Fetal, but Not Postnatal, Deletion of Semaphorin-Neuropilin-1 Signaling Affects Murine Alveolar Development Stephen Joza1,2, Jinxia Wang1, Irene Tseu1, Cameron Ackerley1, and Martin Post1,2 1 Physiology and Experimental Medicine Program, Hospital for Sick Children, Toronto, Ontario, Canada; and 2Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada
The disruption of angiogenic pathways, whether through genetic predisposition or as a consequence of life-saving interventions, may underlie many pulmonary diseases of infancy, including bronchopulmonary dysplasia. Neuropilin-1 (Nrp1) is a transmembrane receptor that plays essential roles in normal and pathological vascular development and binds two distinct ligand families: vascular endothelial growth factor (Vegf) and class 3 semaphorins (Sema3). Although Nrp1 is critical for systemic vascular development, the importance of Nrp1 in pulmonary vascular morphogenesis is uncertain. We hypothesized that Sema3–Nrp1 and Vegf–Nrp1 interactions are important pathways in the orchestration of pulmonary vascular development during alveolarization. Complete ablation of Nrp1 signaling would therefore lead to interruption of normal angiogenic and vascular maturation processes that are relevant to the pathogenesis of bronchopulmonary dysplasia. We have previously shown that congenital loss of Sema3-Nrp1 signaling in transgenic Nrp1Sema2 mice resulted in disrupted alveolar–capillary interface formation and high neonatal mortality. Here, pathohistological examination of Nrp1Sema2 survivors in the alveolar period revealed moderate to severe respiratory distress, alveolar hemorrhaging, abnormally dilated capillaries, and disintegrating alveolar septa, demonstrating continued instability of the alveolar–capillary interface. Moreover, consistent with a reduced capillary density and consequent increases in vascular resistance, hypertensive remodeling was observed. In contrast, conditional Nrp1 deletion beginning at postnatal day 5 had only a transient effect upon alveolar and vascular development or pneumocyte differentiation despite an increase in mortality. Our results demonstrate that although Sema3–Nrp1 signaling is critical during fetal pulmonary development, Nrp1 signaling does not appear to be essential for alveolar development or vascular function in the postnatal period. Keywords: alveolarization; vascular development; semaphorin; neuropilin
Although the mechanisms that control pulmonary vascular development during alveolarization are incompletely understood, renewed interest has arisen from studies demonstrating that disruption of angiogenic signaling can affect alveolar development. Clinical and experimental models of bronchopulmonary dysplasia (BPD), a disease characterized by alveolar developmental (Received in original form October 9, 2012 and in final form May 13, 2013) This work was supported by operating grant MOP-77751 from the Canadian Institutes for Health Research, by an infrastructure grant (CSCCD) from the Canadian Foundation of Innovation, by an Ontario Graduate Scholarship (S.J.), and by Restracomp, Hospital for Sick Children (S.J.). Correspondence and requests for reprints should be addressed to Martin Post, Ph.D., Physiology and Experimental Medicine, Hospital for Sick Children, 555 University Avenue, Toronto, ON, M5G 1X8 Canada. E-mail: martin.
[email protected] This article has an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org Am J Respir Cell Mol Biol Vol 49, Iss. 4, pp 627–636, Oct 2013 Copyright ª 2013 by the American Thoracic Society Originally Published in Press as DOI: 10.1165/rcmb.2012-0407OC on May 28, 2013 Internet address: www.atsjournals.org
CLINICAL RELEVANCE Abnormal vessel formation may underlie many pulmonary diseases of infancy, including bronchopulmonary dysplasia. Here we show for the first time that, in contrast to its critical role in fetal pulmonary vascular morphogenesis, class 3 semaphorins-neuropilin-1 signaling during postnatal development does not appear to be essential for alveolar development or vascular function.
arrest, exhibit dysmorphic microvasculature and misexpression of key angiogenic growth factors, including the down-regulation of vascular endothelial growth factor (Vegf) signaling (1–4). Likewise, pharmacological disruption of the canonical Vegf receptor Vegfr2 impairs lung vascular and alveolar development (5, 6), and treatment with recombinant Vegf or Vegf downstream effectors can partially restore normal alveolarization in experimental models (3, 7–9). Conversely, despite its proangiogenic and cytoprotective effects, excessive Vegf signaling is implicated in a variety of lung pathologies, including acute lung injury, asthma, pulmonary edema, and alveolar hemorrhaging (10, 11). Many of the angiogenic and pneumotropic effects of Vegf have been attributed to well-known downstream effectors, such as endothelial nitric oxide synthase, which mediates angiogenesis and vascular tone (12). Yet the pleiotropic effects of Vegf upon physiological and pathological development imply that its individual functions (proliferation, migration, permeability, etc.) may use discrete signaling pathways. Neuropilin-1 (Nrp1) is a transmembrane receptor capable of binding Vegf and enhancing discrete Vegf–Vegfr2 pathways through the formation of an Nrp1/ Vegf/Vegfr2 heterocomplex (13, 14). Nrp1 knockdown or deletion thus results in partial or complete attenuation of certain Vegf functions, including endothelial migration, differentiation, angiogenesis, and vascular permeability, while having minimal impact upon proliferation or survival (13–16). Congenital or endothelial-specific Nrp1 deletion results in serious vascular anomalies incompatible with life in the embryonic period (17, 18). Nrp1 also binds class 3 semaphorins (Sema3), which are secreted growth factors originally identified in axonal guidance that have since been implicated in guiding various aspects of endothelial function, including endothelial cell differentiation, angiogenesis, and permeability, independently of Vegf–Vegfr2 (13, 15, 16, 19–21). Thus, although Vegf–Vegfr2 signaling broadly mediates alveolar and pulmonary vascular development, Vegf–Nrp1 and Sema3–Nrp1 interactions might function as auxiliary pathways that fine tune endothelial development and homeostasis. We have recently established an essential role for Sema3– Nrp1 signaling during fetal pulmonary development (22). Knock-in mice encoding an Nrp1 receptor unable to bind Sema3 ligands (Nrp1Sema2 mice) display severe parenchymal
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abnormalities at birth, including few and dilated airspaces, thickened septal walls, surfactant deficiency, decreased vascular density, and drastically attenuated alveolar–capillary interface formation, consistent with an arrest in midembryonic pulmonary development. Whether the processes of alveolarization (alveolar septation, microvascular maturation, etc.) are sensitive to perturbations in Nrp1 signaling is unclear. Indeed, because Nrp1Sema2 mice harbor a congenital mutation, the alveolar phenotype observed in Nrp1Sema2 survivors may arise from defects encountered during fetal development rather than from alveolarization. A single study has reported the down-regulation of Nrp1, Vegfr1, and Vegf2 in a baboon model of BPD (23). Nevertheless, the functional impact of postnatal Nrp1 disruption upon pulmonary development and function is unknown. The goal of this work was to determine whether the loss of Sema3–Nrp1 signaling or complete Nrp1 deletion in the early postnatal period disrupts alveolar and vascular development and thus whether Nrp1 misexpression contributes toward chronic lung diseases of infancy. Given the impact of deficient Sema3–Nrp1 signaling upon fetal lung development (22), the expression of Sema3 and Vegf ligands and receptors in the lung parenchyma during the postnatal period, and the BPD-like phenotype observed upon disruption of Vegfr2 signaling (5, 24), we hypothesized that partial or complete postnatal ablation of Nrp1 signaling would result in arrested alveolar development and vascular attenuation. Thus, we examined Nrp1Sema2 survivors pathohistologically to assess the impact of attenuated alveolar–capillary interface formation upon later pulmonary development. Furthermore, using conditional transgenic mice, we induced complete Nrp1 deletion during postnatal lung morphogenesis and assessed its impact on alveolarization.
MATERIALS AND METHODS Animals All protocols were in accordance with Canadian Council of Animal Care guidelines and approved by the Animal Care and Use Committee of the Hospital for Sick Children (Toronto, ON, Canada). Timedpregnant female Wistar rats were bred and obtained from Charles River (Oakville, ON, Canada). Nrp1Sema2, Nrp1Flox, and Esr1-Cre mouse lines (#005245, #005247, and #004682) were obtained from The Jackson Laboratory (Bar Harbor, ME). Viable and fertile heterozygous Nrp1Sema2 mice were interbred to produce homozygous mutants. Littermate wild-type (WT) mice were used as controls for all Nrp1Sema2 homozygous mutants analyzed. For conditional Nrp1 deletion, the Esr1-Cre and Nrp1Flox lines were bred together to produce Nrp1Flox/Flox;Esr1-Cre, Nrp11/Flox;Esr1-Cre and their respective Esr1-Cre–negative littermates. Because the Esr1Cre transgene is homozygous lethal, Nrp1Flox/Flox females were bred with Nrp1Flox/1;Esr1-Cre males or vice versa. To induce Nrp1 excision, 100 mg tamoxifen (TM) (#T5648; Sigma-Aldrich, Oakville, ON, Canada) was injected intraperitoneally into pups each day on postnatal day (P)5 to P8 (early deletion) or P10 to P13 (late deletion). Esr1-Cre– negative littermates were injected and used as controls.
Immunohistochemistry, Western Blotting, and Quantitative RT-PCR Immunostaining, blotting, and quantitative RT-PCR were performed as described in the online supplement
Morphometric Analysis of Paraffin-Embedded Lung Lungs from were fixed with 4% paraformaldehyde under constant inflation of 20 or 25 cm H2O (age < P19 or P30, respectively) for 5 minutes. Tissues were then fixed in 4% paraformaldehyde overnight at 48 C. Lungs were embedded in paraffin, cut in 3- to 5-mm sections, and stained with hematoxylin and eosin and Hart’s elastin stain (Rowley
Biochemicals, Danvers, MA). Morphometric assessments were performed on coded images to mask the control and transgenic lungs and are described in detail in the online supplement.
Data Presentation Data are presented as mean 6 SEM. Student’s unpaired t test and oneway ANOVA with Holm-Sidak post hoc analysis was used as appropriate. A P value , 0.05 was deemed statistically significant.
RESULTS Sema3 Ligand and Receptor Expression in the Lung Increases during Alveolarization
Alveolarization in rodents and humans primarily occurs during early postnatal development. Therefore, to determine the potential involvement of Sema3 signaling in alveolarization, postnatal rat and mouse lungs were screened by qPCR for mRNA expression of Sema3 ligands that bind Nrp1 (Sema3a and Sema3c) or the related Nrp2 receptor (Sema3c and Sema3f) (Figures 1A and 1B). Genes up-regulated in both species may indicate ligands or receptors of significance for postnatal lung development. In both species, large (6-fold) increases in the expression of Sema3c and moderate (2-fold) increases in Sema3f, as well as the Sema3 signaling receptor Plexin-A1 (Plxna1), were observed during the course of alveolar development relative to birth. The Sema3c binding receptors Nrp1 and Nrp2 were moderately increased (2-fold) in rats but not in mice. Expression of these ligands and receptors remained elevated in adults (data not shown). Unlike Sema3c and Sema3f, Sema3a levels remained low and invariable. Signaling interactions between the developing endothelium and parenchymal epithelium are essential during alveolarization (5). On the basis of our analysis of isolated fetal lung cells and expression in embryonic tissue (22), we expected Sema3c-Nrp1 expression to persist in the parenchymal epithelium and capillary endothelium, respectively. Indeed, Sema3c protein was strongly expressed in the developing alveoli and appeared to be secreted into the extracellular matrix, becoming increasingly expressed subjacent to a-smooth muscle actin (SMA)-positive myofibroblasts in the secondary crests (Figure 1C). Expression in large airway epithelium was not observed at this time point, although a-SMA–positive vascular smooth muscle cells surrounding pulmonary arterioles were also Sema3c positive (Figure 1D). Unlike most Sema3 ligands, Sema3c is able to bind Nrp1 and Nrp2 receptors (25). Nrp1 coexpressed with CD31 (cluster of differentiation 31; also known as platelet endothelial cell adhesion molecule) in the capillary endothelium and faintly in the endothelium of larger vascular structures throughout alveolarization (Figure 2). Contrary to reports by others (26), Nrp1 was not detectable in bronchiolar or Type I cell epithelium, although expression was noted in the branching epithelium during fetal development (22). In contrast, Nrp2 remained consigned to the large airways, particularly in discrete epithelial cells of bronchioles, corresponding with the bronchiolar expression of Sema3f, an Nrp2-obligate ligand (see Figure E1 in the online supplement). Sema3f staining was also observed in alveolar monocytes, indicating that, unlike Nrp1, Nrp2 may be involved in postnatal bronchiolar epithelial development, homeostasis, and/or pulmonary immunity, but it is not essential for vascular development per se. Reduced Alveolarization and Alveolar Hemorrhaging in Nrp1Sema2 Survivors
Although the vast majority of Nrp1Sema2 mice die within the first days of life, some mutants were viable for an extended period of time and were thus amenable for further study.
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Figure 1. Class 3 semaphorins (Sema3) ligand and receptor expression in postnatal lung development. (A and B) Real-time PCR analyses of postnatal rat and mouse lungs for Sema3 ligands and receptors indicated substantial increases in mRNA expression of Sema3c during postnatal alveolar development. Modest increases were also observed in Sema3f, neuropilin-1 (Nrp1), Nrp2, and Plexin-A1 (Plxna1). e, embryonic. (C) Mouse lungs were triple stained with a-smooth muscle actin (SMA) and T1alpha/podoplanin (T1a) (top row) or CD31 (bottom row). Sema3c is present in the developing alveolar septa adjacent to a-SMA during alveolar septation (arrowheads); insets are higher magnifications of boxed regions. (D) Sema3c also colocalizes with a-SMA–positive vascular smooth muscle mural cells surrounding pulmonary arterioles. Consecutive sections from a postnatal day (P)12 wild-type mouse are shown. Bars: 20 mm.
Consistent with reduced and abnormal surfactant production observed in Nrp1Sema2 mutants at P1 (22), survivors at P8 displayed histological signs of respiratory distress, including inflammation and respiratory epithelial injury, although with varying and focal degrees of severity between and among samples. Moderate to severe pneumonitis was observed, as evidenced by the presence of inflammatory infiltrates in bronchioles and alveolar ducts (chiefly neutrophils) and hemosiderin-laden macrophages with alveolar hemorrhaging in the alveolar spaces (Figure 3A, Figure E2A). Some incorporation of fibrous tissue into the thickened septal interstitium and alveolar duct fibrosis were observed, although, unlike clinical respiratory distress syndrome, hyaline membranes, pneumocyte hyperplasia, and histological signs of pulmonary edema (perivascular cuff formation or interstitial edema) were not apparent. Nonetheless, consistent with pulmonary inflammation, hyperproliferation of a-SMA–positive myofibroblasts within the septal interstitium and medial wall smooth muscle hypertrophy in arteries was apparent (Figures 4A, Figure E3B). Indeed, a morphometric assessment of vessel wall thickness demonstrated that the smooth muscle layers in arteriole and artery walls were significantly thicker in the Nrp1Sema2 mutants than in control animals at P8. The thickness of the arteriole smooth muscle layer was significantly less than that of the artery wall in control animals, unlike in Nrp1Sema2 mutants, where the smooth muscle thicknesses in the arteriole and artery wall were essentially the same (Table E3). Signs of acute inflammation appeared to be resolving in P18 survivors (Figure 3B), and an examination of mutant hearts did not reveal overt histological cardiac defects at these time points (not shown).
Enlarged distal airspaces and significantly reduced alveolar numbers were observed in Nrp1Sema2 mutants at P8 and P18 relative to WT littermates (Figure 3C), which appeared to persist in mice that reached adulthood (Figure E2B). Although control mice and relatively spared regions of Nrp1Sema2 mutants displayed normal elastin deposition at secondary crests, erratic and tortuous elastin distribution was observed in inflamed regions, whereas pulmonary arteriolar elastic laminae appeared unaffected (Figure E3A). Severely reduced alveolarization along with dilated interstitial capillaries was observed at P8 and P18 (Figures 4B–4D). A quantitative morphometric assessment revealed that the alveolar capillary density in Nrp1Sema2 mutants at P8 was reduced by 27% (214 versus 156 capillaries/ mm2 alveolar air space, control mice versus Nrp1Sema2 mutants). Alveolar areas with severe inflammation and hemorrhaging also displayed abnormal reticular fibers, indicative of connective tissue destabilization when compared with relatively spared regions (Figure 4B). Early Postnatal Nrp1 Deletion Displays Haploinsufficiency and High Mortality
The congenital origin of the Nrp1Sema2 mutation suggests that the dramatic phenotype observed in Nrp1Sema2 survivors may result from abnormal fetal lung development rather than from disrupted alveolarization. To assess the effect of postnatal Nrp1 deletion on alveolar development, we generated inducible Nrp1 knockout mice by crossing Nrp1Flox transgenic mice with mice harboring a TM-responsive and ubiquitously expressed Esr1Cre transgene (18, 27). CRE-mediated Nrp1 excision in these compound mice is robustly induced in a variety of tissues after
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Figure 2. Nrp1 expression in control and Nrp1D/D mice. (A) Tamoxifen (TM) was administered intraperitoneally to pups at P5 to P8 or P10 to P13 to induce Nrp1 excision in neonatal mice. Sac, sacrificed. (B) Representative Nrp1 immunoblot and densitometry show partial and complete Nrp1 knockout in Nrp11/D and Nrp1D/D lung lysates, respectively, injected by the early postnatal protocol (n ¼ 4 per group; *P , 0.05). OD, optical density. (C) Nrp1 immunostaining strongly localized to the CD31-positive capillary endothelium throughout alveolarization. Nrp1 expression was completely ablated in P12 Nrp1D/D littermates after early postnatal TM administration. Some autofluorescent erythrocytes are observed, but they do not colocalize with CD31. (D) No Nrp1 expression was observed in T1a-positive epithelium. Arrows indicate T1a-positive lymphatic vessels, which are Nrp1 negative. Insets are magnified from boxed regions. v, vessel. Bars: 25 mm.
TM administration. For simplicity, mice that have undergone excision will be referred to as Nrp1D/D homozygous or Nrp11/D hemizygous knockouts. Littermates that received TM but did not express Esr1-Cre (Nrp1Flox/Flox and Nrp11/Flox mice) were used as controls. TM was administered directly to pups at P5 to P8, with assessments at P12 and P19 (Figure 2A). Immunoblotting and immunofluorescence of lung, kidney, and liver samples indicated essentially complete excision at P12, although expression in P12 brains persisted, suggesting limited recombination outside of visceral organs (Figure 2B, Figure E4). Nrp1 immunostaining in the pulmonary capillary endothelium was completed ablated in Nrp1D/D mice (Figure 2C). Nrp1D/D mice injected at P5 to P8 were significantly growth retarded by P12, and more than half died before assessment at P19, compared with Esr1-Cre–negative littermates, which were viable (Table 1). Nrp11/D hemizygous mice (mice with one functional copy of Nrp1) displayed similar degrees of growth retardation and mortality and expressed significantly reduced levels of Nrp1 mRNA and protein in the lung compared with control littermates (Table 1, Figure 2B). Thus, Nrp1 appears to display haploinsufficiency in the early postnatal protocol because a single functional Nrp1 allele produces inadequate levels of Nrp1 protein for normal viability. Early Postnatal Nrp1 Deletion Results in a Transient Decrease in Alveolarization in Nrp1D/D and Nrp11/D Mice
To determine if Nrp1 knockout affects alveolar development, lungs from Nrp1D/D and Nrp11/D mice were examined histologically. Nrp1D/D and Nrp11/D mice injected at P5 to P8 showed a moderate but significant decrease in alveolar density at P12 relative to controls as assessed by mean linear intercept and
radial alveolar count, although no overt histological signs of respiratory distress were observed (Table 1, Figure 5A). Indeed, secondary crests appeared to form normally as assessed by elastin staining (Figure 5B), although a decrease in alveolar cell proliferation at P12 was noted as assessed by phosphohistone H3–positive staining (2.50 6 0.25%, 1.10 6 0.15%, and 1.23 6 0.59% positive cells in control, Nrp11/D, and Nrp1D/D, respectively; P ¼ 0.016 [n ¼ 3]). Pup weight and alveolar density normalized to controls by P19 (Table 1, Figure 5), suggesting a catch-up in alveolarization and development in Nrp1 knockout survivors. TM-induced knockouts were also performed at a later postnatal period (TM on P10–P13), with analyses performed at P30 to determine the effect on late alveolarization (34). Although Nrp1 excision in lungs appeared essentially complete (Figure E4B), Nrp1D/D mice were of comparable body weight to control littermates, and lungs were histologically and morphometrically normal (Table 2, Figure E5). Microvascular Endothelial and Pericyte-Like Cell Association Is Normal in Nrp1D/D Mice
Enhanced Sema3–Nrp1 signaling can improve pericyte– endothelial cell association and, consequently, vascular normalization in the context of tumor biology (13, 21, 28). On the basis of neuron–glial antigen (NG)2 immunostaining, we (22) and others (29) have observed pericyte-like cells associated with the pulmonary microvasculature. In Nrp1Sema2 survivors at P8, dilated capillaries, consistent with interstitial lung inflammation and alveolar hemorrhaging, were observed, although it was not possible to determine if endothelial–pericyte-like cell association was reduced (Figure 6A). However, we often observed an overlap between inflammatory a-SMA–positive
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Figure 3. Alveolar hemorrhaging and arrested alveolar development in Nrp1Sema2 survivors. (A) Nrp1Sema2 mice at P8 presented with variable degrees of respiratory distress. Moderate to severe alveolar hemorrhaging with activated macrophages (arrowheads in vii) are observed. Neutrophils within fibrosed alveolar ducts were frequently encountered (compare iv with viii). Thickened septa without interstitial edema are observed in mutants. Bars: 50 mm. (B) In P18 survivors, severe inflammation and alveolar hemorrhaging were largely resolved, although residual signs of phagocytic influx are apparent and alveoli remain dilated and fewer in number than in controls. Bars: 50 mm. (C) Reduced alveolar development in Nrp1Sema2 mice at P8 and P18 was quantified by radial alveolar count (n ¼ 3–4 per group; *P , 0.05).
myofibroblasts and NG2 staining, suggesting that, as observed in other forms of acute inflammation (30), pericyte-like cells may express myofibroblast markers and contribute to the excessive extracellular matrix deposition characteristic of fibrosis. In P18 survivors, we observed normal NG2 and absent a-SMA expression in relatively spared regions of the parenchyma, but we observed a similar NG2/a-SMA overlap in focal areas of residual inflammation (data not shown). In contrast, NG2 and a-SMA stained discrete cells within the alveolar spaces in Nrp1D/D mice at P12, with no apparent differences from littermate controls (Figure 6B).
Reduced Vegfr2 Expression in Nrp1 Knockouts
Vegf–Nrp1 and Sema3–Nrp1 signaling plays an essential role in endothelial proliferation and cell differentiation in vitro (15), whereas in the lung, Vegf enhances expression of the essential surfactant protein (Sp)-B in cultured Type II cells and enhances surfactant production (8, 10). Moreover, there has been some suggestion that aberrant Sema3–Nrp1 signaling affects pneumocyte maturation and viability (26, 31). Thus, to determine if ablated Nrp1 signaling affects parenchymal differentiation or function during alveolar development, expression levels of Type
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Figure 4. Hypertensive changes and extracellular and capillary abnormalities in Nrp1Sema2 mice. (A) Intraacinar pulmonary arteries are observed (note well-defined double elastic laminae) by elastin staining. Possible increases in medial wall thickness were qualitatively observed in Nrp1Sema2 mice. Br, bronchiole; Pa, pulmonary artery. Bar: 50 mm. (B) Characteristic reticular fibers in control mice at P8 show an intact septal interstitium. In contrast, Nrp1Sema2 mutants show abnormal interstitial thinning and a beaded appearance of reticular fibers, indicative of septal disintegration. Abnormally dilated capillaries are observed in these areas (bottom panel). Bottom panels are H&E staining of similar fields as middle panels. Bar: 50 mm. (C) CD31 stains the capillary endothelium and T1a stains Type I cell epithelium. Bar: 50 mm. (D) Ultrastructural analysis by transmission electron microscopy of Nrp1Sema2 mutant and control mice at P8. Note the lack of vessels lining the alveoli (arrows) in mutant mice. In areas where the alveolar wall was thickened, numerous lipid containing fibroblasts were seen throughout the interstitium (asterisks). A macrophage (M) is seen in the air space, and a neutrophil (N) is seen in the interstitium. Bars: 2 mm.
or trending increases in mRNA levels of genes essential for normal Type II cell function, including ATP-binding cassette subfamily A member 3 (Abca3), Sp-B, choline-phosphate cytidylyltransferase A (Pcyt1a), and Sp-C, were noted, these changes were less remarkable at the protein level (Figure E6). Significant decreases in Vegfr2 expression were observed in Nrp1D/D and Nrp11/D mice, whereas Vegfr1 and total Vegf transcripts were unchanged (Figure E7).
I and Type II cell marker genes were assessed in Nrp1 knockouts at P12. A moderate but significant increase in mRNA expression of the Type I cell marker aquaporin-5 (Aqp5) was noted in the lungs of Nrp1D/D mice, which was not recapitulated by immunoblotting (Figure E6). Also, the level of transcripts for T1alpha/ podoplanin (T1a), another Type I–specific gene, was unchanged in control and Nrp1D/D mice. Likewise, although some significant
TABLE 1. WEIGHT, MORTALITY, AND LUNG MORPHOMETRY FROM Nrp1D/D AND Nrp11/D MUTANTS AND CONTROL MICE UNDERGOING EARLY POSTNATAL Nrp1 DELETION Weight (g) TM
Genotype
P5-8
Nrp1Flox/Flox Nrp1D/D Nrp1Flox/1 Nrp11/D
5.5 3.9 5.6 3.6
RAC
P12
P19
6 6 6 6
7.0 6 0.5 5.9 6 0.8 nd nd
0.4 0.6* 0.4 0.1*
MLI
P12
P19
6 6 6 6
9.3 6 0.4 9.0 6 0.8 nd nd
9.7 7.7 10.2 7.4
0.2 0.5* 0.4 0.5*
P12
P19
Mortality P19
6 6 6 6
52.3 6 1.5 56.3 6 4.2 nd nd
0/15 12/18 0/7 5/9
56.7 68.6 55.4 62.4
2.2 3.5* 1.5 2.5*
Definition of abbreviations: MLI, mean linear intercept; nd, not determined; P, postnatal day; RAC, radial alveolar count; TM, tamoxifen. * P , 0.05; n ¼ 5–6 per group at P12, and n ¼ 4 per group at P19.
Joza, Wang, Tseu, et al.: Neuropilin-1 in Postnatal Pulmonary Development
Figure 5. Nrp1D/D and Nrp11/D histology at P12 and P19. (A) Transient decreases in alveolar development are observed in Nrp1D/D and Nrp11/D mice at P12 after early postnatal Nrp1 deletion, which are resolved by P19. Bar: 100 mm. (B) Elastin deposition at secondary crests appears normal in control and mutant mice. Bar: 50 mm.
DISCUSSION We describe the effects of disrupted Nrp1 signaling upon postnatal alveolar development. Although congenitally mutated Nrp1Sema2 mice are largely unviable, examination of survivors showed disrupted alveolar and vascular morphogenesis during early postnatal life. Disruption of Vegfr2 signaling after birth causes arrested alveolar development and vascular attenuation similar to BPD (5, 24), and thus we predicted that deletion of a closely associated receptor would phenocopy these results. Moreover, Nrp1 is known to be essential for sprouting angiogenesis (32); thus, we speculated that Nrp1 would be required to promote the postulated microvascular sprouting that occurs during late alveolarization (33, 34). Nevertheless, global Nrp1 deletion during the postnatal period appeared to have a minimal and transient impact upon alveolar development and normal vascular function despite an increase in mortality. Moreover, differences in pneumocyte differentiation did not appear to be severely affected, thus demonstrating that Nrp1 does not significantly contribute to alveolarization. Complete Nrp1 knockout during alveolarization did not recapitulate the severe inflammation, dramatically reduced alveolarization, or attenuated capillary density observed in Nrp1Sema2 survivors, suggesting that the Nrp1Sema2 phenotype primarily arises from defects encountered during fetal development. However, it is possible that the postnatal Nrp1 knockout was not induced early enough to disrupt key aspects of Nrp1 signaling in the initiation of
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alveolarization or that, by knocking out Sema3–Nrp1 and Vegf–Nrp1 signaling, we inadvertently rescued the Nrp1Sema2 phenotype. Nrp1 Sema2 survivors exhibited destabilization and disintegration of the septal walls with resultant alveolar hemorrhaging, consistent with abortive alveolar–capillary interface formation and endothelial detachment from the basal lamina observed in P1 mutants (22). Neutrophil and macrophage accumulations in the distal lung were also observed, particularly in areas of tortuous elastin fiber deposition and septal disintegration. In the context of BPD, phagocytic influx can contribute toward ventilator-induced lung injury and developmental arrest through the release of reactive oxygen and nitrogen species, proteolytic enzymes, and proinflammatory cytokines (35, 36) and may thus have played a similar role in Nrp1Sema2 mice. Macrophages are also essential for inflammatory resolution via the clearance of debris and apoptotic neutrophils (37); hence, the presence of hemosiderin-laden macrophages is consistent with a physiological response to alveolar hemorrhaging. Due to the rarity of Nrp1Sema2 mutants, we were unable to fully assess the mechanisms that may have contributed to septal wall destruction and alveolar hemorrhage. Nevertheless, it appears that lung immaturity and abnormal vascular development led to respiratory distress in Nrp1Sema2 neonates at birth, followed by a progressive inflammatory response with phagocytic recruitment in Nrp1Sema2 survivors. Because transient increases in inflammatory cytokines during early alveolarization may have lifelong consequences upon alveolar morphology and function (38), it is possible that developmental arrest in Nrp1Sema2 survivors was due to the inflammatory cascade and microvascular immaturity rather than deficient postnatal Nrp1 signaling. Alveolar simplification and vessel attenuation would be expected to cause hypoxemia, decreased vascular compliance, and increased pulmonary vascular resistance (12). Indeed, extensive hypertensive wall remodeling that manifests with pulmonary hypertension (39) was observed in the arterioles and arteries of Nrp1Sema2 survivors. Furthermore, alveolar hemorrhaging with hemosiderin-laden macrophages is indicative of noncardiogenic pulmonary edema, possibly arising from a hypertensive crisis and/or alveolar–capillary instability (40). Fluid accumulation in perivascular cuffs or within the septal interstitium was not apparent, arguing against cardiogenic pulmonary edema. Alveolar edemal fluid may have been washed out upon tissue fixation, explaining its absence upon histological examination; an analysis of bronchoalveolar lavage fluid would be required for this finding to be conclusive. One previous study described postnatal conditional Nrp1 deletion in the airway epithelium, which resulted in a mild increase in airspace size and enhanced parenchymal susceptibility to chronic cigarette smoke exposure (26). This was attributed to increased pneumocyte apoptosis, suggesting that Nrp1 contributes to epithelial homeostasis, particularly because Nrp1 expression may be down-regulated in smokers with chronic obstructive pulmonary disease (41). However, that study did not show conclusive Nrp1 localization to the epithelium, and the degree of Nrp1 deletion in total lung lysates appeared miniscule, especially in comparison to the complete excision in our protocol. Moreover, despite using the same primary antibody, we have been unable to observe Nrp1 expression in the parenchymal epithelium during postnatal alveolarization despite clear expression in the branching epithelium during fetal morphogenesis (22). Although we did note Nrp2 and Sema3f expression in bronchiolar epithelium, we have consistently observed strong Nrp1 expression in the pulmonary endothelium throughout fetal and postnatal development, which was completely abrogated after excision. Thus, it may be that any Nrp1-mediated
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TABLE 2. WEIGHT, MORTALITY, AND LUNG MORPHOMETRY FROM Nrp1D/D MUTANTS UNDERGOING LATER POSTNATAL Nrp1 DELETION* TM
Genotype Flox/Flox
P10-13 Nrp1 Nrp1D/D
Weight (g) P30 15.7 6 0.4 14.6 6 0.7
RAC P30
MLI P30
10.8 6 0.2 60.6 6 5.2 10.9 6 0.3 61.7 6 5.2
Mortality P30 0/13 0/6
Definition of abbreviations: MLI, mean linear intercept; P, postnatal day; RAC, radial alveolar count; TM, tamoxifen. * For morphometry, n ¼ 3–4 per group from two litters; no significant differences were observed.
epithelial homeostasis arises as an indirect effect of endothelial Nrp1 expression. Nrp1 haploinsufficiency during early postnatal development precluded the study of Sema3–Nrp1 signaling in isolation using a transgenic approach. That is, because one normally functioning Nrp1 allele is insufficient for normal development, it was not possible to conditionally knock out Nrp1 in a hypothetical Nrp1Sema2/Flox mouse line without introducing haploinsufficiency as a confounder. However, the moderate and transient impact of total Nrp1 deletion upon alveolarization argues against a significant contribution of Sema3–Nrp1 signaling for normal alveolar septation. Although Nrp1 haploinsufficiency has not been previously reported, embryonic lethality (42) and postnatal growth retardation (43) have been observed in Vegf, but not in Vegfr1 or Vegfr2, hemizygotes (44, 45). Significantly reduced Vegfr2 expression in the lungs of Nrp1D/D and
Nrp11/D mice at P12 were observed, and Nrp1 knockdown is associated with Vegfr2 down-regulation, possibly through enhanced endocytosis and degradation (46). Because Vegfr2 signaling is essential for pulmonary vascular growth and alveolarization, it is possible that decreased Vegfr2 expression in Nrp1 mutants contributed to the delay in alveolar development. Alternatively, it is possible that reduced Vegfr2 expression is a consequence of a transient reduction in capillary density coincident with the transient decrease in alveolarization. Because Vegf–Nrp1 and Sema3–Nrp1 signaling enhances Vegfr2-mediated endothelial differentiation in vitro (15) and because Vegf enhances surfactant production (8, 47), we postulated that Nrp1 deletion would attenuate these effects during development. However, despite the moderate arrest in alveolar development and alveolar cell proliferation, Nrp1 knockout had only a minimal effect upon pneumocyte differentiation at P12, based upon the expression of Type I and Type II cell markers. We here, and others (29), have observed pericyte-like cells associated with the pulmonary microvasculature on the basis of NG2 immunostaining. In Nrp1Sema2 survivors, we noted increased a-SMA expression, indicative of inflammatory myofibroblasts, with overlap between a-SMA and NG2 staining, suggesting that, as observed in other forms of inflammation (30, 48), NG2-positive pericytes may differentiate into myofibroblasts and contribute to excessive extracellular matrix protein deposition. Whether such pericyte-like cells are involved in the inflammatory process and whether this observation constitutes actual transdifferentiation is not certain. We observed normal a-SMA/NG2/CD31 expression patterns in Nrp1D/D
Figure 6. Pericyte-like cell staining in Nrp1Sema2 and Nrp1D/D mutants. (A) Pericyte-like cells, immunostained with neuron–glial antigen (NG)2, associate with the capillary endothelium and are distinct from a-SMA–positive myofibroblasts in P8 littermate lungs. Pericyte-like cells in Nrp1Sema2 mutants at P8 show possibly reduced association with dilated capillaries (arrowheads) and appear to coexpress a-SMA. (B) Normal pericyte, myofibroblast, and endothelial architecture is observed in control and Nrp1D/D mice at P12 after early postnatal Nrp1 deletion. Bars: 25 mm. RBC, autofluorescent red blood cells; WT, wild type.
Joza, Wang, Tseu, et al.: Neuropilin-1 in Postnatal Pulmonary Development
mice, suggesting that the association between pericyte-like cells and capillary endothelium in the lung is not affected by early postnatal Nrp1 deletion despite the possible involvement of Sema3–Nrp1 signaling in the context of tumor vascular normalization (13, 21, 28). We have been unable to determine the physiological basis for the high mortality and growth retardation observed in early postnatal Nrp1 deletion despite additional examination of mutant hearts, kidneys, intestines, and livers (data not shown). Subcutaneous injection of Vegf inhibitors during postnatal development reduces overall body weight and Vegfr2 protein expression (3), similar to that observed in the early postnatal protocol. Although Nrp1 deletion had only a moderate impact upon alveolar development, Nrp1 may still be essential for normal pulmonary function or homeostasis. Functional measurements during normal development and during the response to pathological stress, such as in models of acute respiratory distress or pulmonary fibrosis, may be required to tease out less conspicuous roles for Sema3–Nrp1 and Vegf-Nrp1 signaling. Beyond axonal guidance, Sema3–Nrp1 signaling has been implicated in fetal cardiac, renal, and peripheral vascular morphogenesis (19, 49). Additional mechanisms have recently been uncovered in the regulation of endothelial permeability and tumor angiogenesis (16, 28). However, in contrast to its critical role in fetal pulmonary vascular morphogenesis (22), Sema3– Nrp1 signaling during postnatal development does not appear to be essential for alveolar development or vascular function. Persistent Nrp1 and Sema3 expression in the lung throughout postnatal life may reflect a constitutive homeostatic role in the lung parenchyma outside of alveolar development. Author disclosures are available with the text of this article at www.atsjournals.org.
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Acknowledgments: The authors thank Angie Griffin-Aizic for excellent technical support in mouse colony maintenance.
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