Published ahead of print on March 6, 2008, doi:10.1165/rcmb.2007-0231OC
© 2008 American Thoracic Society DOI: 10.1165/rcmb.2007-0231OC Spatiotemporal Expression of flk-1 in Pulmonary Epithelial Cells during Lung Development1 University of Giessen Lung Center, Department of Internal Medicine, University Hospital Giessen, Giessen, Germany; 2 Department of Pathology, University of Regensburg, Regensburg, Germany; 3 Department of Anatomy and Histology, Flinders University Adelaide, Adelaide, South Australia; 4 Department of Pathology, University of Dresden, Dresden, Germany Correspondence and requests for reprints should be addressed to Katrin Ahlbrecht, M.D., Medical Clinic 2, University Clinic Giessen, Klinikstrasse 36, 35392 Giessen, Germany. E-mail: Katrin.Ahlbrecht{at}innere.med.uni-giessen.de
Vascular endothelial growth factor-A (VEGF-A) responsive effects mediated via the receptors fetal liver kinase-1 (flk-1) and fms-like tyrosine kinase (flt-1), are key processes of pulmonary vascular development. Flk-1 has been shown to be involved in early embryonic lung epithelial to endothelial crosstalk and branching morphogenesis. Recent reports suggested a role of VEGF-A in lung epithelial cell function. Based on these observations, we hypothesize that epithelial flk-1 has a unique function in pulmonary development. Thus, the aim of this study is to elucidate spatiotemporal expression of flk-1 during lung development with respect to the epithelial system. Embryonic lungs were screened for flk-1 messenger RNA and protein at daily intervals, including postnatal stages. From Embryonic Day (ED) 12.5 through ED 15.5, flk-1 expression was restricted to the early vascular primitive network, while from ED 16.5 on flk-1 was detectable in the epithelial system and persisted there postnatally. At postnatal stages, flk-1 expression was increasingly restricted to individual cells in the alveolar septa. Isolation and in vitro cultivation of alveolar epithelial cells confirmed flk-1 expression and showed VEGF secretion into the supernatant. To our knowledge, this is the first murine study characterizing epithelial flk-1 expression at different stages throughout lung organogenesis until birth and at postnatal stages. To confirm epithelial flk-1 expression, we performed reporter gene analysis of the flk-1 promoter in vivo. Investigations on transgenic mouse strains, containing either a complete or incomplete flk-1 promoter driving expression of the lacZ reporter gene, suggested differential flk-1 regulation in endothelial and epithelial cells.
Key Words: VEGF • VEGF receptor-2 • alveolarization • alveolar epithelial cells • lung development
The development of the vascular system is a key process in lung development. Angiogenesis and vasculogenesis are initiated at early stages of lung development (1, 2). The homodimeric glycoprotein vascular endothelial growth factor (VEGF)-A is a central growth factor regulating physiological and pathological blood vessel formation (3–7). VEGF-A belongs to the VEGF platelet-derived growth factor (PDGF) family of structurally related mitogenic growth factors (7). Alternative splicing leads to four different isoforms consisting of 121, 165, 189, and 206 amino acids in humans (8). The VEGF-A isoforms primarily differ in their capacity to bind to the extracellular matrix and cell surface via heparin-binding domains and thereby generate gradients for angiogenic sprouting (7, 9). VEGF-A is secreted by peripheral respiratory epithelial cells at the tip of developing respiratory tubes (10), creating a VEGF gradient that guides the vascular network to follow and surround the growing bronchi (11). The key receptors mediating VEGF-A signaling in vascular development are fms-like tyrosine kinase (flt-1) or VEGF-receptor 1 and fetal liver kinase-1 (flk-1) or VEGF-receptor 2 (12). Targeted deletion of VEGF-A, flk-1, or flt-1 have demonstrated the importance of these ligand receptor systems in lung development: Loss of a single VEGF-A allele leads to a lethal phenotype due to defective blood vessel formation (13, 14). Analogous effects were observed in homozygous flk-1 mutants (15), while flt-1 deletion resulted in a disorganized vascular network with a lethal phenotype (16). As the majority of developmental studies concerning the VEGF/VEGF-receptor system have focused on vascular development, little is known about VEGF-related tissue interactions between mesenchymal and endodermal cells. Nevertheless, recent studies have suggested that the VEGF-A/VEGF-receptor system mediates a linkage between the processes of branching morphogenesis, development of the vascular system, and morphogenesis of the pulmonary epithelial system. Thus, Del Moral and coworkers have reported increased epithelial branching morphogenesis on whole lung cultures in response to VEGF-A treatment at Embryonic Day (ED) 11 (17). Cell culture experiments showed different effects on isolated endodermal and isolated flk-1–positive mesenchymal cells, respectively (17). Further studies to explore vascular epithelial interactions were done by van Tuyl and colleagues, and suggested that inhibition of vascularization reduced epithelial branching morphogenesis (18). Furthermore, Hara and coworkers demonstrated that expression levels of total pulmonary VEGF-A, flt-1, and flk-1 vary during lung development in rats (19). Data from Marszalek and colleagues revealed highest flk-1 expression in rats at ED 19, which then declined until Postnatal Day (P) 14 (20). Since little is known about extraendothelial flk-1 expression, Gebb and Shannon focused on the spatial patterning of flk-1 expression in the developing rat lung. They postulate that due to spatial juxtaposition of mesenchymal flk-1–positive and epithelial cells, signals from the epithelium may be directing pulmonary vascularization. Furthermore, flk-1 messenger RNA (mRNA) expression was detected postnatally in the alveoli of rats (1). This led to investigations by Raoul and coworkers, which showed flk-1 expression on isolated ED 19 rat alveolar epithelial type two cells. Furthermore, these studies revealed an increase in surfactant protein B transcripts in response to VEGF-A stimulation (21). There are further studies suggesting a functional role of VEGF-A for the epithelial system (22–25), but little is known about the underlying mechanism. We hypothesize that epithelial flk-1 has a unique function in lung development. Even though Hara and colleagues provided evidence for higher mRNA expression of flt-1 as compared with flk-1 in the lung during development, we postulate that VEGF in the lung epithelial system primarily signals through flk-1. Flk-1 has been shown to mediate the majority of the developmental VEGF-A functions (26, 27), in particular the mitogenic and chemotactic effects (28, 29). Nevertheless, flt-1 might be of relevance to the epithelial system. For example, Lassus and coworkers were able to show bronchial epithelial expression of flt-1 in lungs of preterm and term infants. Flt-1 was detectable in alveolar type 2 cells of patients suffering from bronchopulmonary dysplasia and/or primary pulmonary hypertension (30). Further studies by Zhao and colleagues showed epithelial expression of flt-1 and flk-1 in cells of the saccular walls of ED 18.5 mice on mRNA level (31), but nevertheless the majority of VEGF-related effects on the pulmonary epithelial system has been suggested to be mediated by flk-1 (21, 23, 32). Moreover, the studies done by Del Moral and coworkers point out a critical role of VEGF-A signaling through flk-1 in early embryonic lung epithelial and endothelial crosstalk and branching morphogenesis (17). Therefore, in the present study we focused on flk-1 expression. We characterized spatiotemporal expression of flk-1 throughout lung development with special respect to the epithelial system. Widespread epithelial expression of flk-1 in mice commenced at ED 17 and persisted postnatally. Flk-1 phosphorylation in response to VEGF-A treatment demonstrated the functional activity of flk-1 in isolated adult alveolar epithelial cells (AEC). To confirm epithelial flk-1 expression in vivo, investigations on transgenic mouse strains, containing either a complete or incomplete flk-1 promoter driving expression of the lacZ reporter gene were performed. LacZ expression in these mice suggested differential flk-1 regulation in endothelial and epithelial cells.
In Situ Hybridization mRNA in situ hybridization for flk-1 was performed as described previously (33) on frozen sections using 35 S  UTP-labeled antisense mRNA probes. The probes were generated from the murine flk-1 cDNA.
Immunohistochemistry and Immunocytochemistry
Cell Culture and Reagents
Immunoprecipitation
Western Blot Analysis
Quantitative VEGF-A Measurement
Reporter-Gene Expression of flk-1 lacZ Knock-In and flk-1 lacZ5 Transgenic Mice
Flk-1 mRNA Expression during Lung Development To characterize flk-1 mRNA expression during lung development, we analyzed embryonic mouse lungs of C57Bl6 mice at ED 12.5 to ED 18.5 as well as at P 1 and P 14 by radioactive 35 S UTP-labeled mRNA-in situ hybridization. On ED 12.5, flk-1 expression was restricted to the pulmonary vascular network (Figure 1, black and white arrowheads). From ED 16.5 onward, flk-1 mRNA was detected in the epithelial cells of the branching epithelial tubes (Figure 1, black and white arrows), persisted in the primordial bronchi at ED 17.5, and spread to the emerging gas exchange regions at ED 18.5. At P 1 and P 14, flk-1 expression was maintained in the bronchial epithelium, while in the alveolar septa expression was increasingly restricted to individual cells (Figure 1, black and white arrows). Along with maturation of the vascular system, endothelial flk-1 mRNA expression regressed over time (Figure 1, ED 17.5 to P 14).
Flk-1 Protein Expression during Lung Development The localization of flk-1 protein expression was assessed by immunohistochemistry and confocal laser scanning microscopy. Embryonic mouse lungs of C57Bl6 mice at ED 14.5 and ED 18.5 as well as adult mouse lung tissue (12 wk of age) were investigated. Cellular phenotyping for specific endothelial and epithelial cell protein expression in combination with flk-1 detection confirmed the flk-1 mRNA expression pattern derived from the in situ hybridization experiments. During the early embryonic stages (ED 14.5–16.5) flk-1 protein expression was exclusively co-localized with the endothelial marker CD 31 in the vascular networks (Figure 2A), whereas no flk-1 immunoreactivity could be detected in cytokeratin-positive epithelial cells (Figures 2D–2F). From ED 17.5 onward, co-expression of flk-1 and cytokeratin was localized to the bronchial tubules and primordial gas exchange regions (Figures 2G–2I). In adult lung tissue, flk-1 was predominantly expressed in airway epithelial cells (Figures 2J–2L). Staining with respective IgG controls did not show any immunoreactivity.
Flk-1 Expression and Receptor Phosphorylation in Isolated Alveolar Epithelial Cells To elucidate the functional role of flk-1 expression, we isolated alveolar epithelial cells (AEC) from adult C57Bl6 mice. Endothelial cell contamination of the AEC culture was excluded by absence of cells immunoreactive for CD 31 (a specific endothelial cell marker) and revealed a purity of over 95% (data not shown). Immunocytochemistry of AEC, 48 and 72 hours after isolation, revealed co-expression of flk-1 with the epithelial cell markers proSPC and cytokeratin (Figures 3A–3F). Immunoreactivity of respective IgG controls was not detectable. Immunoprecipitation of flk-1 from isolated AEC lysates followed by Western blot analysis for flk-1 further confirmed VEGFR-2 expression in these cells (Figure 3G). To investigate the functional activity of flk-1 in AEC, isolated AEC were treated with 100 ng/ml VEGF-A. Immunoprecipitation of flk-1 from cell lysates and Western blot analysis for phosphotyrosine showed a clear increase of the phosphorylation state of the receptor compared with the nonstimulated control (Figure 3H).
Freshly Isolated Alveolar Epithelial Cells Constitutively Secrete VEGF-A To examine whether AEC also secrete VEGF-A, supernatants were collected from isolated AEC after 72 hours of culture and subjected to VEGF-A ELISA (R&D Systems). The VEGF-A concentration in the supernatant was 128.3 ± 5.6 pg/ml (Figure 3I; mean ± SEM; n = 3; P < 0.01).
Differential Epithelial Reporter Gene Expression by Two Different flk-1 Promoter Constructs In Vivo
The novelty of this study is the characterization of murine flk-1 expression at daily intervals throughout lung development, with a special focus on the epithelial system. The VEGF-A/VEGF-receptor system has been extensively studied during vascular development (6, 8, 26, 37). Targeted deletions of VEGF-A, flk-1, or flt-1 have demonstrated the relevance of this ligand receptor system in vascular development. Homozygous flk-1 or VEGF-A deficiency leads to lack of mature endothelial cells and inhibits blood vessel development (15, 38, 39). Even heterozygous VEGF-A deficiency induces early embryonic lethality due to defective blood vessel formation (13, 14, 39). This indicates an essential minimum of VEGF-A/VEGF-receptor interaction needed for endothelial cell generation and function. Furthermore, deletion of flt-1 resulted in a lethal phenotype at mid-somite stages, due to defective blood vessel formation (16). Though vascularization of the lung has been extensively studied, little is known about interactions between mesenchymal and endodermal cells during lung organogenesis and the specific processes of lung epithelial morphogenesis with respect to the VEGF-A/VEGF-receptor system. However, recent studies postulate that VEGF-A signaling via flk-1 mediates an epithelial to endothelial crosstalk influencing epithelial branching morphogenesis (17). In line with these observations, Gebb and Shannon suggest that signals from the epithelium may be directing pulmonary vascularization (1). Studies by Raoul and coworkers show flk-1 expression in isolated rat fetal alveolar type 2 cells and direct functional effects of VEGF-A on these cells (21). Moreover, further studies have demonstrated effects of VEGF-A on the pulmonary epithelial system (22, 24, 25). Taking these observations into account, we hypothesize that epithelial flk-1 expression has a unique role in pulmonary development and is involved in the VEGF-A related effects on the epithelium. Studies by Lassus and colleagues and by Hara and coworkers have shown epithelial flt-1 expression (30) and increased mRNA expression of flt-1 in whole lung lysates during lung development (19). However, we postulate that VEGF-A signaling in the epithelial system is mediated via flk-1 similar to that observed in the endothelial system (26, 29). Furthermore, Del Moral and colleagues showed that flk-1 is an essential mediator of early embryonic VEGF-A signaling (17). In addition, neutralizing antibodies against flk-1 injected intra-amniotically impaired fetal lung maturation, whereas anti–flt-1 antibodies were ineffective (32). Therefore, in the present study we focused on the spatiotemporal expression pattern of flk-1 at different stages throughout lung development until the postnatal stage and also in adult mice. Using mRNA in situ hybridization, we found flk-1 expression during the early investigated murine stages from ED 12.5 to 15.5 in the primitive vascular network in the mesenchyme but not in the epithelial system, supporting existing literature (27). These findings are also in line with investigations by Gebb and Shannon, which show flk-1 expression at the same stages in rat embryonic lungs in mesenchymal cells in a population that is closely apposed to the developing epithelial system (1). Our investigations on the later stages starting from ED 16.5 revealed the novelty of our study findings in comparison with detailed investigations on rat tissue by Gebb and Shannon or Marszalek and coworkers. Epithelial expression of flk-1 commenced around ED 16.5 and persisted throughout the later stages until birth and postnatally in our study. In contrast, Gebb and Shannon did not show epithelial expression at stages of the pseudoglandular rat lung. However, at P 7, flk-1 expression was found in the alveoli where gas exchange takes place (1). In contrast, Marszalek and colleagues showed that flk-1 expression at P 1 is restricted to the vascular areas (Figure 1B in Ref. 20). These different expression patterns may be due to different stages analyzed, P 7 versus P 1. With respect to our findings, we speculate that these discrepancies may also be due to interspecies differences between rats and mice. However, this assumption has to be further investigated because it is highly speculative. To confirm our mRNA expression data, we localized flk-1 by immunohistofluorescence. These data on protein level revealed co-expression with CD 31 at ED 14.5 (Figure 2A) but not with cytokeratin (Figure 2F). At later stages, however, flk-1 was co-expressed with cytokeratin at ED 18.5 (Figure 2I) and not with CD 31 (Figure 2B). Thus, to our knowledge, these findings are the first murine data describing epithelial expression of flk-1 on both RNA and protein levels, throughout lung development, at birth and postnatally. To gain further evidence to support our hypothesis that flk-1 has a unique role in the pulmonary epithelial system, we demonstrated activation of flk-1 in isolated AEC from adult mice. These findings support the suggestions of Gebb and Shannon that signals from the epithelial system may contribute to pulmonary vascularization. However, our investigation is limited by the lack of functional evidence that epithelial flk-1 mediates reciprocal effects on the endothelial system. Thus, further investigations are needed to show this interaction. Nevertheless, we were able to show that VEGF-A is produced by isolated AEC of adult mice (Figure 3I), which again may contribute to the postulated intercellular crosstalk. The above-mentioned studies by Raoul and coworkers and by Brown and colleagues show epithelial flk-1 expression and effects of VEGF-A on fetal rat and human isolated AEC and lung explants (21, 23). Taking these data into account, the constitutive VEGF-A secretion by adult isolated AEC as shown in our data might contribute to direct effects on the epithelial system. Moreover, investigations by Marszalek and coworkers and by Hara and colleagues concentrated on the expression levels of VEGF, flk-1, and flt-1 during lung development. Marszalek and coworkers showed highest VEGF expression levels in capillaries by morphometric estimation in fetuses. This expression decreased postnatally, with a single exception at P 3. Moreover, they describe that the main source of VEGF is cells in the immediate vicinity of the proliferating vessels. Thus they suggest a paracrine mechanism for the stimulation of angiogenesis. This is supported by their finding that VEGF and flk-1 expression significantly decreased with increasing age of the fetuses. This phenomenon was accompanied by decreasing proliferative activity of the developing endothelial system (20). In contrast, Hara and colleagues showed a permanent increase of expression levels of the receptors flt-1 and flk-1 throughout lung development and until adulthood in rats (19). These differences might be due to the different methods employed. Hara and coworkers measured mRNA as percentage of 18S RNA of total lung lysates, whereas Marszalek and colleagues evaluated morphometric estimations (19, 20). In addition, the use of whole lung total mRNA may explain the higher expression levels of flt-1 compared with flk-1 by Hara and coworkers, due to the possible inclusion of mRNA from circulating cell types (19). Even though both studies do not focus directly on the epithelial system itself, they support our hypothesis in the importance of the VEGF–VEGF-receptor system during lung development and in particular during the later stages, which are crucial for the development of the epithelial system. In line with this suggestion, Ema and colleagues showed that expression of VEGF-A in total lung rises in the last days of murine fetal development, coinciding with the epithelial flk-1 expression observed in our data (40). Again, this data suggests a role of VEGF-A in the critical window of lung epithelial maturation. Thus, due to the novelty of our data, we wanted to confirm the epithelial flk-1 expression by an in vivo reporter gene analysis using the endogenous or a truncated flk-1 promoter. The endogenous flk-1 promoter revealed LacZ staining in the developing vasculature at ED 12.5 (Figure 4A). Analogous results were observed in the animals carrying the truncated promoter at this stage (Figure 4B). In contrast, at P 20 the endogenous flk-1 promoter drove LacZ expression in the epithelial and vascular endothelial system (Figure 4C), whereas the truncated promoter exclusively induced LacZ expression in the vascular endothelial cells (Figure 4D). At 9 months the endogenous flk-1 promoter revealed LacZ staining in the bronchial epithelial system (Figures 4E and 4G). No LacZ staining was observed in the mice carrying the truncated flk-1 promoter at this stage (Figures 4F and 4H). CD 31 immunohistological staining revealed positive signals in pulmonary arteries and parenchyma in both strains at this stage (Figures 4G and 4H). In addition, flk-1 immunohistological staining showed a signal in bronchial epithelial cells (Figure 4F). Expression patterns of the endogenous flk-1 promoter are in line with our results obtained by mRNA in situ hybridization and immunohistological staining for flk-1 in wild-type mice.
In contrast, the truncated flk-1 promoter did not induce epithelial expression of LacZ. Reduced endothelial LacZ staining in later developmental stages was shown in both strains. Due to the different expression patterns of both constructs, we postulated that flk-1 is regulated differently in endothelial and epithelial cells. Mice carrying the truncated flk-1 promoter construct (flklacZ5 mice) contain a transgenic construct consisting of the flk-1 promoter (–640 bp/+299 bp, calculated from the expression initiation site) and an enhancer taken from the first flk-1 intron (+1,677 bp/+3,947 bp) (35). This construct is not sufficient to direct epithelial cell reporter gene expression. Though our investigations show these differences, they do not show the underlying mechanism of regulation. However, the transcription factors scl/tal, GATA, HIF-2 Studies by van Tuyl and coworkers support the hypothesis of an intercellular crosstalk. They suggest that pulmonary vascular development has a rate-limiting role in epithelial branching morphogenesis, and demonstrate that inhibition of vascularization by antisense oligodeoxynucleotides against VEGF reduced epithelial branching morphogenesis (18). Similar effects are shown in the above-mentioned studies by Del Moral and colleagues, which focused on the role of flk-1–mediated VEGF-A signaling pathways during lung morphogenesis. They showed that VEGF-A did not induce branching of ED 14 isolated lung endoderm, but did induce differentiation of flk-1–positive cells in isolated mesenchyme from heterozygous flk-1 LacZ knock-in mice. In contrast, VEGF-A led to increased epithelial branching morphogenesis in ED 11 whole lungs (17). These findings are in line with our data, because they demonstrate the same flk-1 expression pattern in mesenchymal and epithelial cells at the early developmental stages. Moreover, they show a functional effect of VEGF-A on epithelial branching morphogenesis in the absence of epithelial flk-1 expression in early whole lung cultures. Thus, they suggest an indirect effect of VEGF-A on epithelial branching morphogenesis. This suggestion does not contradict our hypothesis, as we observed epithelial flk-1 expression commencing at ED 16.5, whereas they investigated earlier stages. Thus there might be indirect and direct effects regulating lung morphogenesis but in different time frames. Nevertheless, the lack of functional analysis on the direct effect of VEGF-A on the epithelial system is a central limitation of our study. Further studies are clearly needed to show this effect. Though there are studies showing an effect of VEGF-A on the epithelium in vitro (21, 23) and studies showing that pulmonary epithelial cell specific overexpression of VEGF-A in vivo induced a phenotype of airway dilation and epithelial branching defects (22, 25, 43), they do not demonstrate direct effect of flk-1 activation. However, flk-1 knock-down in embryonic lung explants reduced endothelial and epithelial cell proliferation (44). Postnatal flk-1 tyrosine kinase inhibition arrested alveolarization and induced alveolar septal cell apoptosis in rats in vivo, leading to emphysema formation (45, 46). But these investigations are not specific for the epithelial system. Accordingly, targeted tissue-specific deletion in vivo is needed to investigate direct flk-1 effect. Yamamoto and coworkers have shown that deletion of epithelial VEGF-A led to a reduction of flk-1 mRNA in ED 18.5 mice with decreased endothelial cell development. Moreover, saccular walls without capillaries did not give rise to septum formation. Thus they postulate that primary septum formation depends on reciprocal interactions between the respiratory epithelium and the underlying vasculature. Furthermore, the development of the vasculature is dependent on reciprocal epithelium-derived VEGF-A (47). Studies by Zhao and colleagues support these suggestions. They postulate that VEGF co-ordinates epithelial and vascular development. Furthermore, the vasculature is necessary for epithelial proliferation and morphogenesis, but not for cell differentiation (31). Though these data support the hypothesis of the importance of the VEGF-A/VEGF-receptor system for the intercellular crosstalk, they do not focus directly on epithelial flk-1 function. However, our study reveals the first murine data elucidating spatiotemporal flk-1 expression and in particular the expression pattern in the epithelial system daily from ED 16.5 in the late stages of lung development. This is of relevance to establish further functional investigations on the underlying mechanisms of the role of epithelial flk-1 expression in the intercellular crosstalk during lung morphogenesis.
The authors thank Leigh Marsh for critical reading of the manuscript.
Originally Published in Press as DOI: 10.1165/rcmb.2007-0231OC on March 6, 2008
Conflict of Interest Statement: W.S has received/receives grant and contract support by the following companies: In 2005 Lung Rx $120,000, Encysive Received in original form July 18, 2007 Accepted in final form January 24, 2008
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Abstract
CLINICAL RELEVANCE
77,414.23, Schering AG