Published online before print April 29, 2004, doi: 10.1161/01.RES.0000130656.72424.20
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© 2004 American Heart Association, Inc.
Molecular Medicine |
Paired-Related Homeobox Gene Prx1 Is Required for Pulmonary Vascular Development
From Department of Pediatrics (K.I.-S., D.M.M., J.V., P.L.J.) and Department of Medicine (S.A.G., R.N.), University of Colorado Health Sciences Center, Denver, Co; Institute of Biosciences and Technology (J.F.M.), Texas A&M University, Houston, Tex; Center for Lung Biology (T.S.), University of South Alabama, Mobile, Ala; and Cincinnati Children’s Hospital Medical Center (A.A.), Cincinnati, Ohio.
Correspondence to Peter Lloyd Jones, PhD, University of Colorado Health Sciences Center, Department of Pediatrics, 4200 East 9th Avenue, B-131, Denver, CO 80262. E-mail Peter.Jones{at}UCHSC.edu
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Abstract |
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Herein, we show that the paired-related homeobox gene, Prx1, is required for lung vascularization. Initial studies revealed that Prx1 localizes to differentiating endothelial cells (ECs) within the fetal lung mesenchyme, and later within ECs forming vascular networks. To begin to determine whether Prx1 promotes EC differentiation, fetal lung mesodermal cells were transfected with full-length Prx1 cDNA, resulting in their morphological transformation to an endothelial-like phenotype. In addition, Prx1-transformed cells acquired the ability to form vascular networks on Matrigel. Thus, Prx1 might function by promoting pulmonary EC differentiation within the fetal lung mesoderm, as well as their subsequent incorporation into vascular networks. To understand how Prx1 participates in network formation, we focused on tenascin-C (TN-C), an extracellular matrix (ECM) protein induced by Prx1. Immunocytochemistry/histochemistry showed that a TN-C–rich ECM surrounds Prx1-positive pulmonary vascular networks both in vivo and in tissue culture. Furthermore, antibody-blocking studies showed that TN-C is required for Prx1-dependent vascular network formation on Matrigel. Finally, to determine whether these results were relevant in vivo, we examined newborn Prx1–wild-type (+/+) and Prx1-null (–/–) mice and showed that Prx1 is critical for expression of TN-C and lung vascularization. These studies provide a framework to understand how Prx1 controls EC differentiation and their subsequent incorporation into functional pulmonary vascular networks.
Key Words: lung • vascular development • homeobox gene • extracellular matrix
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Introduction |
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To ensure efficient gas exchange in the postnatal lung, fetal pulmonary vessels form in parallel to the developing airways and as capillary networks surrounding the alveoli. Pulmonary endothelial cells (ECs) can be detected as early as embryonic day (E) 9.5 to 10 in the developing mouse lung.1–6 Although multiple mechanisms lead to colonization of the lung by ECs, it is generally accepted that vasculogenesis (ie, differentiation of ECs from the uncommitted distal mesoderm and organization of endothelial progenitors into a primitive vascular plexus) and angiogenesis (ie, sprouting of pulmonary vessels from pre-existing central trunk vessels) represent principal processes that give rise to the lung vasculature.2–4 By E14, differentiating proximal and distal pulmonary ECs form a 3-dimensional array of arteries, veins, and capillaries, which become invested by surrounding mesenchymal cells that have the potential to give rise to pericyte, smooth muscle, and adventitial cell layers.5 Concurrently, a complex extracellular matrix (ECM) forms around the vessel wall, providing structural and functional cues to adjacent cells.1 Adding to the complexity of generating a vascular network, the lung epithelium controls the development of the adjacent pulmonary vasculature and vice versa.6–8 Thus, gene expression within the lung needs to be precisely coordinated in time and space. Because homeobox genes encode transcription factors that dictate tissue patterning and morphogenesis throughout development,9 it is likely that these genes play a role in controlling pulmonary vascular development.
Prx1 (also known as Mhox10) encodes a divergent, paired-related homeobox gene. In the developing chick cardiovascular system, Prx1 is first evident in the endocardial cushions and valves, the epicardium, and in the walls of the great arteries and veins.11 In the embryonic pulmonary vasculature, Prx1 appears in the primary vessel wall of muscularized vessels from early stages onward, and later in the adventitial and medial cell layers, indicating that this homeobox gene may promote differentiation and patterning of pulmonary vessels. In support of this, Prx1-null (–/–) mice exhibit a variety of vascular anomalies, including abnormal positioning and awkward curvature of the aortic arch and an elongated ductus arteriosus.11 Furthermore, Prx1–/– mice are cyanotic and die soon after birth from respiratory distress, which has been suggested to arise from palatal defects.12 Alternatively, it is possible that Prx1 affects respiratory function, because it is required for 1 or more stages of lung vascular development, yet this idea has not been explored.
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Materials and Methods |
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Tissues
Lung tissues either were paraformaldehyde-fixed, before sectioning, or were used to extract total RNA.
Cell Culture
Cells were maintained at 37°C in 5% CO2 in DMEM supplemented with 10% fetal bovine serum, antibiotics, and amino acids.
Generation of CVM-HA and Prx1-Transformed Cell Lines
Cells were cotransfected either with pPrx1-HA or with pCMV-HA plus pMSCVpuro vector. Puromycin-resistant colonies were isolated as clones after 1 week.
RT-PCR
cDNA was synthesized using Superscript II RT. Primer pairs were designed to detect Prx1, GAPDH, tenascin-C (TN-C), Flk-1, VCAM-1, and 18S rRNA.
Immunohistochemistry/Cytochemistry
Prx1, eNOS, TN-C, or von Willebrand factor (vWF) antibodies were applied to sections. Antigen detection was performed using an ABC kit. Cells on Matrigel were fixed and sequentially incubated with either a control IgG or an anti–TN-C IgG, and then with FITC-conjugated or Texas Red-conjugated antibodies.
Western Immunoblotting
Membranes were incubated with either an anti–TN-C antibody or an anti–PECAM-1 antibody, followed by a horseradish peroxidase (HRP)-conjugated secondary antibody. For Prx1-HA detection, blots were incubated with an HRP-conjugated anti-HA antibody. Proteins were visualized via ECL.
Fluorescence-Activated Cell Sorting
EC antibodies used for fluorescence-activated cell sorting (FACs) analysis included anti–PECAM-1, anti-vWF, and anti–VE-cadherin. Secondary antibodies were FITC-conjugated. Normal IgG and appropriate secondary antibodies were used as negative controls.
Matrigel Assays
To assess network formation, cells were seeded onto Matrigel. For function-blocking experiments, cells were preincubated with rabbit polyclonal anti–TN-C antibody or with an IgG control.
Transmission EM
Semi-thin sections were stained with toluidine blue for light microscopy, whereas ultra-thin sections were stained with uranyl acetate and lead citrate.
TN-C Gene Promoter-Luciferase Reporter Assays
For luciferase assays, RFL-6 cells were transfected with pTN7 in the presence of 2 µg pCMV–ß-galactosidase and 2 µg pCMV-HA or 2 µg of pPrx1-HA. After normalizing for ß-galactosidase activity, luciferase assays were performed.
EMSAs
Nuclear extracts were prepared from cells transfected with either pPrx1-HA or pCMV-HA plasmid, together with pCMV–ß-gal plasmid. TN-C gene promoter wild-type and mutated probes were radiolabeled with 
-[32P] dATP. EMSAs and supershift assays were then performed.
Gene Microarrays
Total RNA was extracted from newborn Prx1 +/+ and –/– mouse lung tissue. Gene expression profiles were compared using an Affymetrix mouse expression chip 430A (MOE430A).
Statistics
Statistical assessments were made using an unpaired Student t test. P<0.05 was considered statistically significant. Values are represented as means±SEM.
An expanded Materials and Methods section can be found in the online data supplement available at http://circres.ahajournals.org.
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Results |
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Prx1 Expression in the Developing Lung Vasculature
To begin to define a function for Prx1, we assessed the mRNA expression profile for this gene in the developing lung from E13 through postnatal day 1 (p1). RT-PCR assays demonstrated that Prx1 mRNA is expressed at all stages of lung development examined, albeit at different levels (Figure 1A). Next, to determine which cell types express Prx1, immunohistochemistry experiments were performed. At E12, Prx1 was expressed in cell aggregates located within the distal lung mesenchyme (Figure 1B). Immunostaining of serial sections for eNOS suggested that Prx1-positive cells were endothelial in nature (Figure 1B). By E17, Prx1 expression became more widespread and was evident throughout the lung mesenchyme, in the smooth muscle layer beneath the branching airways, as well as in and around eNOS-positive capillaries (Figure 1B). In contrast, control IgG produced no immunoreactivity in either E12 or E17 lung tissue (data not shown). Collectively, these data indicate that Prx1 might support EC differentiation within the early distal lung mesenchyme, whereas at later stages of development, it may promote incorporation of ECs into functional vascular networks.
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Prx1 Promotes Morphological and Functional Differentiation of Fetal Lung Mesodermal Cells
To determine whether Prx1 has the potential to control lung EC differentiation, we made use of RFL-6 cells, originally isolated from E18 fetal rat lung mesoderm. FACs analysis showed that these cells express a number of EC markers including PECAM-1, vWF, and VE-cadherin (Figure 2A). In addition, RFL-6 cells express mesenchymal markers, including -actin and vimentin, as well as low levels of Prx1 mRNA and protein (data not shown). Despite these characteristics, RFL-6 remained morphologically primitive, resembling fibroblasts (Figure 2B; left panel). To evaluate whether increasing levels of Prx1 expression could affect this phenotype, we cotransfected RFL-6 cells with either an HA epitope-tagged Prx1-expression vector (ie, pPrx1-HA), or a CMV-HA control vector (ie, pCMV-HA), together with an expression vector encoding resistance to puromycin. After puromycin selection, 30 individual cell clones were isolated from pPrx1-HA-transfected cultures, 50% of which acquired an endothelial-like phenotype, characterized by a cobblestone morphology in monolayer culture (Figure 2B; middle panel). In contrast, all stable clones generated by transfection with pCMV-HA retained a fibroblast-like phenotype (Figure 2B; right panel), identical to parental RFL-6 cells. Thus, it is the levels of Prx1 in RFL-6 cells that appear to be critical for promoting their morphological differentiation to a more endothelial-like phenotype.
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To determine whether Prx1-dependent alterations in RFL-6 morphology were accompanied by changes in cell function, we focused on 2 stable cell lines, designated Prx1-HA (pPrx1-HA-transfected) and CMV-HA (pCMV-HA-transfected control). Western immunoblotting using total cell lysates and an anti-HA antibody showed that Prx1-HA cells expressed an expected 
30-kDa Prx1-HA fusion protein (Figure 2C), whereas no equivalent HA-positive band was detected in control CMV-HA cells (Figure 2C). FACs analysis was then used to evaluate EC markers in Prx1-HA cells, and this showed that the expression levels of PECAM-1, vWF, and VE-cadherin were the same in Prx1-HA, CMV-HA, and parental RFL-6 cells (data not shown). Thus, Prx1-dependent changes in RFL-6 cell morphology do not appear to depend on alterations in expression of these particular EC markers. Furthermore, Western immunoblotting for PECAM-1 confirmed that expression of this endothelial cell marker was unchanged after stable overexpression of Prx1-HA (Figure 2D). Even so, it is possible that the basal levels of Prx1 observed in RFL-6 cells contribute to the expression of this and other EC-associated proteins.
Having shown that Prx1 overexpression promotes morphological differentiation of RFL-6 cells, we next compared the ability of CMV-HA and Prx1-HA cells to form vascular networks on exogenous basement membrane proteins (ie, Matrigel). After plating onto Matrigel, CMV-HA cells interacted with one another to form aggregates, yet these cells were unable to generate networks (Figure 3A, upper panels). Parental RFL-6 cells behaved in an identical manner (data not shown). In contrast, both Prx1-HA (Figure 3A, lower panels) and adult pulmonary artery ECs (used as a positive control; data not shown) not only aggregated on Matrigel but also went on to form extensive networks (Figure 3A). Furthermore, light and electron microscopy revealed that Prx1-HA cell networks were 3-dimensional and polarized in nature, evidenced by the presence of a central lumen (Figure 3B), and the formation of intercellular junctions (Figure 3C). Collectively, these results indicate that Prx1 promotes both morphological and functional differentiation of pulmonary ECs, at least in tissue culture.
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To ensure that RFL-6 lung mesodermal cells are not unique in their ability to respond to Prx1, we repeated key experiments using MFLM-4 cells. This cell line, originally isolated from E14.5 mouse lung mesoderm, has already been described as a model for lung vasculogenesis and angiogenesis.13,14 As with RFL-6 cells, MFLM-4 cells already express certain EC markers, but they too remain morphologically primitive.13,14 Identical to RFL-6 cells, MFLM-4 cells stably transfected with the pPrx1-HA expression vector, lost their fibroblastic appearance and gained a more endothelial-like phenotype, whereas control-transfected cells remained unchanged (Figure 4A). Fluorescence microscopy showed that when compared with CMV-HA-transfected cells, Prx1-HA-transformed MFLM-4 cells express Prx1-HA in their nuclei, as determined by costaining for DAPI and HA (Figure 4B). Next, we determined whether Prx1-transformed MFLM-4 cells are capable of forming vascular networks on Matrigel. Whereas CMV-HA expressing cells only formed aggregates on Matrigel (Figure 4C), Prx1-transformed MFLM-4 cells rapidly assimilated into capillary networks within 2 hours (Figure 4C). Therefore, morphological and functional differentiation of fetal lung mesodermal cells by Prx1 is not unique to RFL-6 cells.
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Prx1-Dependent Vascular Network Formation Relies on Tenascin-C
To define a potential mechanism whereby Prx1 promotes vascular network formation, we focused on TN-C. This ECM glycoprotein was chosen for a number of reasons: (1) Prx1 colocalizes with TN-C in remodeling pulmonary arteries;15,16 (2) Prx1 activates TN-C gene transcription in vascular smooth muscle cells and embryonic fibroblasts;16,17 (3) studies with TN-C heterozygous and null mice show that wild-type levels of TN-C are essential for normal lung development;18 (4) studies examining TN-C–null mice show that this protein is essential for tumor angiogenesis via its ability to regulate VEGF expression;19 and (5) TN-C directly controls a number of processes that are crucial for vascular network formation, including the promotion of EC sprouting and migration.20–23
Consistent with the idea that TN-C participates in lung vascular network formation, immunostaining experiments using E17 tissue showed that in addition to being expressed in the fetal lung mesenchyme surrounding the branching airways, TN-C was deposited around eNOS-positive pulmonary vessels (Figure 5A). Because Prx1 is also expressed within eNOS-positive vascular networks (Figure 1B), we determined whether Prx1 is able to support TN-C gene transcription. Accordingly, RFL-6 cells were cotransfected with either pCMV-HA or pPrx1-HA expression vectors, together with a TN-C gene promoter-luciferase reporter construct, and a ß-galactosidase expression vector, which showed that overexpression of Prx1 significantly increased TN-C gene promoter activity (Figure 5B).
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Our next question was whether Prx1 promotes TN-C gene transcription by directly interacting with the TN-C gene promoter. To answer this, we performed EMSAs using a radiolabeled 122-bp TN-C gene promoter element containing a homeodomain-binding site (HBS), a sequence which not only has the potential to interact with Prx1 but also is essential for TN-C gene transcription in vascular smooth muscle cells.16 Nuclear extracts derived from Prx1-HA-transfected RFL-6 cells incubated with a radiolabeled wild-type TN-C promoter probe formed 1 major DNA:protein complex in EMSAs (Figure 5C), whereas incubation of nuclear extracts from CMV-HA transfected cells probe failed to support complex formation (Figure 5C). Similarly, DNA:protein complex formation did not occur when nuclear extracts from Prx1-HA-transfected cells were incubated with a TN-C promoter probe containing a mutated HBS (Figure 5C). To determine whether Prx1 protein was present in the DNA:protein complex, wild-type probe and Prx1-HA nuclear extracts were incubated with an antibody that recognizes Prx1. Incubation with the Prx1 antibody super-shifted the DNA:protein complex, whereas incubation with a control IgG had little effect (Figure 5C). In addition, incubation with a cold competitor probe also inhibited complex formation, whereas a mutated probe had no such effect (Figure 5C).
Having shown that Prx1-expressing vascular networks in the developing lung are surrounded by a TN-C–rich ECM, and that Prx1 is capable of driving TN-C gene transcription, we went on to evaluate whether vascular network formation by Prx1-HA cells relies on interactions with TN-C. Consistent with this idea, laser confocal microscopy revealed that after 1 hour, Prx1-HA cells cultured on Matrigel synthesize significantly greater amounts of TN-C protein when compared with CMV-HA cells (Figure 6A). In addition, immunostaining of Prx1-HA cells maintained on Matrigel for 3 hours showed that developing vascular networks subsequently become enveloped by extracellular TN-C protein (Figure 6B). Immunostaining with a control IgG produced no comparable signal (Figure 6B).
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To assess whether extracellular TN-C plays an active role in vascular network formation, Prx1-HA cells plated on Matrigel were treated with either a function-blocking antibody against TN-C or an isotype-matched control IgG. Within 3 hours of plating, capillary network formation by Prx1-HA cells was markedly attenuated by the anti–TN-C IgG, but not by the control IgG (Figure 6C, upper panels). Because TN-C may support vascular network formation by promoting pulmonary EC spreading, we evaluated whether TN-C–dependent network formation by Prx1-transformed ECs relies on this function. Indeed, a closer examination of Prx1-HA cell cultures plated on Matrigel, either in the presence of a control IgG or with the function-blocking TN-C IgG, indicated that TN-C is required for EC cell spreading on Matrigel. Whereas control IgG-treated networks were composed of interconnecting ECs that possessed well-developed lamellipodia, blockade of cellular interactions with TN-C inhibited the formation of these structures (Figure 6C, lower panels). It is important to note that blockade of Prx1-HA cell interactions with TN-C did not lead to their morphological reversion to the parental RFL-6 phenotype. In addition, cultivating RFL-6 cells on Matrigel supplemented with exogenous TN-C protein did not result in vascular network formation (data not shown). Thus, although TN-C appears to be essential for vascular network formation by Prx1-transformed RFL-6 cells, it does not appear to be involved in the initial conversion of parental RFL-6 cells to a more endothelial-like phenotype. These experiments reinforce the idea that Prx1 might play a dual role in pulmonary vascular development, ie, it promotes EC differentiation and vascular network formation via induction of TN-C.
Prx1 Is Required for TN-C Expression and Vascular Network Formation In Vivo
To establish whether our tissue culture studies were relevant to pulmonary vascular development in vivo, we made use of Prx1–/– mice. As reported,24 these animals were smaller and cyanotic when compared with their wild-type littermates (Figure 7A); their breathing was exaggerated, and they died soon after birth from respiratory distress, which has been suggested to result from cleft palate.12 To evaluate whether Prx1–/– mice exhibit alterations in TN-C expression and lung vascular development, we performed RT-PCR and immunohistochemical assays to assess the levels of TN-C mRNA and protein expression. In keeping with a role for Prx1 in regulating TN-C–dependent vascular network formation, newborn Prx1–/– mouse lungs expressed considerably less TN-C than wild-type mice (Figure 7B and 7C). Immunostaining studies also showed that Prx1–/– mice contained markedly reduced numbers of vWF-positive blood vessels (Figure 7C), which were situated within hypoplastic lung tissue (Figure 7C).
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To determine whether expression of other EC-specific genes, besides vWF, was affected in Prx1–/– mice, gene microarray analyses were performed. These studies indicated that expression of Flk-1 and VCAM-1 is also significantly suppressed in Prx1–/– mouse lungs (data not shown). To evaluate these findings further, we performed semiquantitative RT-PCR assays and confirmed that the steady-state levels of Flk-1 and VCAM-1 mRNAs are indeed reduced in Prx1–/– mouse lungs (Figure 7B).
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Discussion |
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We have shown that Prx1 promotes morphological differentiation of fetal pulmonary EC precursors and that these cells also acquire the ability to form vascular networks in a manner that relies on TN-C, a known Prx1 target. Thus, we hypothesize that Prx1 may play at least 2 roles in pulmonary vascular development (Figure 8). Consistent with this, newborn Prx1–/– mice, which are cyanotic and die soon after birth from respiratory distress, possessed severe lung defects, characterized by decreased expression of TN-C and a paucity of pulmonary vessels.
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Vasculogenesis occurs as early as E9.5 to 10 in the distal mouse lung.3 Because Prx1 is able to transform cells derived from the lung mesoderm into a more endothelial-like phenotype, and because the number of ECs is reduced in the mesenchyme of Prx1–/– mouse lungs, we suggest that Prx1 might be involved in the selection and/or differentiation of mesodermal precursors that give rise to differentiated ECs (Figure 8). Recent evidence indicates that the divergent homeobox gene, Hex, promotes vasculogenesis. Hex is expressed in a range of multipotent progenitor lines and is generally down-regulated during terminal differentiation.25 In the mouse embryo, Hex first materializes in the primitive endoderm of the implanting blastocyst. Subsequently, it appears in the foregut endoderm and within the mesoderm, where it is transiently expressed in the nascent blood islands of the yolk sac, and later in the embryonic angioblasts and endocardium.26 In the embryonic mouse lung, the highest levels of Hex are observed in the distal lung mesenchyme, a principal site for vasculogenesis.27 Thus, Hex appears to be well-positioned to drive vasculogenesis. In fact, a comparison with flk-1 expression indicates that Hex represents an early marker of EC precursors.26 Unlike flk-1, however, expression of Hex is transient, being downregulated once EC differentiation commences. Furthermore, whereas flk-1–null mice fail to generate a vascular network and lack hematopoietic progenitor cells,28 disruption of Hex expression has no effect on either of these processes. All the same, the overlap in flk-1 and Hex expression implicates this homeobox protein in the initial stages of EC selection and/or differentiation within the mesoderm. This idea is substantiated by the finding that expression of the Hex homologue, hHex in zebra fish, leads to early and ectopic expression of flk-1.29 Similar approaches involving Prx1 will be needed to determine whether this gene truly plays a role in promoting EC differentiation.
Angiogenesis also contributes to pulmonary vascular development.1,30 As is the case with the later stages of vasculogenesis, vascular network formation during angiogenesis relies on changes in EC shape and migration.31 Even though little is known about how homeobox genes control either of these processes in the lung, our tissue culture studies indicate that TN-C, a gene regulated by Prx1 in the lung, may be involved (Figure 8). A number of possibilities exist to explain how TN-C participates in vascular network formation. First, we showed that blockade of cellular interactions with TN-C attenuates EC spreading on Matrigel, an essential event for network formation. These findings are consistent with previous studies showing that TN-C promotes both EC shape changes22,23 and migration.20 In addition, expression of vascular endothelial growth factor (VEGF) and tumor angiogenesis are suppressed in TN-C–/– mice when compared with wild-type animals.19 Therefore, TN-C might promote lung vascularization through induction of VEGF. If this is so, then it might be expected that TN-C–/– mice would also possess lung defects. Indeed, a comparison of TN-C+/+, TN-C+/–, and TN-C–/– mouse lungs has shown that TN-C influences fetal lung branching morphogenesis.18 Because fetal lung branching relies on the adjacent vasculature,32 it is conceivable that lung hypoplasia observed in TN-C–/– mice arises from defects in the pulmonary vasculature.
This study shows that vascular development is compromised in Prx1–/– mouse lungs. In addition, by demonstrating that Prx1 is required for TN-C expression in the newborn lung, and that Prx1 and TN-C are essential for network formation in tissue culture, it is reasonable to hypothesize that the previously observed vascular patterning defects observed in Prx1–/– mice,12 may arise because of decreased TN-C production. It is unlikely, however, that TN-C is the only Prx1-regulated gene required for lung vascular development. The notion that homeobox genes simultaneously control the expression of multiple genes required for vascular morphogenesis has been established in other tissues. For example, HOXD3 promotes the invasive phenotype of human umbilical vein and dermal microvascular ECs by stimulating expression of both urokinase plasminogen activator and integrin vß3,33 recognized components of the angiogenic cascade. As well, Hoxb-7, which is expressed in E11.5 lung,34 upregulates a variety of angiogenic stimuli, including FGF-2, VEGF, and MMP-9.35
Immunohistochemistry experiments using Prx1+/+ and Prx1–/– lung tissue indicated that Prx1 is not only essential for lung vascular development but also controls tissue interactions. For example, although Prx1 is expressed in the lung mesenchyme, Prx1–/– mice possess hypoplastic lungs. Thus, mesenchymal Prx1 likely controls the behavior of the adjacent epithelium. Alternatively, because pulmonary vessels are deemed to be critical for normal alveolar development,32 it is plausible that alveolar defects in Prx1–/– mice arise because of a lack of pulmonary vessels. One way in to which Prx1 might control lung tissue interactions is through induction of TN-C, given that this Prx1 target gene is required for lung branching morphogenesis.18,36
Although Prx1 appears to be essential for TN-C expression in cultured cells, our studies with Prx1–/– mice demonstrate that residual TN-C is present in these animals. This may reflect functional compensation by Prx2. For example, it is already appreciated that whereas the Prx1–/– mouse exhibits vascular and skeletal anomalies, these defects are exaggerated in the Prx1/Prx2 double knockout mouse.37 Because Prx2 also transactivates the TN-C gene promoter,16 it is possible that residual TN-C in Prx1–/– mice depends on Prx2. This observation raises the question as to whether the amount of TN-C is important for its function. In fact, recent studies comparing TN-C+/+, TN-C+/–, and TN-C–/– lungs has shown that TN-C+/– mice have a lung branching defect that represents an intermediate phenotype between normal lungs and hypoplasia observed in TN-C–/– mice.18
Because Prx1 is essential for normal lung development, it will be important to determine how it is regulated. Recently, we showed that the nonreceptor tyrosine kinase, focal adhesion kinase (FAK), is essential for expression of Prx1 and TN-C, both in cultured embryonic fibroblasts and during embryogenesis.17 Furthermore, FAK-dependent embryonic fibroblast spreading and migration relies on the ability of Prx1 to stimulate TN-C expression.17 Because vascular network formation fails to occur in FAK–/– mice,38 it is plausible that FAK controls Prx1-dependent vascular network formation by stimulating TN-C production, which would subsequently support EC spreading and migration. Work is underway to determine how Prx1 is regulated in the pulmonary vasculature.
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Acknowledgments |
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P.L.J. is supported by NIH/NHLBI grants 1 RO1 HL68798-01 and NIH 2 P50 HL57144-06, and by the 2003 American Physiological Society Giles Filley Memorial Award. K.I.-S. was supported, in part, by NIH training grant 5 T32 HL07171. We appreciate the excellent technical help of Dr Ray Hester, Tray Hamil (University of South Alabama, Mobile), Dr Nihal Kaplan-Albuquerque and Lila Sisbarro (UCHSC, Denver, Col).
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Footnotes |
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Original received December 1, 2003; revision received April 20, 2004; accepted April 22, 2004.
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References |
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