Delta-like 4 is required for pulmonary vascular arborization and alveolarization in the developing lung

Sheng, Xia; Menden, Heather; Townley, Nick; Mabry, Sherry M; Johnston, Jeff; Nyp, Michael F.; Heruth, Daniel P.; Korfhagen, Thomas R.; Sampath, Venkatesh

doi:10.1172/jci.insight.134170

Cited by 7 publications

(48 citation statements)

References 50 publications

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“…To validate our mouse data in human lung EC, we used an immortalized fetal human pulmonary microvascular endothelial cells (HPMEC-Im) that we had generated before 67 . We exposed HPMEC-Im to HOX (85%) or normoxia for 7 days in the presence or absence of immortalized fetal human pulmonary fibroblasts (HPF-Im) to test cell-autonomous changes in gene expression in HOX.…”

Section: New Subpopulation Of Car4+ Cap2 Endothelial Cells Emerges In...mentioning

confidence: 99%

“…scRNA-seq 12 C57BL/6 pups were euthanized using 100 mg/kg intraperitoneal injection of pentobarbital at P14. Fresh lung single cell digests furnished > 10-20x10 6 cells/mouse with >80% viability.12,000 cells were loaded into a 10X Genomics Chromium Single Cell A Chip (10X Genomics; PN-120236), and scRNA-seq was performed using Chromium Single Cell 3' Library & Gel Bead Kit v2 (10X Genomics; PN-120237) following the manufacturer's protocol 67 . Briefly, single-cell gel beads-in-emulsion (GEMs) were produced by running a loaded Chromium Single Cell A Chip on a Chromium Controller instrument (10X Genomics).…”

Section: Cell Dissociationmentioning

confidence: 99%

“…The copyright holder for this preprint (which this version posted July 14, 2022. ; https://doi.org/10.1101/2022.07.12.499826 doi: bioRxiv preprint Immortalized embryonic human pulmonary microvascular endothelial cells (HPMEC-Im) was generated and grown as done in previous studies 67,102 . Primary fetal human pulmonary fibroblasts (HPF) and primary fetal pulmonary alveolar epithelial cells (HPAEpiC) were purchased from ScienCell (Carlsbad, CA).…”

Section: Ipa Analysismentioning

confidence: 99%

“…Immunoblotting for quantifying changes in protein expression was performed as previously described 67 . In brief, mouse lung tissue was homogenized in RIPA lysis buffer containing commercially available protease and phosphatase inhibitors (Sigma) after RA and HOX treatment, with the clarified lysates used for western blotting (WB).…”

Section: Immunoblottingmentioning

confidence: 99%

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Neonatal hyperoxia induces sex-dependent pulmonary cellular and transcriptomic changes in an experimental mouse model of bronchopulmonary dysplasia

Sheng

Ellis

Winkley

et al. 2022

Preprint

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Hyperoxia (HOX) disrupts lung development in mice and causes bronchopulmonary dysplasia (BPD) in neonates. To investigate sex-dependent molecular and cellular programming involved in HOX, we surveyed the mouse lung using single cell RNA sequencing (scRNA-seq), and validated our findings in human neonatal lung cells in vitro. HOX-induced inflammation in alveolar type (AT) 2 cells gave rise to damage associated transient progenitors (DATP). It also induced a new subpopulation of AT1 cells with reduced expression of growth factors normally secreted by AT1 cells, but increased mitochondrial gene expression. Female alveolar epithelial cells had less EMT and pulmonary fibrosis signaling in HOX. In the endothelium, expansion of Car4+ EC (Cap2) was seen in HOX along with an emergent subpopulation of Cap2 with repressed VEGF signaling. This regenerative response was increased in females exposed to HOX. Mesenchymal cells had inflammatory signatures in HOX, with a new distal interstitial fibroblast subcluster characterized by repressed lipid biosynthesis and a transcriptomic signature resembling myofibroblasts. HOX-induced gene expression signatures in human neonatal fibroblasts and alveolar epithelial cells in vitro resembled mouse scRNA-seq data. These findings suggest that neonatal exposure to HOX programs distinct sex-specific stem cell progenitor and cellular reparative responses that underpin lung remodeling in BPD.

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Section: New Subpopulation Of Car4+ Cap2 Endothelial Cells Emerges In...mentioning

confidence: 99%

Section: Cell Dissociationmentioning

confidence: 99%

Section: Ipa Analysismentioning

confidence: 99%

Section: Immunoblottingmentioning

confidence: 99%

See 2 more Smart Citations

Neonatal hyperoxia induces sex-dependent pulmonary cellular and transcriptomic changes in an experimental mouse model of bronchopulmonary dysplasia

Sheng

Ellis

Winkley

et al. 2022

Preprint

Self Cite

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“…Hypomorphic Notch2 affected the formation of specific vascular beds such as the hyaloid vasculature of the eye, the glomerular capillary tuft and the myocardium [ 56 ]. Endothelial inactivation of Notch1 or CSL resulted in enlarged malformed vessels in the liver [ 57 ], and Dll4 haploinsufficiency led to a dysmorphic microvasculature in the lung [ 58 ].…”

Section: Notch and Vascular Developmentmentioning

confidence: 99%

Notch signalling in healthy and diseased vasculature

2022

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Notch signalling is an evolutionarily highly conserved signalling mechanism governing differentiation and regulating homeostasis in many tissues. In this review, we discuss recent advances in our understanding of the roles that Notch signalling plays in the vasculature. We describe how Notch signalling regulates different steps during the genesis and remodelling of blood vessels (vasculogenesis and angiogenesis), including critical roles in assigning arterial and venous identities to the emerging blood vessels and regulation of their branching. We then proceed to discuss how experimental perturbation of Notch signalling in the vasculature later in development affects vascular homeostasis. In this review, we also describe how dysregulated Notch signalling, as a consequence of direct mutations of genes in the Notch pathway or aberrant Notch signalling output, contributes to various types of vascular disease, including CADASIL, Snedden syndrome and pulmonary arterial hypertension. Finally, we point out some of the current knowledge gaps and identify remaining challenges in understanding the role of Notch in the vasculature, which need to be addressed to pave the way for Notch-based therapies to cure or ameliorate vascular disease.

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Delta like 4 regulates cerebrovascular development and endothelial integrity via DLL4‐NOTCH‐CLDN5 pathway and is vulnerable to neonatal hyperoxia

Ke,

Xia,

et al. 2024

The Journal of Physiology

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The mechanisms governing brain vascularization during development remain poorly understood. A key regulator of developmental vascularization is delta like 4 (DLL4), a Notch ligand prominently expressed in endothelial cells (EC). Exposure to hyperoxia in premature infants can disrupt the development and functions of cerebral blood vessels and lead to long‐term cognitive impairment. However, its role in cerebral vascular development and the impact of postnatal hyperoxia on DLL4 expression in mouse brain EC have not been explored. We determined the DLL4 expression pattern and its downstream signalling gene expression in brain EC using Dll4+/+ and Dll4+/LacZ mice. We also performed in vitro studies using human brain microvascular endothelial cells. Finally, we determined Dll4 and Cldn5 expression in mouse brain EC exposed to postnatal hyperoxia. DLL4 is expressed in various cell types, with EC being the predominant one in immature brains. Moreover, DLL4 deficiency leads to persistent abnormalities in brain microvasculature and increased vascular permeability both in vivo and in vitro. We have identified that DLL4 insufficiency compromises endothelial integrity through the NOTCH‐NICD‐RBPJ‐CLDN5 pathway, resulting in the downregulation of the tight junction protein claudin 5 (CLDN5). Finally, exposure to neonatal hyperoxia reduces DLL4 and CLDN5 expression in developing mouse brain EC. We reveal that DLL4 is indispensable for brain vascular development and maintaining the blood–brain barrier's function and is repressed by neonatal hyperoxia. We speculate that reduced DLL4 signalling in brain EC may contribute to the impaired brain development observed in neonates exposed to hyperoxia. imageKey points The role of delta like 4 (DLL4), a Notch ligand in vascular endothelial cells, in brain vascular development and functions remains unknown. We demonstrate that DLL4 is expressed at a high level during postnatal brain development in immature brains and DLL4 insufficiency leads to abnormal cerebral vasculature and increases vascular permeability both in vivo and in vitro. We identify that DLL4 regulates endothelial integrity through NOTCH‐NICD‐RBPJ‐CLDN5 signalling. Dll4 and Cldn5 expression are decreased in mouse brain endothelial cells exposed to postnatal hyperoxia.

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Delta-like 4 is required for pulmonary vascular arborization and alveolarization in the developing lung

Cited by 7 publications

References 50 publications

Neonatal hyperoxia induces sex-dependent pulmonary cellular and transcriptomic changes in an experimental mouse model of bronchopulmonary dysplasia

Neonatal hyperoxia induces sex-dependent pulmonary cellular and transcriptomic changes in an experimental mouse model of bronchopulmonary dysplasia

Notch signalling in healthy and diseased vasculature

Delta like 4 regulates cerebrovascular development and endothelial integrity via DLL4‐NOTCH‐CLDN5 pathway and is vulnerable to neonatal hyperoxia

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