Loss of Semaphorin-Neuropilin-1 Signaling Causes Dysmorphic Vascularization Reminiscent of Alveolar Capillary Dysplasia
2012; Elsevier BV; Volume: 181; Issue: 6 Linguagem: Inglês
10.1016/j.ajpath.2012.08.037
ISSN1525-2191
AutoresStephen Joza, Jinxia Wang, Emily Fox, Valerie Hillman, Cameron Ackerley, Martin Post,
Tópico(s)Angiogenesis and VEGF in Cancer
ResumoRespiratory diseases of the newborn can arise from the disruption of essential angiogenic pathways. Neuropilin-1 (NRP1), which is a critical receptor implicated in systemic vascular growth and remodeling, binds two distinct ligand families: vascular endothelial growth factor (VEGF) and class 3 semaphorins (SEMA3). Although the function of VEGF-NRP1 interactions in vascular development is well described, the importance of SEMA3-NRP1 signaling in systemic or pulmonary vascular morphogenesis is debated. We sought to characterize the effect of deficient SEMA3-NRP1 signaling on fetal pulmonary vascular development in a mouse model. Temporospatial expression of Nrp1 and Sema3 mRNA and protein during murine fetal lung development was investigated, and the development of the pulmonary vasculature in transgenic mice deficient in Sema3-Nrp1 signaling was examined by histology, immunostaining, and electron microscopy. Loss of Sema3-Nrp1 signaling resulted in acute respiratory distress and high neonatal mortality. Pathohistological examination of mutants revealed immature and atelectatic regions in the lung, severely reduced capillary density, thickened alveolar septa containing centrally located dilated capillaries, hypertensive changes in arteriolar walls, anomalous and misaligned pulmonary veins, and reduced pulmonary surfactant secretion. Notably, many features are reminiscent of the fatal pulmonary disorder alveolar capillary dysplasia. These findings indicate a critical role for Sema3-Nrp1 signaling in fetal pulmonary development, which may have clinical relevance for treatment of various neonatal respiratory disorders, including alveolar capillary dysplasia. Respiratory diseases of the newborn can arise from the disruption of essential angiogenic pathways. Neuropilin-1 (NRP1), which is a critical receptor implicated in systemic vascular growth and remodeling, binds two distinct ligand families: vascular endothelial growth factor (VEGF) and class 3 semaphorins (SEMA3). Although the function of VEGF-NRP1 interactions in vascular development is well described, the importance of SEMA3-NRP1 signaling in systemic or pulmonary vascular morphogenesis is debated. We sought to characterize the effect of deficient SEMA3-NRP1 signaling on fetal pulmonary vascular development in a mouse model. Temporospatial expression of Nrp1 and Sema3 mRNA and protein during murine fetal lung development was investigated, and the development of the pulmonary vasculature in transgenic mice deficient in Sema3-Nrp1 signaling was examined by histology, immunostaining, and electron microscopy. Loss of Sema3-Nrp1 signaling resulted in acute respiratory distress and high neonatal mortality. Pathohistological examination of mutants revealed immature and atelectatic regions in the lung, severely reduced capillary density, thickened alveolar septa containing centrally located dilated capillaries, hypertensive changes in arteriolar walls, anomalous and misaligned pulmonary veins, and reduced pulmonary surfactant secretion. Notably, many features are reminiscent of the fatal pulmonary disorder alveolar capillary dysplasia. These findings indicate a critical role for Sema3-Nrp1 signaling in fetal pulmonary development, which may have clinical relevance for treatment of various neonatal respiratory disorders, including alveolar capillary dysplasia. The lung parenchyma is formed through extensive branching, subdivision, and maturation of the terminal airways and microvasculature. Essential to this process is an intimate signaling relationship between epithelial and vascular endothelial cells, which culminates in the formation of the alveolar-capillary interface by the fusion of their basal laminae.1Galambos C. deMello D. Molecular mechanisms of pulmonary vascular development.Pediatr Dev Pathol. 2007; 10: 1-17Crossref PubMed Scopus (46) Google Scholar Renewed interest in epithelial-endothelial interplay has arisen from evidence that disruption of angiogenic pathways or endothelial cell function results in dysplastic pulmonary development,2Le Cras T.D. Markham N.E. Tuder R.M. Voelkel N.F. Abman S.H. Treatment of newborn rats with a VEGF receptor inhibitor causes pulmonary hypertension and abnormal lung structure.Am J Physiol Lung Cell Mol Physiol. 2002; 283: L555-L562Crossref PubMed Scopus (300) Google Scholar, 3McGrath-Morrow S.A. Cho C. Zhen L. Hicklin D.J. Tuder R.M. 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Class 3 semaphorins control vascular morphogenesis by inhibiting integrin function [Erratum appeared in Nature 2003, 424:974].Nature. 2003; 424: 391-397Crossref PubMed Scopus (490) Google Scholar Combinations of the transmembrane receptors NRP1 and NRP2, together with type A and D plexins, form ligand binding and signal transduction complexes with differing SEMA3 affinity and downstream effects.13Pellet-Many C. Frankel P. Jia H. Zachary I. Neuropilins: structure, function and role in disease.Biochem J. 2008; 411: 211-226Crossref PubMed Scopus (290) Google Scholar In addition, NRP1 and NRP2 bind members of the VEGF family, which mediate proangiogenic events through enhanced VEGF interactions with its canonical receptor, VEGFR-2, as well via VEGFR-2–independent signaling.14Pan Q. Chanthery Y. Liang W.C. Stawicki S. Mak J. Rathore N. Tong R.K. Kowalski J. Yee S.F. Pacheco G. Ross S. Cheng Z. Le Couter J. Plowman G. Peale F. Koch A.W. Wu Y. Bagri A. Tessier-Lavigne M. Watts R.J. Blocking neuropilin-1 function has an additive effect with anti-VEGF to inhibit tumor growth.Cancer Cell. 2007; 11: 53-67Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar, 15Soker S. Miao H.Q. Nomi M. Takashima S. Klagsbrun M. VEGF165 mediates formation of complexes containing VEGFR-2 and neuropilin-1 that enhance VEGF165-receptor binding.J Cell Biochem. 2002; 85: 357-368Crossref PubMed Scopus (377) Google Scholar, 16Salikhova A. Wang L. Lanahan A.A. Liu M. Simons M. Leenders W.P. Mukhopadhyay D. Horowitz A. Vascular endothelial growth factor and semaphorin induce neuropilin-1 endocytosis via separate pathways [Erratum appeared in Circ Res 2010, 107: e14].Circ Res. 2008; 103: e71-e79Crossref PubMed Scopus (96) Google Scholar Given the ability of NRP1 to bind two distinct ligand families, its expression in the vascular endothelium during development, and the severe cardiac and peripheral vascular defects observed in certain Nrp1 and Sema3 transgenics,17Behar O. Golden J.A. Mashimo H. Schoen F.J. Fishman M.C. Semaphorin III is needed for normal patterning and growth of nerves, bones and heart.Nature. 1996; 383: 525-528Crossref PubMed Scopus (511) Google Scholar, 18Feiner L. Webber A.L. Brown C.B. Lu M.M. Jia L. Feinstein P. Mombaerts P. Epstein J.A. Raper J.A. Targeted disruption of semaphorin 3C leads to persistent truncus arteriosus and aortic arch interruption.Development. 2001; 128: 3061-3070PubMed Google Scholar, 19Gu C. Rodriguez E.R. Reimert D.V. Shu T. Fritzsch B. Richards L.J. Kolodkin A.L. Ginty D.D. Neuropilin-1 conveys semaphorin and VEGF signaling during neural and cardiovascular development.Dev Cell. 2003; 5: 45-57Abstract Full Text Full Text PDF PubMed Scopus (574) Google Scholar the idea that SEMA3-NRP1 signaling could mediate opposing or additive functions to VEGF-mediated angiogenesis and vascular homeostasis is attractive. Indeed, recent studies have indicated essential roles for SEMA3 in both normal and pathological vascular development, many of which mirror or directly oppose the known functions of VEGF.12Serini G. Valdembri D. Zanivan S. Morterra G. Burkhardt C. Caccavari F. Zammataro L. Primo L. Tamagnone L. Logan M. Tessier-Lavigne M. Taniguchi M. Püschel A.W. Bussolino F. Class 3 semaphorins control vascular morphogenesis by inhibiting integrin function [Erratum appeared in Nature 2003, 424:974].Nature. 2003; 424: 391-397Crossref PubMed Scopus (490) Google Scholar, 20Reidy K.J. Villegas G. Teichman J. Veron D. Shen W. Jimenez J. Thomas D. Tufro A. Semaphorin3a regulates endothelial cell number and podocyte differentiation during glomerular development.Development. 2009; 136: 3979-3989Crossref PubMed Scopus (76) Google Scholar, 21Bates D. Taylor G.I. Minichiello J. Farlie P. Cichowitz A. Watson N. Klagsbrun M. Mamluk R. Newgreen D.F. Neurovascular congruence results from a shared patterning mechanism that utilizes Semaphorin3A and Neuropilin-1.Dev Biol. 2003; 255: 77-98Crossref PubMed Scopus (148) Google Scholar In contrast, other researchers have maintained that SEMA3-NRP1 interactions are uninvolved in vascular development.19Gu C. Rodriguez E.R. Reimert D.V. Shu T. Fritzsch B. Richards L.J. Kolodkin A.L. Ginty D.D. Neuropilin-1 conveys semaphorin and VEGF signaling during neural and cardiovascular development.Dev Cell. 2003; 5: 45-57Abstract Full Text Full Text PDF PubMed Scopus (574) Google Scholar, 22Vieira J.M. Schwarz Q. Ruhrberg C. Selective requirements for NRP1 ligands during neurovascular patterning.Development. 2007; 134: 1833-1843Crossref PubMed Scopus (102) Google Scholar In the present study, we determined the role of Sema3-Nrp1 signaling during fetal pulmonary development in a mouse model. Selective loss of Sema3-Nrp1 signaling while maintaining Vegf-Nrp1 interactions in transgenic Nrp1Sema− mice resulted in respiratory distress and high neonatal mortality. Histological examination revealed vascular anomalies reminiscent of ACD, including severely reduced capillary density, thickened alveolar septa containing centrally located capillaries, and hypertensive changes in arteriolar walls. We concluded, therefore, that the early postnatal respiratory distress and mortality observed in the majority of Nrp1Sema− mice arises from aberrant pulmonary microvascular development and disruption of parenchymal morphogenesis. All protocols were in accordance with Canadian Council of Animal Care guidelines and were approved by the Animal Care and Use Committee of the Hospital for Sick Children, Toronto, ON, Canada. Heterozygous Nrp1Sema− mice19Gu C. Rodriguez E.R. Reimert D.V. Shu T. Fritzsch B. Richards L.J. Kolodkin A.L. Ginty D.D. Neuropilin-1 conveys semaphorin and VEGF signaling during neural and cardiovascular development.Dev Cell. 2003; 5: 45-57Abstract Full Text Full Text PDF PubMed Scopus (574) Google Scholar were obtained from the Jackson Laboratory (005245; Bar Harbor, ME) and interbred. Wild-type littermates were used as controls. Primary culture of embryonic day 19.5 (E19.5) fetal rat lung cells was performed as described previously.23Caniggia I. Tseu I. Han R.N. Smith B.T. Tanswell K. Post M. Spatial and temporal differences in fibroblast behavior in fetal rat lung.Am J Physiol. 1991; 261: L424-L433PubMed Google Scholar Purity was confirmed by cytokeratin-18 and vimentin RT-PCR (see Supplemental Figure S1 at http://ajp.amjpathol.org). For the isolation of conditioned medium, epithelial and mesenchymal cells were grown to confluence, rinsed, and then starved in Dulbecco's modified Eagle's medium (DMEM) for 16 hours. After another rinse, 5 mL of fresh DMEM was added for each T-75 flask, and the cells were incubated for 24 hours. Conditioned medium was then pooled from approximately three flasks and was isolated from floating cells via centrifugation. For immunoblotting, conditioned medium was concentrated approximately 100-fold using Amicon Ultra-15 centrifugal filters with a 10-kDa minimum cutoff (Millipore, Billerica, MA). Tissues were homogenized in TRIzol reagent (Invitrogen; Life Technologies, Burlington, ON, Canada). RNA was reverse transcribed using SuperScript III (Invitrogen; Life Technologies) and quantitative real-time PCR (qPCR) was performed using a StepOne real-time PCR system (Applied Biosystems; Life Technologies, Foster City, CA). Fold change was calculated with normalization to 18S.24Livak K.J. Schmittgen T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method.Methods. 2001; 25: 402-408Crossref PubMed Scopus (123392) Google Scholar Primer sequences are given in Table 1.Table 1Primer Sequences Synthesized for qPCR and RT-PCRTargetPrimer or ProbeSequence or catalog number18SForward5′-GTAACCCGTTGAACCCCATT-3′Reverse5′-CCATCCAATCGGTAGTAGCG-5′Aqp5Forward5′-TATCCATTGGCTTGTCGGTCAC-3′Reverse5′-TCAGCGAGGAGGGGAAAAGCAAGTA-3′Cytokeratin-18Forward5′-CATCCACACGAAGACCACCAGTG-3′Reverse5′-GCTGGTACTCTGGCTGGTCCCTA-3′eNOSForward5′-TCCGGAAGGCGTTTGATC-3′Reverse5′-GCCAAATGTGCTGGTCACC-3′Foxf1Forward5′-AGCAGCCATACCTTCACCAA-3′Reverse5′-TAAGATCCTCCGCCTGTTGT-3′Nrp1Forward5′-CACCAAAGATGTCTGAGATAATCCT-3′Reverse5′-GCTGTAGTTGGCTGAGAAACCTTCC-3′Sema3cForward5′-ATCTGGCAAAGGACGATGCTCTTTC-3′Reverse5′-GTGCGTCCACAAACATGGGTTCACT-3′Sema3fForward5′-GCTTCCAGCCACACCTAGAGTC-3′Reverse5′-TAGTCCTTGCTGCCCACATACA-3′Sp-CForward5′-TGGAGAGTCCACCGGATTAC-3′Reverse5′-GAGCAGAGCCCCTACAATCA-3′T1αForward5′-CAAGAAAACAAGTCACCCCAATAGAGATA-3′Reverse5′-GAAGATCCCTCCGACGAAGCCAATG-3′Total VegfForward5′-TGTACCTCCACCATGCCAAGT-3′Reverse5′-CACAGGACGGCTTGAAGATG-3′Vegfr1Forward5′-GAAGCAAGGAGGGCCTCTGATGGTG-3′Reverse5′-GCCAGGTCCCGATGAATGCACTTC-3′Vegfr2Forward5′-CATCGAGCCCTCATGTCTGAA-3′Reverse5′-GCGTGCCCCTTTGCTCTTATA-3′VimentinForward5′-CTCCTACGGTTCACAGCCACTG-3′Reverse5′-GTGTTCTTGAACTCGGTGTTGAT-3′Abca3Qiagen Primer AssayQT00143822Sema3aQiagen Primer AssayQT00192941Sp-BQiagen Primer AssayQT01537529Ang1Forward5′-GCAAATGCGCTCCTCATGCTA-3′Reverse5′-GGAGTAACTGGGCCCTTTGAA-3′ProbeFAM-AGGTTGGTGGTTCCATCCCTGTGG-TAMRAAng2Forward5′-TGACAGCCACGGTCAACAAC-3′Reverse5′-ACGGATAGCAACCGAGCTCTT-3′ProbeFAM-CAGCAGCATGACCTAATGGAGACCGTC-TAMRATie2Forward5′-AACATCCCTCACCTGCATTGC-3′Reverse5′-TTTCGGCCATTCTCTGGTCAC-3′Vegf120Forward5′-AGCAGATGTGAATGCAGACCAA-3′Reverse5′-CTCCTTCCTGCCAGCCTG-3′ProbeFAM-ACAAAGCCAGAAAAATGTGACAAGCCAA-BHQVegf164Forward5′-CATAGAGAGAATGAGCTTCCTACAGC-3′Reverse5′-TGCTTTCTCCGCTCTGAACA-3′ProbeFAM-AGAACAAAGCCAGAAAATCACTGTGAGCCTT-BHQVegf188Forward5′-CGAAAGCGCAAGAAATCCC-3′Reverse5′-TGCTTTCTCCGCTCTGAACA-3′ProbeFAM-TAAATCCTGGAGCGTTCACTGTGAGCC-BHQ Open table in a new tab Details of antibodies are given in Table 2. Immunohistochemistry was performed as described previously.27Groenman F.A. Rutter M. Wang J. Caniggia I. Tibboel D. Post M. Effect of chemical stabilizers of hypoxia-inducible factors on early lung development.Am J Physiol Lung Cell Mol Physiol. 2007; 293: L557-L567Crossref PubMed Scopus (48) Google Scholar Heat-induced antigen retrieval was performed by incubation in 10 mmol/L sodium citrate, pH 6.0, in a microwave pressure cooker; enzymatic antigen retrieval was performed by incubating in 20 μg/mL proteinase K solution.Table 2Antibodies and Dilutions Used for IHC and IBPrimary antibody (host)Source (identifier)Antigen retrievalMethod and dilutionABCA3 (goat)Santa Cruz Biotechnology (sc-48444)IB 1:200β-Actin (mouse)Sigma-Aldrich (A2228)IB 1:50,000CTα (rabbit)As described by Yang et al25Yang J. Wang J. Tseu I. Kuliszewski M. Lee W. Post M. Identification of an 11-residue portion of CTP-phosphocholine cytidylyltransferase that is required for enzyme-membrane interactions.Biochem J. 1997; 325: 29-38Crossref PubMed Scopus (24) Google ScholarIB 1:14,000Pan-cytokeratin (rabbit)Dako (Z0622)Proteinase KIHC 1:200FAS (rabbit)Cell Signaling Technology (3180)IB 1:1000Ki67Dako (M724029)Sodium citrateIHC 1:100NG2 (rabbit)Millipore (AB5320)Sodium citrateIHC 1:200NRP1 (rabbit)Gift of Dr. David D. Ginty26Kolodkin A.L. Levengood D.V. Rowe E.G. Tai Y.T. Giger R.J. Ginty D.D. Neuropilin is a semaphorin III receptor.Cell. 1997; 90: 753-762Abstract Full Text Full Text PDF PubMed Scopus (1000) Google ScholarProteinase KIHC 1:2000; IB 1:7500NRP2 (rabbit)Santa Cruz Biotechnology (sc-5542)Sodium citrateIHC:100xCD31 (rabbit)Santa Cruz Biotechnology (sc-1506R)Sodium citrateIHC 1:2000; IB 1:15,000CD31 (goat)Santa Cruz Biotechnology (sc-1506)Sodium citrateIHC 1:400CD31 (rat)BD Pharmingen (MEC13.3)Proteinase KIHC 1:100SEMA3C (goat)Santa Cruz Biotechnology (sc-27796)Proteinase KIHC 1:100; IB 1:500α-SMA (mouse)Neomarkers (MS-113-R7)Proteinase KIHC 1:400SP-B (rabbit)Chemicon (AB3426)IB 1:4000ProSPC (rabbit)Abcam (ab40879)Sodium citrateIHC 1:2000T1α (Syrian hamster)DSHB (8.1.1)Sodium citrateIHC 1:200VEGFR2 (rabbit)Santa Cruz Biotechnology (sc-315)IB 1:2500Vimentin (mouse)Dako (M7020)NoneIHC 1:200For IHC, antigen retrieval was performed with either 10 mmol/L sodium citrate or 20 μg/mL proteinase K, as described in Materials and Methods.DSHB, Developmental Studies Hybridoma Bank (University of Iowa, Iowa City, IA); IB, immunoblotting. Open table in a new tab For IHC, antigen retrieval was performed with either 10 mmol/L sodium citrate or 20 μg/mL proteinase K, as described in Materials and Methods. DSHB, Developmental Studies Hybridoma Bank (University of Iowa, Iowa City, IA); IB, immunoblotting. After exsanguination, anesthetized mice were tracheally intubated and lungs filled with 2.5% glutaraldehyde under constant pressure (10 cm H2O) for 5 minutes. Tissues were then processed as described previously.28Ridsdale R. Tseu I. Roth-Kleiner M. Wang J. Post M. Increased phosphatidylcholine production but disrupted glycogen metabolism in fetal type II cells of mice that overexpress CTP: phosphocholine cytidylyltransferase.J Biol Chem. 2004; 279: 55946-55957Crossref PubMed Scopus (11) Google Scholar Ultrathin sections were stained in uranyl acetate and lead citrate and examined under a transmission electron microscope (TEM) (JEM1011; JEOL USA, Peabody, MA). All slides and images were analyzed in a masked manner. Images of toluidine blue–stained 1-μm sections were captured using a Nikon digital camera and a 100× oil immersion lens. Images were analyzed using ImageJ software (NIH, Bethesda, MD) and the total area (mm2) of alveolar space was calculated. Both lamellar body-containing type II cell and vessel profiles were counted within these areas and the density of either profile expressed as type II cells or alveolar vessels per area (mm2). A minimum of 50 fields from each animal and two animals from each group were analyzed. vSMC-vessel association was determined from analyzing >10 mm of pulmonary artery and >5 mm of pulmonary vein. Length of arterioles and venules were measured, and the number of vSMCs per millimeter were counted. Capillary-pneumocyte adhesion was determined from TEM images. Endothelial and type I cells were measured along the length of the basal lamina. Any portion with breaks in association of the basal lamina between any of the components was excluded. The percentage of cell adhesion to the basal lamina was calculated by dividing the length of cell adhesion by the total length of basal lamina associated with the endothelial cell/type I pneumocyte. For tissue-to-air ratio, 40 nonoverlapping and nonatelectatic images were captured at ×400 magnification from three sections per animal; sections were >200 μm apart, and the operator was masked to genotype. Images were batch-processed using ImageJ software by color-thresholding to 8-bit black-and-white, eliminating background noise using fine particle analysis, and dividing thresholded tissue area by airspace area. For CD31/cytokeratin expression in lung at E16.5, five nonoverlapping images were captured at ×100 magnification. Fluorescent channels were thresholded and total pixel area of expression was quantified using ImageJ software. For Ki-67 and proSPC measurements, positive chromogenic staining was quantified in a batch by color threshold analysis and was expressed relative to total tissue area using ImageJ software. Bronchoalveolar lavage fluid was obtained by intubating anesthetized mice at postnatal day 1 (P1) with a blunted 27.5-gauge needle and then instilling and withdrawing a single 100-μL bolus of PBS three times before withdrawing and collecting the lavage fluid. Samples were pelleted at 300 × g for 10 minutes and the supernatant was isolated. Samples were spiked with 1 μg of deuterated 16:0/16:0 phosphatidylcholine (Avanti Polar Lipids, Alabaster, AL) as an internal standard and then lipids were extracted. Lipids were analyzed using an API4000 mass spectrometer (MDS Sciex, Concord, ON, Canada).28Ridsdale R. Tseu I. Roth-Kleiner M. Wang J. Post M. Increased phosphatidylcholine production but disrupted glycogen metabolism in fetal type II cells of mice that overexpress CTP: phosphocholine cytidylyltransferase.J Biol Chem. 2004; 279: 55946-55957Crossref PubMed Scopus (11) Google Scholar Immunoblotting was performed as described previously.28Ridsdale R. Tseu I. Roth-Kleiner M. Wang J. Post M. Increased phosphatidylcholine production but disrupted glycogen metabolism in fetal type II cells of mice that overexpress CTP: phosphocholine cytidylyltransferase.J Biol Chem. 2004; 279: 55946-55957Crossref PubMed Scopus (11) Google Scholar All samples were reduced, except those for anti–SP-B. Antibodies used are listed in Table 2. Fluorescent microangiography was performed essentially as described previously.29Dutly A.E. Kugathasan L. Trogadis J.E. Keshavjee S.H. Stewart D.J. Courtman D.W. Fluorescent microangiography (FMA): an improved tool to visualize the pulmonary microvasculature.Lab Invest. 2006; 86: 409-416Crossref PubMed Scopus (24) Google Scholar After the left atrium was transected, warm PBS was infused into the pulmonary circulation via the right ventricle, followed by a 250-μL infusion of AlexaFluor 488-labeled fluorescent microspheres (Invitrogen; Life Technologies) diluted in low-melt agarose. After being held on ice for 10 minutes, lungs were inflated with 4% paraformaldehyde under 10 cm H2O pressure for 5 minutes. After overnight incubation in paraformaldehyde at 4°C, lungs were washed and then stored in 70% ethanol. Left lung lobes were sectioned at 100 μm using a vibrating microtome and then were mounted onto slides. Confocal z-stacks were imaged at ×100 magnification using 5-μm steps and processed using Improvision Volocity version 4.3.2 software (PerkinElmer, Waltham, MA). Lung buds were isolated from E11.5 embryos as described previously.27Groenman F.A. Rutter M. Wang J. Caniggia I. Tibboel D. Post M. Effect of chemical stabilizers of hypoxia-inducible factors on early lung development.Am J Physiol Lung Cell Mol Physiol. 2007; 293: L557-L567Crossref PubMed Scopus (48) Google Scholar Branching was assessed by counting terminal buds every 24 hours. After 72 hours of culture, whole-mount CD31 immunostaining was performed.30Dong X.R. Maguire C.T. Wu S.P. Majesky M.W. Chapter 9. Development of coronary vessels.Methods Enzymol. 2008; 445: 209-228Crossref PubMed Scopus (8) Google Scholar Confocal z-stacks were imaged at ×100 magnification, and a composite picture of the vasculature was generated using Photoshop CS5 software (Adobe Systems, San Jose, CA). Composites were skeletonized, and measurements of vascular patterning relative to total explant surface area were calculated using ImageJ software. Primary cultures of adult male rat pulmonary microvascular endothelial cells (PMVECs) were a gift from Dr. Judy Creighton (University of South Alabama Center for Lung Biology). Cells were grown in DMEM with 10% fetal bovine serum beginning at passage 9 and were used until passage 15. For cell chemotaxis, PMVECs were seeded to 5 × 106 in a T-75 flask and incubated for 36 hours to achieve ∼70% confluency. Cells were then serum-starved for 5 to 6 hours in DMEM, harvested using TrypLE Express enzyme (Invitrogen; Life Technologies), and resuspended in DMEM to 4 × 105 cells/mL. Transwell inserts (24 wells, 8.0-μm pore size) were coated with 5 μg/mL fibronectin (F4759; Sigma-Aldrich, St. Louis, MO) for 1 hour at 37°C and then rinsed. Next, 250 μL of cell suspension (105 cells total) was pipetted into the inner insert chamber, and 750 μL of DMEM with chemoattractant was pipetted into the outer well. Inserts were incubated at 37°C for 22 hours, then rinsed in PBS and fixed with 4% paraformaldehyde. Nonmigrated cells (on the inner insert surface) were removed using a cotton swab, and migrated cells (on the outer insert surface) were stained with 0.6 μmol/L DAPI and imaged using an inverted epifluorescence microscope; five random images at ×100 magnification were captured for each insert. Images were uniformly thresholded, and particle analysis was performed in a batch to count nuclei using ImageJ software. The experiments (both conditioned medium-induced and individual factor-induced migration) were performed three times in triplicate with similar results. Recombinant protein chemoattractants used were 50 ng/mL human VEGF165 and 500 ng/mL mouse Sema3c; 500 ng/mL mouse nerve growth factor receptor (Ngfr) was used as a negative control (all from R&D Systems, Minneapolis, MN). Data are expressed as means ± SEM. Student's unpaired t-test and one-way analysis of variance with Holm-Šidák post-hoc analysis were performed using SigmaPlot version 11 software (Systat Software, San Jose, CA). P < 0.05 was deemed statistically significant. Murine lung development begins at E9.5 and proceeds through defined stages that expand airway and vascular complexity and establish the epithelial-endothelial interface. For preliminary assessment of Sema3 signaling in these processes, gene expression of several Sema3 ligands and receptors in the fetal mouse lung was measured by qPCR (Figures 1A and 2A). Large relative increases in expression were observed for all genes as gestation proceeded, particularly in gene expression of Nrp1, a critical receptor involved in vascular patterning throughout fetal development.19Gu C. Rodriguez E.R. Reimert D.V. Shu T. Fritzsch B. Richards L.J. Kolodkin A.L. Ginty D.D. Neuropilin-1 conveys semaphorin and VEGF signaling during neural and cardiovascular development.Dev Cell. 2003; 5: 45-57Abstract Full Text Full Text PDF PubMed Scopus (574) Google Scholar, 31Kawasaki T. Kitsukawa T. Bekku Y. Matsuda Y. Sanbo M. Yagi T. Fujisawa H. A requirement for neuropilin-1 in embryonic vessel formation.Development. 1999; 126: 4895-4902Crossref PubMed Google ScholarFigure 2NRP1 expression during murine fetal lung development. A: qPCR analysis of prenatal lungs showed increased expression of the Nrp receptors, particularly Nrp1, relative to E11.5. B–E: NRP1 protein expression (green) was examined by costaining smooth muscle cells with α-SMA (pink), the vasculature with CD31 (red), and DAPI nuclear stain (blue). NRP1 expression was observed along the abluminal edge of branching airway epithelium (asterisks, B–D
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