonic life. It was not possible to discriminate presumptive veins from arteries by their expression of EphB4 and ephrinB2.
Human fetuses aged 34 (n = 2), 38 (n = 2), 44 (n = 1), 47 (n = 1), 56 (n = 1), 84 (n = 4), and 98 (n = 1) d were obtained from the Medical Research Council Human Embryo Collection (London, UK). The fetuses were staged by limb bud and facial features (7). In addition, lungs from three fetuses aged 105, 119, and 140 d were obtained from the Anatomy Department of the University of Leiden (Leiden, the Netherlands), and their collection and use was approved by the Medical Ethics Committee of the Leiden University Medical Centre. The whole fetus or lung of all but two 84-d-old fetuses were fixed in 4% paraformaldehyde for 24 h at 4°C and then rinsed in several changes of 70% alcohol before being processed for wax histology. In addition, we examined postmortem lung biopsies taken from three neonates who died with normal lungs. This tissue was taken within 24 h of death and fixed in 10% buffered formaldehyde for a minimum of 24 h before processing for routine histology. Serial 5-μm wax sections were cut through the thoracic region of each fetus or lung tissue sample.
The sections were used for immunohistochemical staining with antibodies specific for either vascular endothelial or smooth muscle cells (2), for the receptor EphB4 and its ligand ephrinB2, and for tenascin, a matrix glycoprotein associated with angiogenesis (8, 9) (Table 1). In addition, tissue sections were stained with hematoxylin and eosin and with Miller's elastic van Gieson stain. Sections were also stained for replicating cells by expression of Kiel University-raised antibody number 67 (Ki67), which labels all cells not in the Go phase of mitosis (Table 1). Before incubation with the primary antibody, the sections were first dewaxed, and antigen unmasking was performed by autoclaving at 121°C for 14 min in citric acid buffer (2.1 g citric acid in 1 liter distilled water, pH adjusted to 6.0). Endogenous peroxidase was then blocked by incubation in 0.3% hydrogen peroxide in methanol and nonspecific binding blocked by incubation with serum-free protein block (Dako UK, Cambridgeshire, UK). Sections were incubated in 100 μl of primary antibody diluted in phosphate buffered saline with 0.02% bovine serum albumin for 1 h at room temperature. Antibody binding was visualized with diaminobenzidine using a biotin-conjugated relevant secondary antibody and avidin streptavidin amplification (StrepABComplex, Dako UK). Sections were lightly counterstained with hematoxylin and eosin. For each gestational age group, at least eight sections were studied with each antibody.
Antibody | Clone | Dilution | Source | |||
---|---|---|---|---|---|---|
Endothelial specific protein | ||||||
Monoclonal anti-human CD31 | JC/70A | Prediluted | Dako (Ely, Cambs, UK) | |||
Rabbit anti-human von Willebrand factor | A 0082 | 1:200 | Dako (Ely) | |||
Rabbit polyclonal anti-ephrinB2 human | P20 | 1:1,000 (wax) 1:100 (frozen) | Santa Cruz Biotechnology (Santa Cruz, CA) | |||
Goat polyclonal anti-EphB4 human | C16 | 1:100 | Santa Cruz Biotechnology | |||
Smooth muscle specific proteins | ||||||
Monoclonal anti-α-SM actin | 1A4 | 1:3,000 | Sigma (Poole, Dorset, UK) | |||
Monoclonal anti-γ-SM actin | B4 | 1:100 | Dr. J. Lessard (University of Cincinnati, OH) | |||
Monoclonal anti-MHC SM-1 | MCA MH-01 | 1:1,000 | Yamasa (Tokyo, Japan) | |||
Monoclonal anti-calponin | hCP | 1:1,000 | Sigma | |||
Monoclonal anti-caldesmon | hHCD | 1:400 | Sigma | |||
Monoclonal anti-desmin | ZC18 | Prediluted | Zymed Labs (San Francisco, CA) | |||
Matrix protein | ||||||
Monoclonal anti-human tenascin | BC-24 | 1:500 | Sigma | |||
Cell replication marker | ||||||
Monoclonal anti-Ki67 | 7B11 | Prediluted | Zymed Labs |
The lungs of two 84-d-old fetuses were mounted on cork discs in Cryo-M-Bed (Bright Instrument, Cambridge, UK) and snap frozen in isopentane cooled in liquid nitrogen. Serial 10-μm sections of lung tissue were cut in a transverse plane, air dried, and fixed briefly with paraformaldehyde and stained for ephrinB2 (Table 1).
In addition to conventional microscopic study, three-dimensional reconstructions were made from images acquired using a Zeiss Axioskop microscope, ×10 objective, Hamamatsu camera, and the OpenLab Program (Improvision, Warwick, UK).
Reconstruction of serial sections showed that at 34 d of gestation the early lung bud was entirely enclosed within the splanchnopleural mesenchyme, which surrounds the foregut (Figures 1a and 1b). The trachea lay ventral to the esophagus and gave rise to two bronchi, which curved dorsally to lie on either side of the esophagus. The heart lay ventral to the lung bud, and at the level of the atria the dorsal mesocardium was continuous with the splanchnopleural mesenchyme. CD31-labeled endothelial tubes in the mesenchyme around each lung bud merged on the ventral side of the lung bud and joined to form the pulmonary venous confluence, which opened into the prospective left atrial cavity (Figures 1a and 1b). Endothelial tubes also merged on the lateral side of each bronchus to form a vessel that was in continuity with paired pulmonary arteries connecting with the aortic sac at the cephalic end of the trachea. Erythrocytes were present in all vessels, suggesting that blood could circulate through the lung bud from at least 34 d gestation.
From 38–56 d gestation, the lung buds gradually separated from the oesophageal mesenchyme, and distally in each lung bud the mesenchyme expanded progressively around an increasing number of airway generations. This resulted in a relative narrowing of the hilar region. The extrapulmonary veins lengthened with age as the heart (left atrium) differentiated and separated from the base of the lung. Within the mesenchyme surrounding the terminal airway buds (Figures 1c and 1d), a plexus of CD31-labeled endothelial tubes or capillaries could be identified. Cell replication, as assessed qualitatively by the number of cells reacting positively to Ki67, was greatest in the undifferentiated peripheral mesenchyme surrounding these capillaries (Figure 1e). Beyond the capillary bed, the endothelial tubes merged to form venules that were separated from the artery by the airway bud. Serial reconstruction showed that these venules and capillaries were continuous proximally with the main pulmonary veins and distally with capillaries that were in continuity with pulmonary arteries (Figure 1c). By 84 d of gestation, veins ran midway between airways where the mesenchymal cell density was low (Figure 1f). At all ages between 38 and 98 d gestation, during the pseudoglandular phase of development (10), continuous coalescence of capillaries at the periphery lengthened the veins and increased the number of pulmonary vein generations. Thus, the veins appeared to be derived by vasculogenesis.
There was a marked change in the appearance of the lung after 84 d gestation, when the lung periphery appeared to be divided into lobules. In the areas of low cell density between the airways, there were large clear spaces lined by CD31-positive cells. These were presumptive lymphatic channels (Figure 1g) forming the prospective septa. A small amount of collagen was visible in the septa by 105 d. The lymphatic channels formed an extensive but incomplete sheath around the larger and more proximal pulmonary veins, isolating them partially from the surrounding mesenchyme and airways, and were larger than the lymphatic channels seen postnatally. The lymphatic sheath ended approximately two generations of vein proximal to the capillary plexus. Small veins and venules draining the terminal airways bridged the sheath and joined the more proximal veins (Figure 1h).
At 119 d of gestation (canalicular phase [10]), capillaries close to the cuboidal epithelium of terminal buds were continuous with the venules and then veins. The Ki67 labeling showed that at this age the endothelial cells of these capillaries had a high replication rate, whereas the surrounding undifferentiated mesenchyme was more sparse and now had few replicating cells (Figure 1i). This appearance was retained until at least 140 d gestation.
In the neonatal lungs, the capillaries from the alveolar region drained into venules ∼ 5 μm in diameter, which in turn opened into veins 16–20 μm in diameter (Figure 1j, inset). As noted earlier, small veins ran directly from the alveolar walls and connected with the larger veins lying within the connective tissue septa (Figure 1j). The lymphatic sheaths around the peripheral veins were still present two generations from the capillary bed, now within the alveolar region of the lung.
Tenascin is a matrix protein that is expressed in regions of both cell migration and angiogenesis (11). From 38–98 d gestation, tenascin was expressed in the mesenchyme around all airways except the terminal buds (Figure 2a) and was detectable in the subendothelium of proximal, muscularized veins but not in the arteries. By 105–140 d gestation, it was found also at the base of the epithelium of the terminal buds (Figure 2b). This distribution was still seen in the newborn lungs where tenascin was strongly expressed on the subendothelium of the capillaries and intraacinar veins (Figure 2c) and was now present in the media of preacinar veins. Throughout fetal life and at birth, tenascin was not detectable in the subendothelium or media of any precapillary pulmonary artery (Figure 2c).
A distinct layer of smooth muscle cells, distinguished by expression of αSM actin, outside the endothelium was seen only from 47 d of gestation and at this time only in the extrapulmonary vein. These smooth muscle cells were flattened and closely applied to the endothelium. This single layer later became surrounded by a loose meshwork of elongated but poorly oriented mesenchymal cells that also stained for α-SM actin, presumptive smooth muscle cells (Figure 3a). By 56 d, a single layer of flattened smooth muscle cells was found in the intrapulmonary veins. With age and increase in vein size, the number of muscle layers increased progressively, and there were six layers in the hilar veins at 98 d of gestation, whereas the more peripheral veins, lying 2–3 generations postcapillary, had a single layer of smooth muscle cells. All these smooth muscle cells appeared to derive from the surrounding mesenchyme.
From 44–140 d of gestation, some endothelial cells of the smallest venules expressed α-SM actin (insets in Figures 1d and 3b) and from 84 d α-SM actin positive endothelial cells were seen in the capillaries. By birth, no endothelial cells stained with α-SM actin.
Smooth muscle cells of the subendothelial layer gradually acquired other proteins in the order characteristic of maturating smooth muscle cells (12). First to appear was the smooth-muscle–specific myosin heavy chain isoform SM-1 (SM-1), followed by γ-SM actin (Figure 3) and the actomyosin regulatory protein calponin. The outermost layers acquired these proteins slightly later than the inner layer (Table 2). Caldesmon, another actomyosin regulatory protein, was not expressed in any pulmonary vein, although it was present in the pulmonary arteries at this time. At 98–140 d, SM-1 immunostaining was no longer present in proximal veins > 70–100 μm in diameter (Figure 3d), although α-SM actin, γ-SM actin, and calponin were still present. The transient disappearance of SM-1 from the proximal veins was associated with compression of the smooth muscle cell layers and an increase in lumen size. In the neonatal lung, all the venous smooth muscle cells again expressed SM-1 in addition to α- and γ-SM actin. Calponin was present in some cells in all generations of veins, but caldesmon was still absent. Thus, all venous smooth muscle was derived directly from the mesenchyme. No α-SM actin positive cells were seen migrating from the airway smooth muscle to the veins, as has been described for the arteries (2). This may be as a result of the greater distance between the veins and airways. Maturation of the smooth muscle was different in veins and arteries.
Gestational Age (d) | α-SM Actin | MHC SM1 | γ-SM Actin | Calponin | ||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Hilar– Midlung (subend outer) | Peripheral 2–3 Generations | First Post- capillary Generation | Hilar– Midlung(subend outer) | Peripheral 2–3 Generations | First Post- capillary Generation | Hilar– Midlung(subend outer) | Peripheral 2–3 Generations | First Post- capillary Generation | Hilar– Midlung(subend outer) | Peripheral 2–3 Generations | First Post- capillary Generation | |||||||||||||
38 | −† | − | − | − | − | − | − | − | − | − | − | − | ||||||||||||
44–47 | +‡ − | + | + | + − | + | − | + − | − | − | − − | − | − | ||||||||||||
56–84 | + + | + | + | + − | + | + | + − | + | − | (+)§ − | (+) | − | ||||||||||||
98–140 | + + | + | + | − − | − | + | + + | + | − | (+) (+) | (+) | − | ||||||||||||
Neonate | + + | + | + | + + | + | + | + + | + | − | (+) (+) | (+) | − |
In addition to vascular smooth muscle cells, myocardial muscle was found in the extrapulmonary veins, identified by the presence of desmin, an intermediate filament protein expressed only in mature muscle cells and not found in the pulmonary vasculature during fetal life. Desmin was detected in the atrial myocardium from 38 d gestation, and discreet dense bundles of desmin-positive cells were present in the outer part of the wall of the extrapulmonary veins continuous with the left atrial myocardium from 56 d of gestation onward (Figure 3e). Staining for α-SM actin on adjacent tissue sections revealed that these desmin-containing cells also contained striated actin filament bundles.
In the systemic circulation, the ligand ephrinB2 was strongly expressed in the aortic endothelium and large coronary arteries of all the embryos and fetuses examined (38–98 d gestation) but was weaker in small arteries branching from the aorta (Figure 3f) and in small coronary arteries (the endocardium was positive). Conversely, the receptor EphB4 was not expressed in the aortic endothelium in any of the embryos and fetuses examined, but it was expressed in small arteries branching from the dorsal aorta (Figure 3g). It was also seen in small coronary arteries and veins (the endocardium was negative) and other small systemic arteries and veins. Endothelial cells of large cardinal veins were positive for ephrinB2 but not EphB4.
For the pulmonary circulation, at 38–47 d gestation the ligand ephrinB2 was expressed in the endothelium of both prospective extrapulmonary and hilar intrapulmonary arteries but not in the peripheral generations or in the capillary plexus (Table 3). By 84–140 d gestation, it was found in both the endothelium and medial smooth muscle cells of large proximal intrapulmonary arteries (Figure 3h) and extended in both to 3–4 generations proximal to the periphery. EphrinB2 was not detectable in the pulmonary veins at any age before or after birth (Figure 3i). However, it was found in endothelial cells lining the adjacent lymphatic channels from 98–140 d.
ephrinB2 | EphB4 | |||||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Arteries | Veins | Arteries | Veins | |||||||||||||||||||||||||
Gestational Age (d ) | Extra- pulmonary, Hilar | Mid- lung | Peripheral 2–3 Generations | Capil- laries | Peripheral 2–3 Generations | Mid- lung | Extra- pulmonary, Hilar | Extra- pulmonary, Hilar | Mid- lung | Peripheral 2–3 Generations | Capil- laries | Peripheral 2–3 Generations | Mid- lung | Extra- pulmonary, Hilar | ||||||||||||||
34–38 | +† | − ‡ | − | − | − | − | − | − | − | − | ||||||||||||||||||
44–47 | + | − | − | − | − | + | + | − | − | + | ||||||||||||||||||
56–84 | + | + | + | − | − | − | − | − | + | + | (+)§ | + | + | − | ||||||||||||||
98–140 | + | + | + | − | − | − | − | − | (+) | + | + | + | (+) | − | ||||||||||||||
Neonate | − | − | − | − | − | − | + | + | + | − |
The receptor EphB4 was not detected in the lung buds before 44 d gestation. At 44–47 d it was expressed in the endothelium of the prospective extrapulmonary vein (Table 3). In the 56- to 98-d-old fetuses, the endothelium of the extrapulmonary vein no longer showed EphB4 expression. However, expression was now strong in all intrapulmonary veins and arteries and was also detectable in proximal capillaries (Figure 3j and inset). By 105–140 d gestation, strong expression of EphB4 was found in the endothelial cells of peripheral veins and arteries and throughout the capillary plexus around the terminal buds. In the neonatal lungs, EphB4 was also detected only in respiratory unit veins and arteries and in the alveolar capillaries (Figure 3k). Thus, throughout gestation, EphB4 was present in the peripheral 2–3 generation of arteries and veins (up to 25–30 μm) and was not expressed as the vessels increased in size. It colocalized with ephrinB2 in the endothelium of arteries approximately three generations from the capillary bed. Thus, expression of EphB4 and ephrinB2 did not discriminate, in humans, between the presumptive venous and arterial endothelia, either pulmonary or systemic.
The findings in the present study suggest that the pulmonary veins of the human embryo form initially by vasculogenesis from the splanchnopleural mesoderm and that the later growth in the intraacinar region is likely to be by angiogenesis. At 34 d gestation, serial reconstruction demonstrated physical continuity between the aortic sac, pulmonary artery, peribronchial capillary plexus, pulmonary veins, and left atrium, suggesting circulation of blood through the lung bud. This continuity was described previously in mice at an equivalent gestational age (13). We used EphB4 and ephrinB2 in an attempt to distinguish presumptive pulmonary veins from pulmonary arteries during vasculogenesis but could not detect either receptor or ligand on the capillary bed in early embryos. EphrinB2 was expressed on pulmonary arteries but not veins, as described in the systemic circulation of mice (5, 14, 15). However, EphB4 did not discriminate human pulmonary veins from arteries (5, 14) and showed temporal and spatial changes in expression in both structures throughout gestation. Pulmonary venous smooth muscle appeared to be derived from the mesenchyme by differentiation from undifferentiated mesenchymal precursors. There was no evidence of smooth muscle derivation from bronchial smooth muscle as we had previously described in the pulmonary arteries, probably because the pulmonary veins did not mature in close proximity to the airways. By mid-gestation, the intrapulmonary veins were closely associated with lymphatic channels, and together they formed rudimentary septa between the airways. The lymphatic profiles could be important in isolating veins from the influence of airway-derived vasculogenic or angiogenic signals (8, 16-18).
In the human embryo, ephrinB2 and its receptor EphB4 were not early molecular markers of the endothelial cells within the lung bud. They were expressed sometime after the cells expressed CD31. Also, as noted above, ephrinB2 and EphB4 expression did not distinguish the endothelium of presumptive pulmonary arteries and veins. EphB4 labeled the capillaries connected to both arteries and veins. The discriminatory expression described in systemic vessels in mice is thought to reflect a complex repulsive interaction between receptor and ligand, which is crucial for the morphogenesis of capillary beds into either arteries or veins (5). The findings in the present study indicate either that such repulsion is not the mechanism for arterial and venous discrimination or that different molecules are responsible for such a mechanism in the human lung.
Our observations suggest that until the end of the pseudoglandular period, the pulmonary veins originate, like the arteries, by vasculogenesis from the splanchnopleural mesenchyme. Induction of vasculogenesis is thought to be mediated by vascular endothelial growth factor (VEGF), which is expressed in the epithelium of terminal buds in the human fetal lung during the late pseudoglandular phase but not in the adjacent capillary bed (19). In cultured murine embryonic lung, beads impregnated with VEGF stimulate a local vasculogenic response at the leading edge of branching airways (20). Formation of blood vessels is abnormal and lethal in VEGF-deficient mice (21). In humans, we found that peripheral expansion of the capillary bed is accompanied proximally by continuous coalescence and maturation of veins until mid-gestation. Previous studies have shown that all preacinar intrapulmonary veins have formed by this time (22). After the pseudoglandular phase, the amount of undifferentiated mesenchyme, the source of the earlier endothelial tubes, had decreased in volume. This suggests that endothelial tubes, which form more peripheral veins, may originate from a different source, possibly by angiogenesis from existing veins. Ki67-labeled replicating cells were identified in venules close to the terminal bud, and tenascin was newly expressed in the subepithelium of the terminal buds. Tenascin expression is known to be abnormally high in angiogenic lesions (11), and studies on the control of angiogenic sprouting of endothelial cells in vitro have shown that tenascin is essential for the formation of endothelial tubes (8, 9). In mouse lungs, a shift in the mechanism of vascular expansion corresponds with a decrease in sonic hedgehog expression at the start of the canalicular phase (23). Transgenic overexpression of sonic hedgehog results in the retention of a high mesenchymal rate of cell replication and inhibition of alveolar development (23). In our later fetal and neonatal cases, EphB4 expression was particularly strong in the capillary endothelium. This may be associated with a change from vasculogenic to angiogenic expansion of the vascular bed. In mouse ephrinB1 and B2, ligands induced capillary sprouting in vitro with a similar efficiency to VEGF (14).
Pulmonary venous smooth muscle was derived initially from undifferentiated mesenchymal cells. No muscle cells originated from bronchial smooth muscle, as in the pulmonary arteries (2). There were other differences in vascular smooth muscle development between pulmonary veins and arteries. The veins were at a greater distance from the capillary bed and closer to the hilum when they acquired a smooth muscle coat than were the arteries. Veins acquired α-SM actin positive cells at 56 d gestation, whereas arteries acquired them from 38 d gestation. Expression of mature smooth muscle specific proteins in the veins also lagged behind that seen in the arteries (2). The first structural sign of smooth muscle cell differentiation in the veins was the acquisition of an elongated shape by the mesenchymal cells lying alongside the endothelium. This was associated with expression of α-SM actin. In undifferentiated mesenchymal cells in vitro, elongation is sufficient for the acquisition of differentiated smooth muscle cell characteristics (24, 25). In the systemic circulation, smooth muscle differentiation from mesenchymal precursors is regulated by platelet-derived growth factor-β released from immature endothelial cells (26).
In late gestation, the endothelial cells of peripheral veins expressed α-SM actin, as did the pulmonary arteries (2). Endothelial cells are thought to give rise to smooth muscle cells in the chick aorta and rat lung (27, 28). Using immunohistochemistry, it is impossible to determine whether the venous endothelial cells actually give rise to smooth muscle cells or if this is only a transient change in the phenotype of the endothelial cells that is perhaps regulated by transforming growth factor β expression (17, 29, 30).
Maturation of pulmonary venous smooth muscle was not a continuous process throughout gestation, as it was in pulmonary arterial smooth muscle (2). The expression of SM-1 disappeared transiently from pulmonary veins greater than 70–100 μm in diameter at 98–140 d gestation. This reduction was associated with an increase in lumen size, which was maintained even when SM-1 reappeared. This suggests that developmentally regulated remodeling may reduce resistance to blood flow. By contrast, developing pulmonary arteries remained thick walled around a narrow lumen, and SM-1 expression was constant (2). Thus, regulating expression of specific smooth muscle contractile proteins influenced vascular wall remodeling and presumably pulmonary blood flow in fetal life.
By the end of gestation, all pulmonary venous smooth muscle had acquired several cytoskeletal proteins characteristic of differentiated vascular smooth muscle. The absence of caldesmon expression is noteworthy because it is present in the pulmonary artery smooth muscle from 56 d of gestation (2). Caldesmon is thought to act as a regulator of contraction in vivo, and tethering of actin to myosin by caldesmon in vitro blocks actomyosin interaction and so may inhibit contraction (31). These observations may help explain why porcine pulmonary veins are more responsive to contractile agonists than arteries at birth (1).
In conclusion, it appears that the pulmonary veins arise by vasculogenesis like the pulmonary arteries, but the molecular distinction between presumptive venous and arterial endothelium is still to be determined. The smooth muscle cells are derived entirely from the surrounding mesenchyme. Unlike the pulmonary arteries, there is no initial contribution from the bronchial muscle. In vitro studies indicate that bronchial epithelium can influence vascular development (20). The influence may be less in venous than in arterial development because of a greater diffusion pathway. The veins are further isolated from airway and mesenchymal mediators by the lymphatic channels. The mechanisms regulating pulmonary vascular development demand an in-depth study including the use of genetically modified mice. Such experimental studies will improve our understanding of human development, but experimental studies must themselves be guided by our present knowledge of human pulmonary vascular development.
This study was supported by The British Heart Foundation.
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Abbreviations: alpha smooth muscle specific actin, α-SM actin; cluster of differentiation number CD31, CD31; gamma smooth muscle specific actin, γ-SM actin; Kiel University-raised antibody number 67, Ki67; smooth-muscle–specific myosin heavy chain SM1, SM-1; vascular endothelial growth factor, VEGF.