Regulation of embryonic lung vascular development by vascular endothelial growth factor receptors Flk-1 and Flt-1.код для вставкиСкачать
Growth & Development THE ANATOMICAL RECORD 290:958–973 (2007) Regulation of Embryonic Lung Vascular Development by Vascular Endothelial Growth Factor Receptors, Flk-1 and Flt-1 YASUTOSHI YAMAMOTO,1,2 ISAO SHIRAISHI,2 PING DAI,1 KENJI HAMAOKA,2 AND TETSURO TAKAMATSU1* 1 Department of Pathology and Cell Regulation, Kyoto Prefectural University of Medicine Graduate School of Medical Science, Kyoto, Japan 2 Department of Pediatric Cardiology and Nephrology, Kyoto Prefectural University of Medicine Graduate School of Medical Science, Kyoto, Japan ABSTRACT The biological effects of vascular endothelial growth factor A (VEGFA) are mediated by fetal liver kinase-1 (Flk-1) and fms-like tyrosine kinase-1 (Flt-1). In lung tissue, VEGF-A is diffusely expressed throughout the embryonic stages, whereas the development of vascular endothelial cells is not uniform. Noting the signaling properties of the two receptors, we hypothesized that Flk-1 and Flt-1 regulate the embryonic development of lung vasculature. We herein show the spatiotemporal expression and experimental inhibition of Flk-1 and Flt-1 of embryonic mouse lung tissue. When Flk-1 was predominantly expressed (embryonic day [E] 9.5– E13.5), then vascular endothelial cells actively proliferated. When Flt-1 was enhanced (E14.5–E16.5), these cells less actively proliferated, thereby constituting organized networks. The treatment of cultured lung buds (E11.5) with antisense oligonucleotides complementary to Flk-1 inhibited branching of capillaries and proliferation of endothelial cells. In contrast, the inhibition of Flt-1 promoted the branching of capillaries and enhanced proliferation of endothelial cells. Of interest, inhibition of Flt-1 promoted Flk-1 expression. These results suggest that the two VEGF-A receptors regulate pulmonary vascular development by modulating the VEGF-A signaling. Anat Rec, 290:958–973, 2007. Ó 2007 Wiley-Liss, Inc. Key words: vascular endothelial growth factor receptors (VEGFRs); Flk-1; Flt-1; angiogenesis; vasculogenesis; lung; mouse; embryo Mouse lung development starts at embryonic day (E) 9.5, when the endodermal foregut evaginates into the surrounding splanchnic mesoderm. Subsequently, the two lung buds elongate and repeat branching in a highly reproducible manner, thus giving rise to the bronchial tree (Ten Have-Opbroek, 1991; Hogan, 1999). Fibroblast growth factor 10 (FGF10) and bone morphogenetic protein 4 (BMP4) and the regulation of these molecules at the transcription level by the sonic hedgehog (shh), patched (ptc), smoothened (smo), and Gli pathways have been clariﬁed (Warburton et al., 2000). On the other hand, abundant mesodermal mesenchymal cells, which surround the developing bronchial tree, differentiate into Ó 2007 WILEY-LISS, INC. This article contains supplementary material available via the internet at http://www.interscience.wiley.com/jpages/19328486/suppmat. Grant sponsor: Japan Society for the Promotion of Science. *Correspondence to: Tetsuro Takamatsu, Department of Pathology and Cell Regulation, Kyoto Prefectural University of Medicine Graduate School of Medical Science, KawaramachiHirokoji, Kamigyo, Kyoto, Japan 602-8566. E-mail: email@example.com Received 3 May 2006; Accepted 14 May 2007 DOI 10.1002/ar.20564 Published online in Wiley InterScience (www.interscience.wiley. com). VEGF RECEPTORS DURING LUNG DEVELOPMENT vascular endothelial cells in alignment but they are signiﬁcantly distant from the developing bronchial airway (Gebb and Shannon, 2000; Schachtner et al., 2000), thus eventually forming lung vasculature. Although the lung vasculature and the bronchial tree develop simultaneously during embryogenesis, very little is known regarding the development of the lung vasculature (Stenmark and Gebb, 2003). Vascular endothelial growth factor-A (VEGF-A) plays a critical role in vasculogenesis and angiogenesis by regulating vascular endothelial cell proliferation and differentiation (Carmeliet et al., 1996; Ferrara et al., 1996). VEGF-A also plays an important role during the pulmonary vascular development, and its splice variants are strictly regulated (Ng et al., 2001). The biological effects of VEGF-A are mediated by two receptor tyrosine kinases, fms-like tyrosine kinase-1 (Flt-1) and fetal liver kinase-1 (Flk-1; Shibuya et al., 1990; de Vries et al., 1992; Terman et al., 1992). These differ considerably regarding their signaling properties, such as the afﬁnity for VEGFA and kinase activity (Waltenberger et al., 1994; Seetharam et al., 1995; Sawano et al., 1996). Flk-1 null mutants lack mature endothelial and hematopoietic cells (Shalaby et al., 1995). In contrast, mice lacking Flt-1 demonstrate a disorganized vasculature and an increased number of endothelial progenitor cells (Fong et al., 1995). These ﬁndings indicate that Flk-1 positively regulates the VEGF-A signals, whereas Flt-1 negatively regulates the VEGF-A signals (Park et al., 1994). Although VEGF-A is predominantly expressed by epithelial cells in the lung (Acarregui et al., 1999; Gebb and Shannon, 2000; Ng et al., 2001), vascular endothelial cells emerge and differentiate in areas signiﬁcantly distant from the developing bronchial airway and acini (Schachtner et al., 2000). Furthermore, Flk-1–positive cells maintain a distinct spatial relationship relative to the branching epithelium and they are only present within a characteristic proximity of the epithelium in the pseudoglandular stage (within approximately three to six cell diameters; Gebb and Shannon, 2000). Factor(s) other than VEGF-A itself might be involved in regulating vascular endothelial cell proliferation and differentiation. Because Flk-1 and Flt-1 transduce the VEGF-A signals in different ways, we hypothesized that the coordinated expression of Flk-1 and Flt-1 may thus be an essential process in the constitution of the highly organized lung capillary networks. By means of morphometry with confocal microscopy, mRNA quantiﬁcation by real-time polymerase chain reaction(PCR), 5-bromo-20 -deoxyuridine (BrdU) incorporation into developing vascular endothelial cells, and target inhibition of mRNA or proteins, we herein demonstrate that Flk-1 and Flt-1 regulate vascular endothelial cell proliferation, differentiation, and the subsequent development of the pulmonary vasculature. When Flk-1 expression is enhanced, the vascular endothelial cells actively proliferated and differentiated. When Flt-1 was dominant, vascular endothelial cells less actively proliferated and differentiated. The treatment of cultured lung buds (E11.5) with antisense oligodeoxynucleotides (AS-ODNs) complementary to Flk-1 thus resulted in an insufﬁcient branching of the capillaries, and an impaired proliferation, while also promoting the apoptosis of endothelial cells and decreased the ephrin-B2–positive arterial cell lineage. In contrast, the inhibition of Flt-1 with AS-ODNs 959 promoted the branching of the capillaries, an enhanced proliferation of endothelial cells, and an increased ephrin-B2–positive arterial cell lineage. Of interest, the inhibition of Flt-1 in the lung buds promoted Flk-1 expression; however, the inhibition of Flk-1 did not enhance Flt-1 expression. These results provide the ﬁrst demonstration that the differential expression of VEGF-A receptors Flk-1 and Flt-1 control the development of the pulmonary vasculature. MATERIALS AND METHODS Animal Preparation Lungs were taken from the embryos (E9.5–E18.5) of pregnant ICR mice. Fetal lung buds taken at E11.5 from pregnant ICR mice were obtained for an in vitro functional study using either AS-ODNs or speciﬁc antibodies to Flk-1 and Flt-1. All animal care and all experimental procedures were approved by the Committee for Animal Research, Kyoto Prefectural University of Medicine. Immunohistochemistry and F-Actin Staining Whole-mount lung immunoﬂuorescence studies using an anti–platelet endothelial cell adhesion molecule-1 (anti– PECAM-1) antibody and subsequent sectioning were performed as follows. To elucidate the spatial relationship between vascular endothelial cells and pulmonary epithelial cells, F-actin staining along with PECAM-1 staining was performed: lungs were ﬁxed with 4.0% paraformaldehyde (PFA), and permeabilized with 0.2% Triton X-100 in phosphate-buffered saline (PBS). For F-actin staining, lung tissues were treated with Texas Red-X phalloidin (1:50; Molecular Probes). Thereafter, the tissue specimens were incubated with anti–PECAM-1 antibody (1:200; Pharmingen). After being rinsed, the tissue specimens were incubated with Alexa 488–conjugated anti-rat IgG (1:200; Molecular Probes). Next, tissue specimens were cover-slipped using 50% glycerin with 10-mg/ml 1,4-diazabicyclo(2,2,2)octane (DABCO). Immunohistochemistry for VEGF, Flk-1, Flt-1, PECAM-1, and ephrin-B2 was performed as follows: The lungs were taken from the embryos and ﬁxed with 4% PFA. They were then embedded in OCT compound. Sections on glass slides were dried and ﬁxed with 4% PFA and then were treated with 100% ethanol. Next, the slides were incubated with primary antibodies. The following antibodies were used: rat anti– PECAM-1 (1:500; Pharmingen), rabbit anti-VEGF (1:500; Santa Cruz), rat anti–Flk-1 (1:1,000; kindly donated by Dr. Shin-Ichi Nishikawa, Center for Developmental Biology, Kobe, Japan), rat anti–Flt-1 (1:500; kindly donated by ImClone system), rabbit anti-ephrin–B2 (1:500; Santa Cruz), and rabbit anti-pro surfactant protein C (pro SP-C; 1:1,000; CHEMICON). Confocal Laser Scanning Microscopy The stained specimens were observed by a confocal laser scanning microscope (CLSM: Olympus FLUOVIEW FVX system; Olympus, Tokyo, Japan) equipped with an oil immersion objective (Plan Apo 360, NA 5 1.4; Olympus) and a dry objective (Uplan Fl 310, NA 5 0.3; Olympus). An argon-krypton laser produced excitation bands at 488 nm for Alexa 488, bands at 568 nm for Alexa 594, and monochromatic light for differential interference contrast (DIC) images. Fluorescent images were collected 960 YAMAMOTO ET AL. with emission ﬁlters for 510–550 nm for Alexa 488 and 585–610 nm for Alexa 594. Simultaneous images (800 3 600 pixels, 12 bits each) of either Alexa 488- or 594-labeled and DIC images were acquired and stored. Serial optical sections (up to 30 sections) were obtained by moving the sample stage at intervals of 1.0 mm. Stereo pair images were generated by reconstructing the series of optical sections using the CLSM software package. Morphometry The numbers of PECAM-1–, Flk-1–, Flt-1–, ephrin-B2–, and pro SP-C–positive cells in each high power ﬁeld (objective lens, 360) were counted using a computerized image analysis system (NIH image). The percentages of PECAM-1–, Flk-1–, or Flt-1–positive cells in total cells were calculated. The percentage of pro SP-C–positive cells out of the total pulmonary epithelial cells was calculated. The ratio of arterial vascular endothelial cells was deﬁned as the percentage of ephrin-B2–positive and PECAM-1–positive cells in all PECAM-1–positive cells. VEGF-positive area for the same ﬁeld (21,590 square pixels) was measured and expressed as mean pixels. For each lung bud, the average number of microvessel branch points was calculated essentially as previous described with minor modiﬁcations (Ruhrberg et al., 2002) and determined by stereogram in 3 randomly chosen areas of the peripheral lung buds. The data were obtained from three to ﬁve separate experiments. Quantitative Real-Time PCR Only the peripheral regions of the lung tissues were dissected under a stereomicroscope and then they were harvested to isolate RNA. Quantitative real-time PCRbased measurements of RNA abundance were carried out using gene-speciﬁc double ﬂuorescent probes (designed by ABI) and real-time PCR instruments (ABI, PRISM 7700). The amounts of PECAM-1, Flk-1, Flt-1, and ephrin-B2 mRNA were standardized by 18S ribosomal RNA. The quantitative data are the average of two independent experiments. Each mRNA-quantiﬁcation was done with the total RNA isolated from 10 to 15 embryos. In Situ Hybridization Total RNA was extracted from E14.5 fetal mouse lung tissue. A 419-bp VEGF cDNA fragment, encoding exon 1 to 5, was generated by the reverse transcriptase-PCR (RT-PCR). The primer pair was designed as follows: forward primer, 50 -GGGGATCCATGAACTTTCTGCTGTC TTGGGTGCACTGG-30 ; reverse primer, 50 -AAAAGCTTC TGGCTTTGTCCTGTCTTTCTTTGGTCTGC-30 (Breier et al., 1992). The PCR products were subcloned into pGEM-3Zf (6) Vector (Promega) and linealized. Then both sense and antisense RNA probes were prepared by in vitro transcription using a DIG RNA Labeling Kit (SP6/T7; Boehringer Mannheim, Germany). Six-micrometer frozen sections of E14.5 embryonic lung were ﬁxed with 4% PFA for 5 min, treated with 1.0 mg/ml proteinases K for 15 min, acetylated for 10 min, and then were dehydrated with graded ethanol. After the pretreatment, the sections were hybridized with 1 mg/ml VEGF RNA probe for 20 hr at 558C. Signals were detected using a DIG Nucleic Acid Detection Kit (Boehringer Mannheim, Germany). Lung Bud Culture The serum-free lung bud culture was done essentially as previously described with minor modiﬁcations (Ohmichi et al., 1998). Fetal lung buds were taken from E11.5 ICR pregnant mice (treated group; n 5 10–15, control group; n 5 10–15) and then were placed in culture plates with 12 separate wells containing 600 ml of medium (Dulbecco’s modiﬁed Eagle’s medium containing 0.38% [w/v] NaHCO3, 100 units/ml penicillin, 100 mg/ml streptomycin). All the explants were incubated in a humidiﬁed chamber with 95% air and 5% CO2. The explants were photographed after 2, 24, 48, and 96 hr and then were ﬁxed with 4% PFA. F-actin and PECAM-1 were detected by immunohistochemistry as previously described. Images were taken using a CLSM (Olympus). Each experiment was repeated at least three times. AS-ODNs Targeted to Flk-1, Flt-1 The inhibition of Flk-1 or Flt-1 during lung development in culture was examined using AS-ODNs (1.0 mM and 2.5 mM, respectively; Proligo, Japan) as described in a previous report with some modiﬁcations (Ohmichi et al., 1998). The sequences are designed to inactivate the initiation codon of the published mouse Flk-1 or Flt-1 mRNA as follows: 50 -AGGUGCAGGAUGGAGAGCAA-30 for Flk-1 (NCBI GenBank X59397), 50 -UUGCUCAC CAUGGUCAGC-30 for Flt-1 (NCBI GenBank D88689). Scrambled sequence oligodeoxynucleotides (S-ODNs) were used as a control. After 48 hr, the explants were ﬁxed and double stained with Texas Red-X–conjugated phalloidin and antibody against PECAM-1 as described. The development of lung vascular endothelial cells was evaluated by using the CLSM. To verify the uptake of oligodeoxynucleotides into endothelial cells, ﬂuorescein isothiocyanate–labeled S-ODNs were used in the same manner. A speciﬁc decrease in the gene copy was conﬁrmed by RT-PCR. RT-PCR RT-PCR was done according to the manufacturer’s recommendations (Roche). Total RNA was extracted from lung explants using RNeasy Mini Procedure kit (QIAGEN). Short- and single-strand cDNA was prepared from total RNA using SuperScriptTMII RNase H-Reverse Transcriptase (Invitrogen). The following sets of primers were designed (Choi et al., 1998; Akeson et al., 2000). b-actin: forward primer, 50 -TGGAATCCTGTGGCATCCATGAA AC-30 ; reverse primer, 50 -TAAAACGCAGCTCAGTAAC AGTCCG-30 . Flk-1: forward primer, 50 -CACCTGGCAC TCTCCACCTTC-30 ; reverse primer, 50 -GATTTCATCCCA CTACCGAAA-30 . Flt-1: forward primer, 50 -CTCTGATGG TGATCGTGG-30 ; reverse primer, 50 -CATGCGTCTGGCC ACTTG-30 . PCR conditions were as follows: denaturation at 948C for 30 sec, followed by annealing at 558C for 60 sec, and extension at 728C for 60 sec. Inhibition of Flk-1 or Flt-1 by Antibodies Angiogenesis inhibitors (DC101; rat anti-mouse Flk-1 antibody, MF-1; goat anti-mouse Flt-1 antibody, kindly VEGF RECEPTORS DURING LUNG DEVELOPMENT gifted from ImClone Systems, 50 mg/ml) were added to the culture medium. After 48 hr, the explants were ﬁxed and double stained with Texas Red-X–conjugated phalloidin and antibody against PECAM-1 as previously described. The development of lung vascular endothelial cells was evaluated by using the CLSM. Each experiment was repeated at least three times. Cell Proliferation Assay Flash labeling with BrdU and the subsequent determination of labeling index was performed as previously described (Ruhrberg et al., 2002). Timed pregnant mice (E10.5, E12.5, E14.5, and E18.5) were intraperitoneally injected with 0.0125 mg/g body weight BrdU (Sigma) in PBS. One hour after the injection, the embryos were dissected from the uterus and the lungs were ﬁxed with 4% PFA and embedded into OCT compound. Vascular endothelial cells were stained with anti–PECAM-1 antibody (1:300; Pharmingen) and then were detected by Alexa 594–conjugated anti-rat IgG (1:300; Molecular Probes). Next, the sections were incubated with 4 N HCl for 30 min at room temperature, rinsed in PBS once, and then incubated with anti-BrdU antibody (1:500; Exalpha Biologicals) for 30 min at 378C. Alexa 488–conjugated antisheep IgG (1:1,000; Molecular Probes) was used for the detection of BrdU. The sections were incubated with 4,6diamidino-2-phenylindole (DAPI) for 30 min at room temperature. The slides were observed by conventional ﬂuorescence microscopy (Olympus AX70; Olympus, Tokyo, Japan), and the images were analyzed using a ﬂuorescence imaging system (IPLab3.5.2). The labeling index of pulmonary vascular endothelial cells was deﬁned as a percentage of the BrdU-positive and PECAM-1–positive cells in total PECAM-1–positive cells. The data were obtained from three separate experiments. Detection of Apoptosis Apoptotic cell death was detected by immunoﬂuorescence with antibody against single-stranded DNA (1:1,000; IBL, Japan; Frankfurt, 2004). The incidence of apoptosis in vascular endothelial cells was deﬁned as the percentage of single-stranded DNA-positive and PECAM-1–positive cells out of all PECAM-1–positive endothelial cells. The data were obtained from three separate experiments. Statistical Analysis All values were presented as the mean 6 SD. Statistical signiﬁcance between the groups was determined by a one-way analysis of variance, followed by either the Fisher or the unpaired t-test, as appropriate. A value of P < 0.05 was considered to be signiﬁcant. RESULTS Fetal Lung Vascular Development Depends on the VEGF Receptors Flk-1 and Flt-1 Based on the relationship between vascular endothelial cells and bronchial epithelial cells and in accordance with literature (Burri, 1984; deMello et al., 1997), we divided lung vascular development into the following four stages: 961 stage I, E9.5–E10.5; stage II, E11.5–E13.5; stage III, E14.5–E16.5; and stage IV, E17.5–E18.5. Stage I (E9.5–E10.5): A few PECAM-1–positive vascular endothelial cells appeared in the distal mesenchyme surrounding the developing airway of the lung bud (Fig. 1A,I; 4.3 6 1.0%). Flk-1–positive cells were distributed surrounding the developing airway of the lung bud. (Fig. 2A,I; 40.5 6 5.6%). Flt-1–positive cells were hardly visible at this stage in the distal mesenchyme surrounding the developing airway of the lung bud (Fig. 2E), except in the pulmonary artery in the proximal portion of the lung bud (Fig. 2J; 5.7 6 4.5%, Supplementary Fig. 1). There were abundant BrdU- and PECAM-1–positive cells surrounding the developing airway of the lung bud (Fig. 1J; 67.0 6 10.4%). Stage II (E11.5–E13.5): PECAM-1–positive cells increased in number (Fig. 1B,I; 11.6 6 2.6%), and they formed immature and various-sized vascular lumina (Fig. 1F, stereograms). Flk-1–positive cells were still abundantly expressed (Fig. 2B,I; 43.0 6 8.6%). A small number of Flt1–positive cells appeared in the distal mesenchyme surrounding the developing airway of the lung bud (Fig. 2F,J; 15.9 6 5.6%). Quantiﬁcation of mRNA by real-time PCR revealed that PECAM-1, Flk-1, and Flt-1 mRNA expression was 3.1-fold, 1.6-fold, 2.1-fold higher than that of stage I, respectively. Abundant BrdU-positive and PECAM-1–positive cells surrounded the bronchial acini (Fig. 1J; 62.8 6 16.8%). Stage III (E14.5–E16.5): PECAM-1–positive cells increased in number and encircled the branching airways (Fig. 1C,I; 19.5 6 1.2%). They ultimately constituted organized capillary networks with uniform-sized lumina (Fig. 1C,G; stereograms). As has been previously reported (Acarregui et al., 1999), in situ hybridization revealed that VEGF mRNA was primarily expressed in bronchial epithelial cells and, to a lesser extent, in mesenchymal cells (Supplementary Fig. 2). Flk-1–positive cells were still abundantly distributed (Fig. 2C,I; 47.0 6 12.8%). Flt1–positive cells (arrowheads in Fig. 1S) were increased in number and surrounded the branching airways (Fig. 2G,J; 31.9 6 10.9%). The quantiﬁcation of mRNA revealed PECAM-1, Flk-1, and Flt-1 mRNA expression to be 5.1fold, 1.9-fold, and 2.3-fold higher than that of stage I, respectively. The ratio for BrdU-positive vascular endothelial cells showed a signiﬁcant decrease in comparison to that for stages I and II (Fig. 1J; 43.5 6 16.0%). Stage IV (E17.5–E18.5): PECAM-1–positive vascular endothelial cells were located closer to alveolar epithelial cells (Fig. 1D) with more uniform-sized lumina (Fig. 1H; stereograms). Although the number of PECAM-1–positive cells increased (Fig. 1D,I; 41.0 6 4.8%), the number of Flk-1–positive cells (Fig. 2D,I; 26.7 6 4.7%) and Flt-1– positive cells (Fig. 2H,J; 7.7 6 1.4%) decreased. In addition, the Flk-1 mRNA expression was found to have decreased in comparison to that of stage III (0.68-fold). The BrdU incorporation into vascular endothelial cells signiﬁcantly decreased in comparison to that for stages I and II (Fig. 1J; 39.8 6 5.6%). A real-time PCR analysis of spliced and soluble form of Flt-1, which also plays a signiﬁcant role as a decoy for Flk-1– VEGF signaling (Kendall and Thomas, 1993), showed a robust up-regulation of soluble Flt-1 at stage IV in comparison to the former stages (0.77-, 1.03-, and a 4.65fold increase of stage I at stages II, III, and IV, respectively). 962 YAMAMOTO ET AL. Fig. 1. Four developmental stages of the fetal lung vasculature based on the expression of platelet endothelial cell adhesion molecule-1 (PECAM-1). A–D: The microscopic appearance of fetal lungs were divided into the following 4 stages: stage I (A, embryonic day [E] 10.5), stage II (B, E12.5), stage III (C, E14.5), and stage IV (D, E18.5). A,B,J: Vascular endothelial cells actively proliferated and differentiated at stage I and II. C,D,J: Vascular endothelial cells less actively prolifer- ated and differentiated after stage III. A–D: The whole-mount observation of embryonic lung immunostained with PECAM-1 antibody (green) and F-actin (red). E–H: Stereograms of PECAM-1–positive vascular endothelial cells. I,J: The quantiﬁcation of percentage of PECAM-1 (1) cells (I) and percentage bromodeoxyuridine (BrdU, 1) PECAM-1 (1) cells (J). *P < 0.05, **P < 0.01. Scale bars 5 50 mm in D (applies to A–D), 20 mm in H (applies to E–H). These ﬁndings indicate that the morphological transition from loose capillary networks (stages I–II) into highly organized ones (stage III) is closely associated with the branch formation of bronchial epithe- lial cells, the spatiotemporal expression of Flk-1 and Flt-1, and the altering proliferation of vascular endothelial cells. It is also suggested that vascular endothelial cell proliferation is promoted by the predomi- Fig. 2. Spatiotemporal expression of Flk-1 and Flt-1. There were abundant Flk-1 (1) cells at stage I–III, and their number decreased at stage IV. Flt-1–positive cells increased in number at stage III and decreased at stage IV. A–H: Differential interference microscopy image and immunoﬂuorescence image of Flk-1 (A–D), and Flt-1 (E–H). I,J: Quantiﬁcation of percentage Flk-1 (1) cells (I) and percentage Flt-1 (1) cells (J). **P < 0.01. Scale bars 5 50 mm in D (applies to A–D) and H (applies to E–H). 964 YAMAMOTO ET AL. nant expression of Flk-1 (stages I–II), while it is downregulated by the transient expression of Flt-1 (stage III) and by the up-regulation of soluble Flt-1 at stage IV. AS-ODNs Targeted to Flk-1 mRNA Inhibit the Branching of Pulmonary Capillaries While Impairing the Proliferation of Vascular Endothelial Cells To inhibit Flk-1 during the pulmonary vascular development, cultured lung buds at E11.5 (Stage II) were treated with AS-ODNs complementary to Flk-1 mRNA. The S-ODNs were used as a control. The down-regulation in speciﬁc gene copy was conﬁrmed by RT-PCR (Fig. 3H). The treatment of the lung buds with AS-ODN targeted to Flk-1 decreased the number of PECAM-1–positive vascular endothelial cells and impaired the formation of capillary lumina (compare Fig. 3C with Fig. 3B). The ratio of PECAM-1–positive cells to the total number of cells was signiﬁcantly lower in the Flk-1 AS-ODN–treated buds (10.5 6 4.4%) than in the S-ODN–treated buds (20.6 6 3.8%; Fig. 3I; P < 0.01). The PECAM-1 mRNA level decreased in Flk-1 AS-ODN–treated lung buds in comparison to those treated with S-ODN (0.58-fold). Interestingly, the inhibition of Flk-1 also impaired the growth of bronchial epithelial cells, characterized by large acinus formation (Fig. 3C). To elucidate the effects of Flk-1 inhibition on the epithelial differentiation, we performed immunostaining using pro SP-C, which is expressed in alveolar Type II cells. The inhibition of Flk-1 by AS-ODN attenuated the number of pro SP-C–positive cells in comparison to S-ODN–treated lung buds (Fig. 3L–N; P 5 0.0012). Stereograms of PECAM-1 demonstrated that the inhibition of Flk-1 impaired the formation of the vascular branching and capillary lumen (Fig. 3F). The number of microvessel branch points was signiﬁcantly smaller in the Flk-1 AS-ODN–treated lung buds (6.9 6 2.2) than in the S-ODN–treated ones (21.5 6 6.8; Fig. 3J; P < 0.05). As shown in Figure 3G, the impaired growth of capillary lumina (mean diameter, 6.40 6 2.38 mm for anti–Flk-1 antibody-treated buds vs. 15.3 6 4.04 mm for control buds, P < 0.01) and branching was also conﬁrmed by the treatment of lung buds with antibody against Flk-1 (DC101). These ﬁndings suggest that Flk-1 plays an important role in both the normal growth of the capillary lumen and the promotion of the pulmonary vascular branching. To elucidate how the inhibition of Flk-1 impaired the capillary formation and the branching morphogenesis of cultured lung buds, we investigated both Fig. 3. A–N: Effects of antisense oligodeoxynucleotides (AS-ODNs) directed to Flk-1 mRNA on fetal lung capillary morphogenesis in culture. The treatment of lung buds with AS-ODN targeted to Flk-1 for 48 hr decreased the number of platelet endothelial cell adhesion molecule-1 (PECAM-1) –positive vascular endothelial cells and impaired the capillary branching (I and J, compare F with D and E). K: The inhibition of Flk-1 signiﬁcantly suppressed the proliferation of vascular endothelial cells. G: The impaired growth of the capillary lumina and branching was also conﬁrmed by treating the lung buds with antibody against Flk-1 (DC101). H: Flk-1 AS-ODN decreased the speciﬁc gene copy (reverse transcriptase-polymerase chain reaction). (2), ODN2; S, SODN; AS, AS-ODN. The inhibition of Flk-1 induced a smaller degree of BrdU incorporation and the detection of apoptotic death of vascular endothelial cells. The ratio of proliferating vascular endothelial cells in Flk-1 AS-ODN–treated lung buds (10.4 6 8.1%) was signiﬁcantly decreased in comparison to those treated with S-ODN (41.0 6 9.5%; Fig. 3K; P < 0.01). The incidence of apoptotic death in PECAM-1– positive vascular endothelial cells was higher in Flk-1 AS-ODN–treated lung buds (5.5 6 4.2%) in comparison to those treated with S-ODN (2.0 6 0.7%; P < 0.05). These ﬁndings suggest that the impaired growth of the capillary lumen and vascular branching in Flk-1–inhibited lung buds was associated with the down-regulated proliferation and enhanced apoptosis of vascular endothelial cells. AS-ODNs Targeted to Flt-1 mRNA Inhibit the Organization of Pulmonary Capillaries During Fetal Morphogenesis While Promoting the Proliferation of Vascular Endothelial Cells To elucidate the role of Flt-1 in fetal pulmonary vascular development, we also inhibited Flt-1 using AS-ODN. The down-regulation of Flt-1 mRNA was conﬁrmed by RT-PCR (Fig. 4H). AS-ODN–treated lung buds showed increased number of PECAM-1–positive vascular endothelial cells (compare Fig. 4C with Fig. 4B). The ratio of PECAM-1–positive cells to total cells was signiﬁcantly higher in Flt-1 AS-ODN–treated buds (40.4 6 11.9%) than those treated with S-ODN (19.6 6 6.0%; Fig. 4I; P < 0.01). PECAM-1 mRNA expression was also higher in Flt-1 AS-ODN–treated lung buds than in S-ODN–treated buds (1.86-fold). Stereograms of lung buds demonstrated that AS-ODN targeted to Flt-1 impaired the normal formation of the capillary lumen and networks, that is, the vascular endothelial cells were increased in number, the capillary lumen was not uniform in size, and the capillary branching was promoted (Fig. 4F). The number of microvessel branch points signiﬁcantly increased in the ASODN–treated lung buds (26.3 6 5.1) in comparison to those treated with S-ODN (17.0 6 4.1; Fig. 4J; P < 0.01). As shown in Figure 4G, the irregular formation of capillary lumina (mean diameter, 11.4 6 6.2mm for anti–Flt-1 antibody-treated buds vs. 15.3 6 4.0mm for control buds, p50.16) and the disorganization of capillary networks were also conﬁrmed by treatment with anti–Flt-1 antibody (MF-1). These ﬁndings suggest that Flt-1 plays an important role in the normal formation of the capillary lumen, the regulation of capillary branching, and organization of capillary networks. To clarify how AS-ODN targeting to Flt-1 impaired the organization of pulmonary pro SP-C expression in comparison to the S-ODN–treated lung buds. N: There are signiﬁcant reduction in the ratio for pro SP-C–positive cells / total epithelial cells (%) in the explants treated with AS-Flk-1. Confocal images of the cultured lung buds without ODN [A, ODN (2)], treated with S-ODN (B, S-ODN), and treated with AS-ODN targeted to Flk-1 mRNA (C, AS Flk-1). PECAM-1–positive vascular endothelial cells are shown in green, F-actin staining in red. D–G: Stereograms of the cultured lung buds treated without ODN (D), with S-ODN (E), with AS Flk-1 (F) or with DC101 (G) labeled with anti-PECAM-1 antibody. *P < 0.05, **P < 0.01. Scale bars 5 50 mm in A–C, 20 mm in D–G, 20 mm in L,M. VEGF RECEPTORS DURING LUNG DEVELOPMENT Figure 3. 965 966 YAMAMOTO ET AL. capillaries, we investigated BrdU incorporation and apoptotic death of vascular endothelial cells. The ratio of proliferating vascular endothelial cells in Flt-1 AS-ODN– treated lung buds (58.5 6 2.9%) was signiﬁcantly higher than those treated with S-ODN (35.4 6 10.6%; Fig. 4K; P < 0.01). The incidence of apoptotic death in PECAM-1– positive vascular endothelial cells was lower in the Flt-1 AS-ODN–treated lung buds (1.6 6 1.3%) than in those treated with S-ODN (3.3 6 0.7%; P < 0.01). These ﬁndings suggest that the disorganization of vascular branching produced by Flt-1 AS-ODN is associated with the up-regulated proliferation of vascular endothelial cells. Flk-1 Expression Is Enhanced by the Inhibition of Flt-1 To examine whether the expression of Flk-1 regulates that of Flt-1 and vice versa, we immunohistochemically studied Flk-1- or Flt-1 AS-ODN–treated lung buds stained with Flt-1 and Flk-1 antibodies, respectively. There were more Flk-1–positive cells in the lung buds treated with Flt-1 AS-ODN (Fig. 5C) than those treated with S-ODN (Fig. 5B). The ratio for Flk-1–positive cells to total cells was signiﬁcantly higher in the lung buds treated with Flt-1 AS-ODN (14.2 6 2.2%) than in those treated with S-ODN (6.5 6 3.3%; P < 0.05, Fig. 5G). Flk-1 mRNA was also increased in lung buds treated with Flt-1 AS-ODN in comparison to those treated with S-ODN (1.3fold). To clarify whether the higher levels of Flk-1 in explants treated with AS-Flt-1 is just due to increased number of Flk-1–positive endothelial cells or whether it is due to increased number of Flk-1 receptors in each endothelial cell, double staining with Flk-1 and PECAM-1 was performed. Almost all the Flk-1–positive cells also expressed PECAM-1 (Supplementary Fig. 4A–D), suggesting that the higher expression of Flk-1 was due to the increased number of Flk-1– and PECAM-1–positive endothelial cells. In contrast, there was no signiﬁcant difference in the number of Flt-1–positive cells (Fig. 5F,H; P > 0.05) and mRNA (0.99-fold) in the lung buds treated with Flk-1 AS-ODN in comparison to those treated with SODN. Inhibition of Flk-1 and Flt-1 Affects the Ephrin-B2 Expression of Vascular Endothelial Cells To clarify whether the inhibition of Flk-1 or Flt-1 inﬂuences the subsequent differentiation of vascular endothelial cells, we investigated the ephrin-B2 expression, which is exclusively expressed in the arterial vascular endothelial cell lineage. There were few ephrin-B2–positive cells in the lung buds treated with Flk-1 AS-ODN (Fig. 6B,F,J) in comparison to those treated with S-ODN (Fig. 6A,E,I). The ratio for ephrin-B2–positive and PECAM-1– Fig. 4. A–K: Effects of antisense oligodeoxynucleotides (AS-ODNs) directed to Flt-1 mRNA on fetal lung capillary morphogenesis in culture. The treatment of lung buds with AS-ODN targeted to Flt-1 for 48 hr increased the number of PECAM-1–positive vascular endothelial cells and capillary branching (I and J, compare F with D and E). K: The inhibition of Flt-1 signiﬁcantly promoted the proliferation of vascular endothelial cells. G: The irregular formation of the capillary lumina and the disorganization of capillary networks were also conﬁrmed by the treatment with anti–Flt-1 antibody (MF-1). H: Flt-1 AS-ODN positive cells to total PECAM-1–positive cells was signiﬁcantly smaller in the Flk-1 AS-ODN–treated buds (46.9 6 12.7%) than in S-ODN–treated buds (76.4 6 20.2%; Fig. 6M; P < 0.01). The Ephrin-B2 mRNA level also decreased more in the Flk-1 AS-ODN–treated lung buds than in the S-ODN–treated buds (0.53-fold). In contrast, there were more abundant ephrin-B2–positive cells in Flt-1 ASODN–treated lung buds (Fig. 6D,H,L) than in the SODN–treated buds (Fig. 6C,G,K). The ratio for ephrinB2–positive and PECAM-1–positive cells to the total number of PECAM-1–positive cells was signiﬁcantly higher in the Flt-1 AS-ODN–treated lung buds (94.7 6 6.1%) than in the S-ODN–treated buds (71.5 6 6.0%; Fig. 6N; P < 0.05). The Ephrin-B2 mRNA level slightly decreased in the Flt-1 AS-ODN–treated lung buds in comparison to those treated with S-ODN (0.81-fold). These results suggest that the inhibition of Flk-1 down-regulates the arterialization of the embryonic pulmonary vasculature, whereas the inhibition of Flt-1 promotes it. DISCUSSION In this study, we present evidence supporting the novel concept that the spatiotemporal expression of Flk-1 and Flt-1 regulates the vascular endothelial cell proliferation and differentiation and subsequent development of the pulmonary vasculature (summarized in Fig. 7). The inhibition of Flk-1 resulted in reduced branching of capillaries and an impaired proliferation of vascular endothelial cells. Conversely, the inhibition of Flt-1 promoted vascular branching and enhanced proliferation of vascular endothelial cells associated with an increased Flk-1 expression. This is the ﬁrst report to show that the differential expression of the two VEGF receptors, Flk-1 and Flt-1, is able to regulate embryonic lung vascular development while the emergence of Flt-1 is a key process for the morphological transition from loose capillary networks into highly organized ones. Flk-1 and Flt-1 Function on Vascular Formation According to the previous reports (Gebb and Shannon, 2000; Schachtner et al., 2000) and our immunohistochemical study in vivo, vascular endothelial cells emerge and differentiate in aligned but they are signiﬁcantly distant from the developing bronchial airway and acini. We, therefore, proposed a novel mechanism of vascular endothelial cell development in addition to the mechanism due to VEGF gradient. Two high afﬁnity receptors of VEGF, Flk-1 and Flt-1, seems to be involved in the ﬁne-tuning mechanisms for the signal transduction of VEGF because their effects on vascular endothelial cells considerably differ in their signaling properties. Flk-1 has also been suggested to be a decreased speciﬁc gene copy (reverse transcriptase-polymerase chain reaction). (2), ODN2; S, S-ODN; AS, AS-ODN. A–C: The confocal images of the lung buds without treatment [A, ODN (2)], treated with S-ODN [B, S-ODN]), and treated with AS-ODN targeted to Flt-1 mRNA (C, AS Flt-1). D–G: Stereograms of the cultured lung buds treated without ODN (D), with S-ODN (E), with Flt-1 AS-ODN (F), or with MF-1 (G) labeled with anit–PECAM-1 antibody. *P < 0.05, **P < 0.01. Scale bars 5 50 mm in A–C, 20 mm in D–G. VEGF RECEPTORS DURING LUNG DEVELOPMENT Figure 4. 967 968 YAMAMOTO ET AL. major mediator of VEGF, which transduces many downstream effects of VEGF, including proliferation, migration, permeability, and survival signals (Gerber et al., 1998). In contrast, the function and signaling properties of Flt-1 appear to be different, depending upon the developmental stages and the cell type (Ferrara et al., 2003). One important property is that Flt-1 negatively modulates vascular growth by reducing endothelial cell proliferation (Fong et al., 1995, 1999; Kearney et al., 2002). Our spatiotemporal observations of embryonic lung tissues could be explained by the differential expression and properties of the two receptors. When Flk-1 was predominantly expressed at stages I–II, vascular endothelial cells actively proliferated. When the Flt-1 expression was enhanced at stage III, these cells less actively proliferated and started to constitute organized vascular networks. These data indicated that the transition of dominant receptors from Flk-1 to Flt-1 induces the maturation of the embryonic lung vasculature. Vascular endothelial cells thereafter become more mature and better organized at stage IV, despite a decreased expression of Flt-1. This phenomenon could be induced by a decreased expression of Flk-1 at stage IV and also by an increased expression of spliced variant and soluble form of Flt-1, which has a decoy effect on the VEGF–Flk-1 signaling as well as the full-length (membrane-bound) form. Our data concerning the inhibition of Flk-1 or Flt-1 conﬁrm the signaling properties of the two receptors in developing lung tissues. The inhibition of Flk-1 induced both hypocellularlity and less branching of vascular endothelial cells, whereas the inhibition of Flt-1 resulted in hypercellularity and more branching of endothelial cells. The inhibition of Flk-1 not only arrests endothelial cell proliferation and prevents vessel growth, but it also induces the regression of existing vessels by endothelial cell death (Carmeliet, 2005). Of interest, the inhibition of Flk-1 also impaired the growth of bronchial epithelial cells. Our data demonstrated that the inhibition of Flk-1 induced a smaller degree of pro SP-C expression in comparison to S-ODN– treated lung buds. A recent report revealed that the Flk-1 inhibition by DC101 disrupted the postnatal alveolar development (McGrath-Morrow et al., 2005), and the treatment of newborn rats with the VEGF receptor inhibitor SU5416 impaired alveolar morphogenesis (Le Cras et al., 2002). These ﬁndings support the idea that VEGF-A signaling through Flk-1 is a critical facilitator of the crosstalk between epithelial and endothelial cells and the resultant alveolar development (Del Moral et al., 2006). Recently, DeLisser et al. (2006) demonstrated that loss of PECAM-1 function resulted in the disruption of both proximal and distal lung development without reducing endothelial cell content. These observations support the idea that vascular endothelial cell lineage directly signal back by means of paracrine growth, survival, or morphogenetic factors to inﬂuence epithelial development as seen in the liver development (Matsumoto et al., 2001). In contrast, the inhibition of Flt-1 did not impair pulmonary epithelial development, although it did induce both the hypercellular and disorganized capillary networks. Our data also demonstrated that the inhibition of Flt-1 did not inﬂuenced pro SP-C expression (Supplementary Fig. 5). This ﬁnding suggests that the up-regulation of the VEGF signals by means of inhibition of Flt-1does not inﬂuence the epithelial cell differentiation, even though down-regulation has a signiﬁcant effect in our organ culture models. The transgenic overexpression of VEGF-A in lung epithelium has been reported to result in abnormal lung development with dilated epithelial tubes and increased peritubular vascularity (Zeng et al., 1998). The overexpression of VEGF164 in distal lung epithelium caused decreased peripheral capillary network formation and decreased distal airspace branching (Akeson et al., 2003). These diverse effects of VEGF on the pulmonary epithelial and vascular endothelial cells thus suggest that the spatiotemporal expression of VEGF and downstream signaling molecules are important not only for the capillary network formation but also for the accompanying bronchial growth and alveolization. A further investigation is, therefore, needed to elucidate the interactions between vascular endothelial cells and bronchial epithelial cells during the development of the pulmonary alveolar system. Negative Regulation of Flk-1 Expression by Flt-1 Although Flk-1 and Flt-1 transduce divergent signal properties of VEGF, the mechanisms that regulate the expression of Flk-1 and Flt-1 genes are still not fully understood. VEGF itself is able to up-regulate both Flk-1 and Flt-1 genes (Barleon et al., 1997; Shen et al., 1998; Wang et al., 2000). Until recently, it remains uncertain whether or not a crosstalk mechanism exists between these two divergent receptors, Flk-1 and Flt-1. It has recently been shown that the vascular overgrowth seen in the absence of Flt-1 can be modulated by preventing tyrosine phosphorylation and downstream signaling, thus suggesting that Flt-1 negatively modulates the blood vessel formation by down-regulating the signaling through Flk-1 (Roberts et al., 2004). A recent report also demonstrated that placental growth factor binds to Flt-1, thus resulting in the intermolecular phosphorylation of Flk-1, and amplifying VEGF-A–driven angiogenesis through Flk-1 (Autiero et al., 2003). According to our results, the up-regulation of Flk-1 in response to the inhibition of Flt1 indicates that Flk-1 is at least in part regulated by Flt1. The result that the inhibition of Flk-1 did not affect the Flt-1 expression raises the possibility that Flt-1 is regulated by an independent mechanism from Flk-1. Therefore, our data raise the fascinating possibility that Flk-1 and Flt-1, and not merely VEGF-A itself, are signiﬁcantly involved in the regulation of the development and organization of pulmonary vascular endothelial cells. VEGF Receptors and Ephrin-B2 Expression The Eph–ephrin system has been shown to be involved in the demarcation of arterial and venous boundaries (Wang et al., 1998). Ephrin-B2 is known to be expressed in arterial endothelial cells, pericytes, and smooth muscle cells, whereas EphB4, a receptor for ephrin-B2, is known to be expressed in only in the veins (Wang et al., 1998). As a result, our ﬁndings that the inhibition of Flk-1 or Flt-1 modulates the proliferation of vascular endothelial cells and the expression of ephrin-B2 in vascular endothelial cell suggests that the proportional VEGF signaling mediated by means of Flk-1 and Flt-1 affects the arterial– venous boundaries. Further investigation is called for to clarify how VEGF and downstream signaling modulate Fig. 5. A–H: Flk-1 expression is promoted by the inhibition of Flt-1. B,C,G: There were more Flk-1–positive cells (C,G) in lung buds treated with Flt-1 antisense oligodeoxynucleotide (AS-ODN) than those treated with S-ODN (B). E,F,H: In contrast, there was no signiﬁcant difference in number of Flt-1–positive cells (F,H) in lung buds treated with Flk-1 AS-ODN compared with those treated with S-ODN (E). A–E: Immuno- ﬂuorescence images of lung buds [embryonic day (E) 11.5, cultured for 48 hr] without ODN (A,D, ODN ), treated with S-ODN (B,E, S-ODN), and treated with AS-ODN targeted to Flt-1 (C, AS-Flt-1) or Flk-1 (F, AS-Flk-1) mRNA labeled with anti–Flk-1 (A–C) or anti–Flt-1 antibody (D–F). **P < 0.01. Scale bars 5 20 mm in A–F. Fig. 6. A–N: Effects of antisense oligodeoxynucleotides (AS-ODNs) directed to Flk-1 or Flt-1 mRNA on ephrin-B2 expression in culture. A,B,E,F,I,J,M: There were few ephrin-B2–positive cells (B,F,J,M) in the lung buds treated with Flk-1 AS-ODN in comparison to those treated with S-ODN (A,E,I). C,D,G,H,K,L,N: In contrast, there were more abun- dant ephrin-B2–positive cells (D,H,L,N) in Flt-1 AS-ODN–treated lung buds than in S-ODN–treated buds (C,G,K). A–L: Immunoﬂuorescence images of ephrin-B2–positive cells (A–D, green), PECAM-1–positive vascular endothelial cells (E–H, red), and merge images (I–L). *P < 0.05, **P < 0.01. Scale bars 5 20 mm. VEGF RECEPTORS DURING LUNG DEVELOPMENT 971 Fig. 7. Schematic diagrams of the embryonic development of lung vasculature (upper panels) and the inhibition of Flk-1 or Flt-1 (lower panels). The embryonic development of the pulmonary vasculature is divided into four stages. The inhibition of Flk-1 impaired branching of capillaries, proliferation, and arterial differentiation in both vascular endothelial cells and their precursors. The inhibition of Flt-1 promotes the branching, proliferation, and arterial differentiation of vascular endothelial cells by means of the up-regulation of Flk-1. arterial–venous differentiation through ehrin-B2/EphB4 signaling at both the cellular and molecular levels. organized capillary network. Despite the diffuse distribution of VEGF protein, the VEGF-receptors (Flk-1 and Flt-1) were found to be spatiotemporally expressed and they were thus found to play a role in both vascular endothelial cell proliferation and capillary branch morphogenesis. These results underscore the importance of Flk-1 and Flt-1 in the regulation of vascular endothelial cell development and pulmonary vascular branching. These Conclusions and Future Perspectives In this study, we focused on the role of Flk-1 and Flt-1 in vascular endothelial cell development and in the vascular remodeling involved in the transition from loose to 972 YAMAMOTO ET AL. ﬁndings will hopefully provide new insight into the mechanism of normal pulmonary vascular development. Further investigations are necessary to elucidate the mechanisms behind the pathological conditions of such human neonatal lung vasculature diseases as bronchopulmonary dysplasia or alveolar capillary dysplasia. ACKNOWLEDGMENTS The authors gratefully acknowledge valuable assistance Mark A. Sussman, Heart Institute and Department of Biology, San Diego State University, for his critical review of this manuscript. LITERATURE CITED Acarregui MJ, Penisten ST, Goss KL, Ramirez K, Snyder JM. 1999. 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