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Pericyte-like cell staining in Nrp1 Sema2 and Nrp1 D/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 Nrp1 Sema2 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 Nrp1 D/D mice at P12 after early postnatal Nrp1 deletion. Bars: 25 mm. RBC, autofluorescent red blood cells; WT, wild type.
Source publication
The disruption of angiogenic pathways, whether through genetic predisposition or in consequence of life-saving interventions, may underlie many pulmonary diseases of infancy, including bronchopulmonary dysplasia (BPD). Neuropilin-1 (Nrp1) is a transmembrane receptor that plays essential roles in normal and pathological vascular development, and bin...
Contexts in source publication
Context 1
... 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 Nrp1 Sema2 survivors at P8, dilated capillaries, consistent with interstitial lung inflamma- tion 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 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 exces- sive extracellular matrix deposition characteristic of fibrosis. ...
Context 2
... 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 Nrp1 D/D mice at P12, with no apparent differences from litter- mate controls ( Figure 6B). ...
Context 3
... moderate but significant increase in mRNA expression of the Type I cell marker aquaporin-5 (Aqp5) was noted in the lungs of Nrp1 D/D mice, which was not recapitulated by immuno- blotting ( Figure E6). Also, the level of transcripts for T1alpha/ podoplanin (T1a), another Type I-specific gene, was unchanged in control and Nrp1 D/D mice. ...
Context 4
... the level of transcripts for T1alpha/ podoplanin (T1a), another Type I-specific gene, was unchanged in control and Nrp1 D/D mice. Likewise, although some significant or trending increases in mRNA levels of genes essential for nor- mal Type II cell function, including ATP-binding cassette subfam- ily A member 3 (Abca3), Sp-B, choline-phosphate cytidylyltransferase A (Pcyt1a), and Sp-C, were noted, these changes were less remark- able at the protein level ( Figure E6). Significant decreases in Vegfr2 expression were observed in Nrp1 D/D and Nrp1 1/D mice, whereas Vegfr1 and total Vegf transcripts were unchanged (Fig- ure E7). ...
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Tumor-specific tissue-penetrating peptides deliver drugs into extravascular tumor tissue by increasing tumor vascular permeability through interaction with neuropilin (NRP). Here we report that a prototypic tumor-penetrating peptide iRGD (amino acid sequence: CRGDKGPDC) potently inhibits spontaneous metastasis in mice. The anti-metastatic effect wa...
Citations
... Also, the expression level of the NRP1 receptor, recently shown to facilitate SARS-CoV-2 cell entry [50], was decreased in groups 2 and 3. In support of this notion, disruption of SEMA-NRP1 signaling in murine lungs was reported to cause instability of the alveolar-capillary interface and hypertensive remodeling reminiscent of ACD [51,52]. Our data on SEMA-NRP1 deregulation further imply the role of this signaling pathway in lung development. ...
Background
Alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) is a rare lethal congenital lung disorder in neonates characterized by severe progressive respiratory failure and refractory pulmonary hypertension, resulting from underdevelopment of the peripheral pulmonary tree. Causative heterozygous single nucleotide variants (SNVs) or copy-number variant (CNV) deletions involving FOXF1 or its distant lung-specific enhancer on chromosome 16q24.1 have been identified in 80–90% of ACDMPV patients. FOXF1 maps closely to and regulates the oppositely oriented FENDRR , with which it also shares regulatory elements.
Methods
To better understand the transcriptional networks downstream of FOXF1 that are relevant for lung organogenesis, using RNA-seq, we have examined lung transcriptomes in 12 histopathologically verified ACDMPV patients with or without pathogenic variants in the FOXF1 locus and analyzed gene expression profile in FENDRR -depleted fetal lung fibroblasts, IMR-90.
Results
RNA-seq analyses in ACDMPV neonates revealed changes in the expression of several genes, including semaphorins ( SEMA s), neuropilin 1 ( NRP1 ), and plexins ( PLXN s), essential for both epithelial branching and vascular patterning. In addition, we have found deregulation of the vascular endothelial growth factor (VEGF) signaling that also controls pulmonary vasculogenesis and a lung-specific endothelial gene TMEM100 known to be essential in vascular morphogenesis. Interestingly, we have observed a substantial difference in gene expression profiles between the ACDMPV samples with different types of FOXF1 defect. Moreover, partial overlap between transcriptome profiles of ACDMPV lungs with FOXF1 SNVs and FENDRR -depleted IMR-90 cells suggests contribution of FENDRR to ACDMPV etiology.
Conclusions
Our transcriptomic data imply potential crosstalk between several lung developmental pathways, including interactions between FOXF1-SHH and SEMA-NRP or VEGF/VEGFR2 signaling, and provide further insight into complexity of lung organogenesis in humans.
... Tnf is a potent pro-inflammatory cytokine increased in both BPD patients and in animal models of BPD 22,23 . Nrp1 is critical for normal branching morphogenesis 24 , while Dag1 plays an important role in airway epithelial wound repair 25 . In addition, the analysis revealed a potential regulation of AT2 cells by Angpt2, which was upregulated by hyperoxia in two capillary clusters (Cap, Cap-a) ( Fig. 2e; Supplementary Fig. 4e). ...
During late lung development, alveolar and microvascular development is finalized to enable sufficient gas exchange. Impaired late lung development manifests as bronchopulmonary dysplasia (BPD) in preterm infants. Single-cell RNA sequencing (scRNA-seq) allows for assessment of complex cellular dynamics during biological processes, such as development. Here, we use MULTI-seq to generate scRNA-seq profiles of over 66,000 cells from 36 mice during normal or impaired lung development secondary to hyperoxia with validation of some of the findings in lungs from BPD patients. We observe dynamic populations of cells, including several rare cell types and putative progenitors. Hyperoxia exposure, which mimics the BPD phenotype, alters the composition of all cellular compartments, particularly alveolar epithelium, stromal fibroblasts, capillary endothelium and macrophage populations. Pathway analysis and predicted dynamic cellular crosstalk suggest inflammatory signaling as the main driver of hyperoxia-induced changes. Our data provides a single-cell view of cellular changes associated with late lung development in health and disease.
... NRP1 binds SEMA3A SEMA3B, SEMA3C and SEMA3D via its a1 and a2 domains ( Figure 1) and forms a holoreceptor by interacting with members of the transmembrane receptors plexins (PLXNs) such as PLXND1 and PLXNA4 [109]. Mouse mutants expressing a form of NRP1 with a point mutation in the a1 domain abrogating SEMA3A binding, show defects in axonal pathfinding, in addition to cardiac and lung vascular abnormalities [110][111][112]. In EC, binding of SEMA3B to NRP1 has anti-angiogenic activity by collapsing the actin cytoskeleton and inhibits VEGF-A signalling resulting in apoptosis and reduced angiogenesis [113] (Figure 1). ...
Endothelial cells drive the formation of new blood vessels in physiological and pathological contexts such as embryonic development, wound healing, cancer and ocular diseases. Once formed, all vessels of the vasculature system present an endothelial monolayer (the endothelium), lining the luminal wall of the vessels, that regulates gas and nutrient exchange between the circulating blood and tissues, contributing to maintaining tissue and vascular homeostasis. To perform their functions, endothelial cells integrate signalling pathways promoted by growth factors, cytokines, extracellular matrix components and signals from mechanosensory complexes sensing the blood flow. New evidence shows that endothelial cells rely on specific metabolic pathways for distinct cellular functions and that the integration of signalling and metabolic pathways regulates endothelial-dependent processes such as angiogenesis and vascular homeostasis. In this review, we provide an overview of endothelial functions and the recent advances in understanding the role of endothelial signalling and metabolism in physiological processes such as angiogenesis and vascular homeostasis and vascular diseases. Also, we focus on the signalling pathways promoted by the transmembrane protein Neuropilin-1 (NRP1) in endothelial cells, its recently discovered role in regulating mitochondrial function and iron homeostasis and the role of mitochondrial dysfunction and iron in atherosclerosis and neurodegenerative diseases.
... A role for Nrp1 in lung development is also supported by an earlier finding that constitutive loss of semaphorin-NRP1 signaling due to knock-in of mutant Nrp1 unable to bind Sema3 ligands led to acute respiratory distress and high neonatal mortality, which was associated with loss of alveolar myofibroblasts at sites of presumptive septal tips (18) phenotype, our findings indicate a time-specific requirement for Nrp1-expressing SMCs in the early stages of postnatal lung development, whereas Nrp1 loss in SMCs at later stages of postnatal lung development may not be required or could be compensated by NRP2 and/or other mechanisms. This conclusion is consistent with the report that postnatal deletion of Nrp1 at P5, using the tamoxifen-inducible Esr1-Cre transgene, only caused a mild, transient alveolar and vascular phenotype, indicating that expression of Nrp1 after P5 is not essential for alveolar development or vascular function (19). Given that PDGF-A is essential for alveolar (septal) myofibroblast development and alveogenesis (7), the impairment of Nrp1-deficient pulmonary myofibroblast migration in response to PDGF-AA in vitro and the impairment of PDGF-BB-induced SMC outgrowth in Nrp1 SMCiKO aortic rings suggest that Nrp1 may be important for PDGF signaling in both SMCs and myofibroblast migration and recruitment to septae during alveolar maturation. ...
Neuropilin 1 (NRP1) is important for neuronal and cardiovascular development due to its role in conveying class 3 semaphorin and vascular endothelial growth factor signaling, respectively. NRP1 is expressed in smooth muscle cells (SMCs) and mediates their migration and proliferation in cell culture and is implicated in pathological SMC remodeling in vivo. To address the importance of Nrp1 for SMC function during development, we generated conditional inducible Nrp1 SMC-specific knockout mice. Induction of early postnatal SMC-specific Nrp1 knockout led to pulmonary hemorrhage associated with defects in alveogenesis and revealed a specific requirement for Nrp1 in myofibroblast recruitment to the alveolar septae and PDGF-AA-induced migration in vitro. Furthermore, SMC-specific Nrp1 knockout inhibited PDGF-BB-stimulated SMC outgrowth ex vivo in aortic ring assays and reduced pathological arterial neointima formation in vivo. In contrast, we observed little significant effect of SMC-specific Nrp1 knockout on neonatal retinal vascularization. Our results point to a requirement of Nrp1 in vascular smooth muscle and myofibroblast function in vivo, which may have relevance for postnatal lung development and for pathologies characterized by excessive SMC and/or myofibroblast proliferation.
... Pulmonary epithelial, endothelial, and interstitial mesenchymal cells express nrp1, and various strategies have been used to disrupt nrp1 in the lung (30,31). In pdgfra-expressing pulmonary mesenchymal cells, nrp1 gene expression is higher at P7 than during the late embryonic stages (17,18). ...
... When NRP1 could not bind SEMA3, mice exhibited immature, fragile, misplaced pulmonary microvessels with reduced PC coverage (30). Inducible promoters (surfactant protein C or a universally expressed estrogen receptor Cre) have been used to disrupt nrp1, yielding reversible air space enlargement (31). ␣-Smooth muscle actin (␣-SMA)-and neural/glial antigen-2 (NG2, CSPG4)-containing mesenchymal cells were displaced from the septal tips (30,31). ...
... Inducible promoters (surfactant protein C or a universally expressed estrogen receptor Cre) have been used to disrupt nrp1, yielding reversible air space enlargement (31). ␣-Smooth muscle actin (␣-SMA)-and neural/glial antigen-2 (NG2, CSPG4)-containing mesenchymal cells were displaced from the septal tips (30,31). Although these studies showed that NRP1 is required for development of the gas-exchange surface, several important questions remain. ...
Secondary alveolar septa are mostly generated after birth in humans and in mice this is completely accomplished postnatally, when mechanical stresses vary as airspace pressure oscillates. Alveolar mesenchymal cells deposit elastic fibers which limit cellular strain, but while the elastic fiber network is incomplete, this function is also served by the intracellular cytoskeleton. Intermediate filament proteins support deformation during cellular division and migration, which occur during septal elongation. Because platelet-derived growth factor-alpha (PDGFRα) signaling is essential for alveolar septation, we hypothesized that neuropilin-1 (NRP1) may link PDGFRα to cytoskeletal deformation. During cell migration, NRP1 links receptor tyrosine kinase signaling to cytoskeletal and focal adhesion remodeling. Therefore, we examined the consequences of nrp1-gene deletion in alveolar mesenchymal cells, including myofibroblasts and pericytes. Neuropilin-1-depletion reduced the proportion of mesenchymal cells which contained nestin and desmin within the sub-population which lacked PDGFRα, but contained PDGFRβ. Desmin was reduced at alveolar entry rings, airspaces were enlarged, and the gas-exchange surface area was reduced after NRP1 depletion. PDGFRα and NRP1 co-localized to membrane lipid rafts, which are known to contain Src-kinase. Neuropilin-1depletion reduced alveolar mesenchymal cell migration and PDGF-A mediated activation of Src-kinase, which may limit the accumulation of desmin at septal tips (alveolar entry rings). Cooperation between Nrp1 and PDGF-signaling is required for secondary septation, and manipulating Nrp1 could promote alveolar regeneration without producing fibrosis.
... In contrast, Sema3A inhibits lung branching morphogenesis (16) and alveolar septation in fetal lung explants (17). Controversially, a recent study revealed the nonessential role of postnatal Sema3-Nrp1 signaling in alveolar development, despite its key role during fetal pulmonary development (18). However, prenatal genetic ablation of Sema3-Nrp1 leads to respiratory distress, alveolar hemorrhaging, abnormally dilated capillaries, and disintegrating alveolar septa in mouse models (18). ...
... Controversially, a recent study revealed the nonessential role of postnatal Sema3-Nrp1 signaling in alveolar development, despite its key role during fetal pulmonary development (18). However, prenatal genetic ablation of Sema3-Nrp1 leads to respiratory distress, alveolar hemorrhaging, abnormally dilated capillaries, and disintegrating alveolar septa in mouse models (18). ...
Semaphorins are a large family of proteins originally identified as axon guidance cues which play a crucial role in neural development. They are also ubiquitously expressed beyond the nervous system and contribute to regulation of essential cell functions such as cell migration, proliferation, and adhesion. Binding of semaphorins to their receptors, including plexins and neuropilins, triggers diverse signaling pathways which are involved in the pathogenesis of various diseases from cancer to autoimmune and allergic disorders. Despite emerging evidence suggestive of non-redundant roles of semaphorins in cellular and molecular mechanisms of the airway biology, their precise expression and function have not been fully addressed. Here, we first provide an overall overview about semaphorin family, their receptors, signaling pathways, and their cellular functions. Then, we highlight the novel findings on the role of semaphorins in airway biology under developmental, homeostatic and pathological conditions. In particular, we discuss the dual roles of semaphorins in respiratory disorders where they can up- or down-regulate processes underlying the pathophysiology of the airway diseases. Next, our recent findings on the expression and function of semaphorin 3E in allergic asthma are further emphasized and its potential mechanism of action in allergic airway inflammation and remodeling is discussed. Finally, we raise some unanswered questions aiming to develop future research directions.
... Sema3A signaling was previously reported to control vascular morphogenesis in zebrafish, 9 chick, 10 and mouse embryos. 8,[17][18][19] We hence investigated whether, similar to Sema3A, Sema3F may also regulate mouse embryonic angiogenesis. We examined by fluorescence confocal microscopy the vasculature of whole-mount endomucin-stained wild type ( Figure 1A through 1D), Sema3f-null ( Figure 1F through 1I), and Sema3a-null ( Figure 1K through 1N) embryos at E9.5. ...
... Sema3 indicates class 3 semaphorin. As previously reported in zebrafish, 9 chick, 10 and mouse embryos, 8,[17][18][19] here we further substantiate how, differently from Sema3F, Sema3A is a crucial driver of embryonic vascular morphogenesis. Our findings that ≈80% of Sema3a knockout embryos die in utero and display a strong growth retardation compared with wild-type embryos reconcile previous apparently contradicting observations that, based on age and stage matching between wild-type and Sema3a-null embryos, concluded that Sema3A does not influence embryonic angiogenesis. ...
Objective:
Molecular pathways governing blood vessel patterning are vital to vertebrate development. Because of their ability to counteract proangiogenic factors, antiangiogenic secreted Sema3 (class 3 semaphorins) control embryonic vascular morphogenesis. However, if and how Sema3 may play a role in the control of extraembryonic vascular development is presently unknown.
Approach and results:
By characterizing genetically modified mice, here, we show that surprisingly Sema3F acts instead as a selective extraembryonic but not intraembryonic proangiogenic cue. Both in vivo and in vitro, in visceral yolk sac epithelial cells, Sema3F signals to inhibit the phosphorylation-dependent degradation of Myc, a transcription factor that drives the expression of proangiogenic genes, such as the microRNA cluster 17/92. In Sema3f-null yolk sacs, the transcription of Myc-regulated microRNA 17/92 cluster members is impaired, and the synthesis of Myc and microRNA 17/92 foremost antiangiogenic target Thbs1 (thrombospondin 1) is increased, whereas vascular endothelial growth factor signaling is inhibited in yolk sac endothelial cells. Consistently, exogenous recombinant Sema3F inhibits the phosphorylation-dependent degradation of Myc and the synthesis of Thbs1 in mouse F9 teratocarcinoma stem cells that were in vitro differentiated in visceral yolk sac epithelial cells. Sema3f(-)(/-) mice placentas are also highly anemic and abnormally vascularized.
Conclusions:
Sema3F functions as an unconventional Sema3 that promotes extraembryonic angiogenesis by inhibiting the Myc-regulated synthesis of Thbs1 in visceral yolk sac epithelial cells.
... The gangliosides identified by MALDI-MSI are listed in S1 Table. Immunohistochemistry Cryostat sections of 10-μm thickness were cut from mouse and rat brains and stained for Cholera Toxin B (CTB) and Neuropilin-1 (NRP-1) as described previously [29]. Briefly, tissue sections were rehydrated and blocked for one hour at room temperature followed by incubation with either the biotin-conjugated CTB subunit (1:500; Sigma, St Louis, MO) or rabbit anti-NRP1 (1:1000; gift of Dr. D.D. Ginty, Harvard University) antibody overnight at 4˚C in a humidified chamber. ...
Matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry (MALDI-MSI) allows us to investigate the distribution of lipid molecules within tissues. We used MALDI-MSI to identify prognostic gangliosides in tissue sections of rat intracranial allografts of rat glioma and mouse intracranial xenografts of human medulloblastoma. In the healthy adult rodent brain, GM1 and GD1 were the main types of glycolipids. Both gangliosides were absent in both intracranial transplants. The ganglioside GM3 was not present in the healthy adult brain but was highly expressed in rat glioma allografts. In combination with tandem mass spectrometry GM3 (d18:1/C24:0) was identified as the most abundant ganglioside species in the glioma allotransplant. By contrast, mouse xenografts of human medulloblastoma were characterized by prominent expression of the ganglioside GM2 (d18:0/C18:0). Together, these data demonstrate that tissue-based MALDI-MSI of gangliosides is able to discriminate between different brain tumors and may be a useful clinical tool for their classification and grading.
... Plexins, together with their co-receptor Neuropilin, bind class-3 Semaphorins, which have been studied in the postnatal lung. Germ-line deletion of Sema3a or of the Plexininteracting portion of Nrp1 potently disrupts saccular formation resulting in dilated saccules and diminished and immature distal epithelial cells (McGowan and McCoy 2015;Joza et al. 2013). Nearly all of the mice bearing the deletion die shortly after birth, limiting the usefulness of these models for studying alveolar development. ...
... Nearly all of the mice bearing the deletion die shortly after birth, limiting the usefulness of these models for studying alveolar development. However, the few mice that survive into the second postnatal week demonstrate a diminished distal airspace surface area and fewer αSMA-expressing alveolar MF (Joza et al. 2013). Because of these limitations, most studies of lung fibroblasts migration have been conducted in vitro. ...
Although pulmonary alveolar interstitial fibroblasts are less specialized than their epithelial and endothelial neighbors, they play essential roles during development and in response to lung injury. At birth, they must adapt to the sudden mechanical changes imposed by the onset of respiration and to a higher ambient oxygen concentration. In diseases such as bronchopulmonary dysplasia and interstitial fibrosis, their adaptive responses are overwhelmed leading to compromised gas-exchange function. Thus, although fibroblasts do not directly participate in gas-exchange, they are essential for creating and maintaining an optimal environment at the alveolar epithelial-endothelial interface. This review summarizes new information and concepts about the ontogeny differentiation, and function of alveolar fibroblasts. Alveolar development will be emphasized, because the development of strategies to evoke alveolar repair and regeneration hinges on thoroughly understanding the way that resident fibroblasts populate specific locations in which extracellular matrix must be produced and remodeled. Other recent reviews have described the disruption that diseases cause to the fibroblast niche and so my objective is to illustrate how the unique developmental origins and differentiation pathways could be harnessed favorably to augment certain fibroblast subpopulations and to optimize the conditions for alveolar regeneration.
... ChIP-seq analysis in E18.5 wild-type lungs identified binding of FOXF1 in the proximity of genes involved in biological processes such as blood vessel development, cardiovascular, and embryonic development. Among the genes associated with multiple FOXF1 binding sites, Nrp1 signaling has previously been shown to be essential for fetal pulmonary development (Joza et al. 2013 ). Interestingly, mice homozygous for a knockout allele of Sdpr (Cav2) exhibit abnormal caveolae formation in the lung endothelium affecting endothelial cell function (Hansen et al. 2013). ...
FOXF1 heterozygous point mutations and genomic deletions have been reported in newborns with a neonatally lethal lung developmental disorder, Alveolar Capillary Dysplasia with Misalignment of Pulmonary Veins (ACDMPV). However, no gain-of-function mutations in FOXF1 have been identified yet in human disease. To study the effects of FOXF1 overexpression in lung development, we generated a Foxf1 overexpression mouse model by knocking in a Cre-inducible Foxf1 allele into the ROSA26 (R26) locus. The mice were phenotyped using micro-computed tomography (micro-CT), head-out plethysmography, ChIP-seq and transcriptome analyses, immunohistochemistry, and lung histopathology. Thirty-five percent of heterozygous R26-Lox-Stop-Lox (LSL)-Foxf1 E15.5 embryos exhibit subcutaneous edema, hemorrhages and die perinatally when bred to Tie2-cre mice, which targets Foxf1 overexpression to endothelial and hematopoietic cells. Histopathological and micro-CT evaluations revealed that R26Foxf1; Tie2-cre embryos have immature lungs with a diminished vascular network. Neonates exhibited respiratory deficits verified by detailed plethysmography studies. ChIP-seq and transcriptome analyses in E18.5 lungs identified Sox11, Ghr, Ednrb, and Slit2 as potential downstream targets of FOXF1. Our study shows that overexpression of the highly dosage sensitive Foxf1 impairs lung development and causes vascular abnormalities. This has important clinical implications when considering potential gene therapy approaches to treat disorders of FOXF1 abnormal dosage, such as ACDMPV.
