Stem cells rapidly proliferate after injury to repair damaged tissue, and chronic injury predisposes to cancer. However, injury-activated mitogens, the mechanisms that keep them inactive until injury, and their role in cancer are not understood. Here we identify Igf2 as the injury-activated mitogen for neuroendocrine stem cells, a facultative airway stem cell and origin of small cell lung cancer. Igf2 is constitutively produced by the stem cells but sequestered in inactive form by co-expressed Igf binding proteins. Injury releases Igf2 and induces proliferation by activating its receptors and repressing Rb tumor suppressor, which normally enforces stem cell quiescence. Persistent pathway activation initiates oncogenesis. Thus, in addition to its classical hormonal roles in physiology, growth, and aging, Igf operates locally with Igf binding proteins and Rb to control injury-induced stem cell activation and cancer. This pathway may also control related stem cells and cancers of the body and brain.

Heterozygous TBX4 variants are the second most common genetic cause of pediatric pulmonary hypertension (PH), yet mechanisms underlying TBX4-related lung disease remain poorly understood. This study developed a lung-mesenchyme-specific Tbx4 loss-of-function (Tbx4cKO) mouse model that bypasses embryonic lethality to investigate this condition. Adult Tbx4cKO mice demonstrated significantly impaired pulmonary flow acceleration consistent with PH. Three-dimensional analysis of embryonic lungs revealed reduced lobe volumes and decreased distance between pleural edges and muscularized vessels. In adult Tbx4cKO lungs, we identified extensive vascular remodeling characterized by medial thickening and the extension of muscularized arteries into normally non-muscularized subpleural parenchymal zones. Contrary to previous reports suggesting vascular simplification, 3-dimensional analysis demonstrated an elaborated pulmonary artery tree in addition to pathologic wall muscularization. Depletion of a single Tbx5 allele in addition to both Tbx4 alleles exacerbated histologic phenotypes, with worsened right ventricular dilation. This model also demonstrated dysregulated airway smooth muscle patterning and prominent subpleural smooth muscle bands, similar to those in human TBX4 syndrome. We identify TBX4 as a critical regulator of smooth muscle differentiation and patterning across multiple lung compartments. Our model recapitulates key features of human TBX4 syndrome and identifies dysregulated smooth muscle differentiation as a potential future therapeutic target.

Human lung development is governed by complex gene regulatory networks that orchestrate cellular differentiation and organogenesis. We present a single cell multiomic atlas of human pulmogenesis, simultaneously capturing both the chromatin accessibility profile and the transcriptome from each cell across fetal lungs spanning from post-conception weeks (PCW) 12 to 23. We identified 44 distinct developing cell clusters and mapped 581,745 candidate cis-regulatory elements and nominated 121,486 non-redundant peak-to-gene linkages. We identify highly regulated genes (HRGs) and the cognate highly regulating peaks (HRPs) that describe the most salient regulatory gene programs and developmental enhancer sites for each cell type. Trajectory analysis along with interpretable cell type specific convolutional neural network models were developed to delineate dynamic regulatory programs driving key developmental transitions, including aerocyte and arterial differentiation and alveolar formation. Furthermore, we identified distinct vascular smooth muscle subpopulations with unique spatial associations to either arterial or venous structures with reciprocal signaling within each niche. We also uncovered the regulatory modules of surfactant production in alveolar progenitors, implicating a direct role for the glucocorticoid receptor alongside novel transcription factors. Finally, using cell type specific models linking DNA sequence to chromatin accessibility we prioritize variants associated with impaired pulmonary function or disease and nominate mechanisms of motif disruption. Overall, our multiomic atlas deepens our understanding of the gene-regulatory architecture underlying human lung development and provides a valuable resource for the community to dissect the cellular and molecular programs of pulmonary physiology and disease at the cellular and nucleotide precision. ### Competing Interest Statement W.J.G. is a consultant and equity holder for 10x Genomics, Guardant Health, Quantapore, and Ultima Genomics and cofounder of Protillion Biosciences and is named on patents describing ATAC-seq. All other authors declare no competing interests. Arc Research Institute, https://ror.org/00wra1b14 Chan Zuckerberg Biohub San Francisco, https://ror.org/00knt4f32 Maternal and Child Health Research Institute Paul & Daisy Soros Fellowships for New Americans, https://ror.org/02320dz84

Endothelial cells (ECs) comprise the pulmonary vascular bed and play a significant role in health and diseases. Consequently, the EC niche represents an attractive therapeutic target for treating a wide range of pulmonary vascular diseases. We have identified a new class of dicationic charge-altering releasable transporters. These single-component transporters selectively deliver mRNA to the lung upon intravenous administration without the use of a targeting ligand. Significantly, the number and spatial array of cationic charges within the repeating units of the CART polymer are found to control both mRNA delivery efficacy and tissue tropism. High-resolution imaging revealed efficient mRNA delivery to endothelial cells in pulmonary arteries, veins, and capillaries. The selective lung tropism of these new CARTs, coupled with the efficient and tunable synthesis of this new family of CART amphiphiles, represents an enabling platform for research and clinical applications.

PURPOSE OF REVIEW:Pulmonary vascular disease is more common in certain genetic developmental lung disorders. This review synthesizes clinical descriptions, molecular analyses, and single-cell transcriptional data to build a conceptual framework to help understand why some variants affect the vasculature while others primarily manifest with parenchymal disease. RECENT FINDINGS:Genes predominantly expressed in endothelial and mesenchymal compartments ( TBX4 , FGF10 , FOXF1 , KDR ) commonly present with both parenchymal and pulmonary vascular disease, while epithelial-restricted genes ( SFTPC , ABCA3 , NKX2.1 ) typically manifest as parenchymal disease. Single-cell analyses reveal that compartment-specific expression patterns correlate with clinical phenotypes. Phenotypic variability, even among individuals sharing identical variants, suggests complex interactions between genetic modifiers, epigenetic factors, and developmental processes that remain poorly understood. SUMMARY:Compartment-specific gene expression patterns fundamentally underlie the differential presence of vascular phenotypes in DEVLDs. Genetic advances and single cell technologies have revolutionized our understanding of these disorders, but we are in the early stages of translating this knowledge into meaningful clinical advances. Future efforts must bridge this gap to transform clinical care from supportive to targeted, disease-modifying treatment based on cell-specific molecular mechanisms.

The fibrous cap of atherosclerotic plaques is essential for plaque stability. Rupture of the fibrous cap leads to heart attacks and strokes, and causes tens of millions of deaths globally every year. Identifying and understanding the cellular origins and plasticity of the fibrous cap is critical to developing therapeutic strategies to stabilize the atherosclerotic plaques. The fibrous cap is thought to arise oligoclonal from medial smooth muscle cells (SMCs), but whether all SMCs can give rise to the fibrous cap is unknown. Furthermore, conflicting data exist regarding whether plaque cells deeper in the lesion can give rise to the fibrous cap or vice vera. Murine SMC-lineage traced scRNAseq data revealed a transcriptomically distinct population of Notch3 and Elastin high population of cells that localizes to the fibrous cap. Utilizing a lineage tracing mouse model driven by endogenous Notch3, we demonstrated that fibrous cap cells arise from a predefined population of SMC that expresses Notch3 at baseline. After pulse-labeling the Notch3 CreERT2 ; ROSA lsl-tdTomato ; Apoe -/- mice with tamoxifen before high fat diet and then feeding them with high fat diet for 16 weeks, Notch3 -lineage traced cells stain positive for SMC markers ( Tagln , Cnn1 ) and are nearly exclusively found at the fibrous cap in multiple atherosclerotic prone beds. Furthermore, Notch3 -lineage traced SMCs and chondrogenic SMCs are mutually exclusive in the plaque, as demonstrated by the minimal overlap of tdTomato with chondrogenic SMC markers, including Col2a1 and Sox9. The Notch3 lineage labeled fibrous cap-SMCs display different inflammatory and extracellular matrix program from the non-labeled SMC progenies, as demonstrated by single cell transcriptomic sequencing. Consistently, Notch3 -lineage traced cells are committed to the fibrous cap fate and excluded from the calcified portions of the lesion and acellular core. Altogether, these lineage tracing studies highlight previously unrecognized medial SMC heterogeneity in healthy vessels. Unique Notch3 + populations of SMCs in normal media are fated to form the lesion cap. Once the Notch3 program is turned on, cells are locked into a fibrous cap fate and do not give rise to osteochondrogenic SMCs. Importantly, Notch3 CreERT2 mice can be used as a cap-specific genetic manipulation tool to further elucidate the role of fibrous cap specific genetic programs.

Abstract Background: Pulmonary hypertension (PH) can occur as a complication of schistosomiasis. In humans, schistosomiasis-PH persists despite antihelminthic therapy and parasite eradication. We hypothesized that persistent disease arises as a consequence of exposure repetition. Methods: Following intraperitoneal sensitization, mice were experimentally exposed to Schistosoma eggs by intravenous injection, either once or three times repeatedly. The phenotype was characterized by right heart catheterization and tissue analysis. Results: Following intraperitoneal sensitization, a single intravenous Schistosoma egg exposure resulted in a PH phenotype that peaked at 7–14 days, followed by spontaneous resolution. Three sequential exposures resulted in a persistent PH phenotype. Inflammatory cytokines were not significantly different between mice exposed to one or three egg doses, but there was an increase in perivascular fibrosis in those who received three egg doses. Significant perivascular fibrosis was also observed in autopsy specimens from patients who died of this condition. Conclusions: Repeatedly exposing mice to schistosomiasis causes a persistent PH phenotype, accompanied by perivascular fibrosis. Perivascular fibrosis may contribute to the persistent schistosomiasis-PH observed in humans with this disease.

HomeCirculationVol. 146, No. 5von Willebrand Factor Is Produced Exclusively by Endothelium, Not Neointima, in Occlusive Vascular Lesions in Both Pulmonary Hypertension and Atherosclerosis Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessLetterPDF/EPUBvon Willebrand Factor Is Produced Exclusively by Endothelium, Not Neointima, in Occlusive Vascular Lesions in Both Pulmonary Hypertension and Atherosclerosis Lea C. Steffes, MD, Paul Cheng, MD, Thomas Quertermous, MD and Maya E. Kumar, PhD Lea C. SteffesLea C. Steffes Department of Pediatrics, Division of Pulmonary Medicine (L.C.S., M.E.K.), Stanford University School of Medicine. , Paul ChengPaul Cheng Department of Medicine, Division of Cardiovascular Medicine (P.C., T.Q.), Stanford University School of Medicine. , Thomas QuertermousThomas Quertermous https://orcid.org/0000-0002-7645-9067 Department of Medicine, Division of Cardiovascular Medicine (P.C., T.Q.), Stanford University School of Medicine. and Maya E. KumarMaya E. Kumar Correspondence to: Maya E. Kumar, PhD, 240 Pasteur Dr, Biomedical Innovations Building, Rm 2300, Stanford University School of Medicine, Stanford, CA 94305. Email E-mail Address: [email protected] https://orcid.org/0000-0001-6754-5660 Department of Pediatrics, Division of Pulmonary Medicine (L.C.S., M.E.K.), Stanford University School of Medicine. Originally published1 Aug 2022https://doi.org/10.1161/CIRCULATIONAHA.121.058427Circulation. 2022;146:429–431vWF (von Willebrand Factor) is a large, secreted, multimeric glycoprotein transcribed by vascular endothelial cells and, to a lesser extent, megakaryocytes throughout the body. vWF is an integral part of the coagulation cascade and is important in hemostasis, vessel wall homeostasis, and repair after injury. After endothelial insult, including increased shear stress or inflammation, vWF is released from storage vesicles within endothelial cells and is both secreted directly into circulation and maintained locally, binding to extracellular matrix (ECM) components in the area of vessel injury, resulting in local platelet aggregation.1 Given its role in vascular injury and repair, vWF has been implicated as a biomarker of endothelial dysfunction, with elevated plasma levels of vWF identified in multiple vascular disease processes, including atherosclerosis, cerebral vascular disease, and pulmonary hypertension (PH), with a direct correlation between increasing vWF levels and risk of adverse events and death.2Pulmonary vascular remodeling, including the formation of occlusive neointimal lesions throughout the pulmonary vascular bed, is a hallmark of advanced PH, and the cellular origin of these lesions in human disease is a topic of ongoing debate. Neointimal lesions are composed of longitudinally oriented mesenchymal cells located directly beneath the artery endothelium that grow to occlude the vessel lumen and are characterized by damaged elastic laminae and increased ECM deposition.3 Endothelial to mesenchymal transition, where endothelial cells undergo a phenotypic and transcriptional conversion to a mesenchymal cell type, has been proposed as a mechanism by which neointimal cells are generated in PH. Colocalization of vWF protein and other canonical endothelial cell markers, with neointimal cell markers (ie, Acta2) by immunohistochemistry within neointimal lesions from patient tissue and animal models, has been cited as evidence of an endothelial origin for neointimal cells through endothelial to mesenchymal transition.4 The origin of neointima cells in humans has yet to be definitively determined. We recently demonstrated in a mouse model of PH that neointima arises not from the endothelium but rather from Notch3-expressing smooth muscle cells of the arterial media, leading us to reexamine the use of the endothelial protein vWF as evidence of the neointima's endothelial origin.5 Because (1) vWF is an ECM-binding secreted molecule, (2) ECM is abundant within neointimal lesions, and (3) endothelial damage and dysfunction, including vWF secretion, is a feature of PH,3 the presence of vWF protein in neointimal lesions does not necessarily mean that neointima cells themselves are producing vWF but rather may reflect binding of vWF protein released by neighboring endothelium. To resolve this controversy, we set out to characterize VWF/Vwf transcription and protein localization within neointimal lesions of both patients with PH and a range of established animal models of PH. We then performed a similar analysis of aortic root neointimal lesions in the ApoE–/– atherosclerosis mouse model to see whether vWF localization and Vwf transcription in systemic arteries parallels that seen in the pulmonary circulation.Using recently developed multiplexed in situ hybridization techniques with single-cell resolution, we show that VWF/Vwf is exclusively transcribed by vascular endothelial cells within remodeled pulmonary arteries (Figure) in tissue isolated from human PH, Sugen/Hypoxia-exposed rat, the endothelial deletion of PHD2 mouse model of PH, and the inflammation-driven house dust mite mouse model of PH with no evidence of transcription in Acta2-positive neointima cells (Figure [B–D, F, and G]). In contrast, vWF protein is detected by immunohistochemistry in both endothelial cells and Acta2-positive neointimal lesions from human and mouse PH tissue (Figure [A and E]). The same is found in atherosclerosis neointimal lesions (Figure [H and I]), suggesting that this pattern of vWF localization and VWF/Vwf transcription is a general characteristic of neointimal lesions in both the systemic and pulmonary vascular beds. This supports a model (Figure [J]) in which vWF protein is produced exclusively in endothelial cells that go on to secrete the protein in the setting of endothelial dysfunction.3 Secreted vWF then binds to the abundant ECM surrounding neointimal cells, making neointimal lesions falsely appear to share Vwf expression with endothelial cells. These findings highlight that in situ vWF antibody staining should not be used as evidence of endothelial to mesenchymal transition and, more generally, that caution is warranted when inferring cellular lineage from canonical marker expression. The cellular origin of the neointima in human PH remains undetermined.Download figureDownload PowerPointFigure. von Willebrand Factor is produced exclusively by endothelium in neointimal lesions of both pulmonary arteries and atherosclerosis aortic root. vWF protein (red) is detected in both the endothelium (white) and neointima (green) of pulmonary arteries with neointimal lesions (bounded by yellow dashed lines) in rodent models of PH (A), human pulmonary arterial hypertension (PAH, E), and in the aortic root of the ApoE–/- mouse model of atherosclerosis (H) by immunohistochemistry. Neointimal vWF protein is indicated by white arrowheads. In situ hybridization identifies transcripts of the VWF/Vwf gene (red) only in endothelial cells (CLDN5/Cldn5 mRNA, white) and not neointima cells (green) in multiple animal models of PH (B, HDM mouse; C, Sugen-hypoxia (Su/Hx) rat; D, Tie2-Cre PHD2 mouse), human PAH pulmonary arteries (F and G), and aortic root atherosclerosis (I). The diameters of pulmonary arteries evaluated for mouse and rat were 20 to 150 µm, and human 50 to 200 µm. J, Schematic of artery cross section with neointimal lesion showing proposed model in which vWF protein (red) is secreted from endothelial cells and bound to neointimal ECM. Neointima cells and smooth muscle media, green; endothelium, red; elastic laminae, heavy gray lines. The atherosclerosis mouse model is a constitutive knockout of ApoE transitioned onto a high-fat (Western) diet at 8 weeks old that was then sustained for 16 weeks. Neointima and vascular smooth muscle cells identified by antibody staining for Acta2/ACTA2 (clone 1A4; Sigma F3777) for A through G and by Myh11-CreER;tdTomato lineage trace for H and I. Endothelium in A and H identified by antibody staining for CD31 (BD Pharmingen, 550274) and in E for VE-cadherin (R&D Systems, AF938). vWF antibody in A, E, and H from AbCam (ab6994). In situ probes (all RNAscope from ACD): human VWF (560461), human CLDN5 (517141), mouse Vwf (858851), mouse Cldn5 (491611), rat Vwf (413401), and rat Cldn5 (1055891). DAPI indicates 4′,6-diamidino-2-phenylindole; ECM, extracellular matrix; HDM, house dust mite; PH, pulmonary hypertension; PHD2, prolyl hydroxylase domain protein 2; and vWF, von Willebrand Factor.All animal experiments received approval from the Stanford University Institutional Animal Care and Use Committee (institutional review board 31869, 27636, and 10054). Patient tissues were obtained under a protocol approved by Stanford University's Human Subjects Research Compliance Office (institutional review board 54182), and informed consent was obtained from each patient before surgery. All experiments followed applicable regulations and guidelines. All image data, analytic methods, and study materials will be made available to other researchers on request.Article InformationAcknowledgmentsDrs Steffes and Kumar conceived, performed, and interpreted experiments, wrote the manuscript, and secured funding. Drs Cheng and Quertermous provided expertise and experimental samples, interpreted experiments, and reviewed and edited the manuscript. The authors wish to thank R. Metzger for providing PHD2 (prolyl hydroxylase domain protein 2) mouse tissue, E. Spiekerkoetter for providing Su/Hx rat tissue, A. Andruska for comments on the manuscript, and Stanford Medicine's Animal Histology Service for paraffin sample preparation.Sources of FundingThis work was supported by Stanford Maternal & Child Health Research Institute Ernest & Amelia Gallo Endowed Postdoctoral Fellowship, National Institutes of Health (NIH) 5T32HL129970-03 and a Parker B. Francis Fellowship to Dr Steffes; NIH K08HL153798 and American Heart Association 20CDA35310303 to Dr Cheng; R01HL134817, R01HL139478, R01HL156846, R01HL151535, R01HL145708, and UM1 HG011972 to Dr Quertermous; and NIH R01HL163013, American Heart Association 16SDG30030006, Stanford Spectrum-Child Health Research Institute seed grants, Bravo Family endowed faculty scholarship, and a Vera Moulton Wall Center for Pulmonary Vascular Disease research grant to Dr Kumar.Nonstandard Abbreviations and AcronymsECMextracellular matrixPHpulmonary hypertensionvWFvon Willebrand FactorDisclosures None.FootnotesCirculation is available at www.ahajournals.org/journal/circFor Sources of Funding and Disclosures, see page 431.Correspondence to: Maya E. Kumar, PhD, 240 Pasteur Dr, Biomedical Innovations Building, Rm 2300, Stanford University School of Medicine, Stanford, CA 94305. Email [email protected]eduReferences1. Wagner DD. Cell biology of von Willebrand factor.Annu Rev Cell Biol. 1990; 6:217–246. doi: 10.1146/annurev.cb.06.110190.001245CrossrefMedlineGoogle Scholar2. Lip G, Blann A. von Willebrand factor: a marker of endothelial dysfunction in vascular disorders?Cardiovasc Res. 1997; 34:255–265. doi: 10.1016/s0008-6363(97)00039-4CrossrefMedlineGoogle Scholar3. Humbert M, Guignabert C, Bonnet S, Dorfmüller P, Klinger JR, Nicolls MR, Olschewski AJ, Pullamsetti SS, Schermuly RT, Stenmark KR, et al. Pathology and pathobiology of pulmonary hypertension: state of the art and research perspectives.Eur Respir J. 2019; 53:1801887. doi: 10.1183/13993003.01887-2018CrossrefMedlineGoogle Scholar4. Good RB, Gilbane AJ, Trinder SL, Denton CP, Coghlan G, Abraham DJ, Holmes AM. Endothelial to mesenchymal transition contributes to endothelial dysfunction in pulmonary arterial hypertension.Am J Pathol. 2015; 185:1850–1858. doi: 10.1016/j.ajpath.2015.03.019CrossrefMedlineGoogle Scholar5. Steffes LC, Froistad AA, Andruska A, Boehm M, McGlynn M, Zhang F, Zhang W, Hou D, Tian X, Miquerol L, et al. A Notch3-marked subpopulation of vascular smooth muscle cells is the cell of origin for occlusive pulmonary vascular lesions.Circulation. 2020; 142:1545–1561. doi: 10.1161/circulationaha.120.045750LinkGoogle Scholar Previous Back to top Next FiguresReferencesRelatedDetails August 2, 2022Vol 146, Issue 5 Advertisement Article InformationMetrics © 2022 American Heart Association, Inc.https://doi.org/10.1161/CIRCULATIONAHA.121.058427PMID: 35914017 Originally publishedAugust 1, 2022 Keywordsmodels, animalvascular remodelinghypertension, pulmonaryvon Willebrand FactorPDF download Advertisement SubjectsAnimal Models of Human DiseaseAtherosclerosisBasic Science ResearchPulmonary BiologyVascular Disease

We recently described four distinct types of plexiform lesions in human idiopathic and familial pulmonary arterial hypertension (PAH) [1], visualising the three-dimensional lesion structure using synchrotron-based phase-contrast micro-computed tomography (SPµCT). Two types, 1 and 2, are shunt-type lesions that connect pulmonary arteries to the bronchial circulation: type 1 to the vasa vasorum, and type 2 to peribronchial vessels. Type 3 lesions are found peripherally in the lung as spherical structures abruptly terminating the distal pulmonary artery/arteriole, and type 4 lesions are characterised by recanalisation of an occluded artery/arteriole. Our observation of type 1 and type 2 lesions in PAH supports previous work that demonstrated intrapulmonary bronchopulmonary anastomoses (IBAs) connected to plexiform lesions in human PAH, suggesting that shunting of blood can occur within lesions in the setting of supra-systemic pulmonary arterial pressure [2]. Further haemodynamic studies of distinct subtypes of plexiform lesions have been hampered by the lack of available animal models with plexiform lesions representative of the full range of lesion types found in human disease. Plexiform lesions have previously been described in the Sugen5416/hypoxia rat model of pulmonary hypertension when time until sacrifice following hypoxia is extended to 13–14 weeks. Initially plexiform lesions were identified within the pulmonary artery, as well as in the form of aneurysm-like lesions projecting outside the vessel lumen [3], and recently the latter type was shown to form in supernumerary arteries [4]. However, neither study observed plexiform lesions communicating with the bronchial circulation, possibly because of methodological limitations of the histological analysis. Human like plexiform lesions identified in the prolonged Sugen5416/hypoxia rat model, visualised by synchrotron tomography imaging https://bit.ly/3KQvDHg We acknowledge the Paul Scherrer Institute, Villigen, Switzerland, for provision of synchrotron radiation beamtime at the X02DA TOMCAT beamline of the Swiss Light Source and Anne Bonnin for excellent technical assistance. We also acknowledge Phan-Kiet Tran for schematic illustrations.

Molecular characterization of cell types using single-cell transcriptome sequencing is revolutionizing cell biology and enabling new insights into the physiology of human organs. We created a human reference atlas comprising nearly 500,000 cells from 24 different tissues and organs, many from the same donor. This atlas enabled molecular characterization of more than 400 cell types, their distribution across tissues, and tissue-specific variation in gene expression. Using multiple tissues from a single donor enabled identification of the clonal distribution of T cells between tissues, identification of the tissue-specific mutation rate in B cells, and analysis of the cell cycle state and proliferative potential of shared cell types across tissues. Cell type-specific RNA splicing was discovered and analyzed across tissues within an individual.

Pulmonary hypertension (PH) is a heterogenous and incurable disease marked by varying degrees of pulmonary vascular remodeling. This vascular remodeling, which includes thickening of the smooth muscle layer (an early finding) and formation of occlusive neointimal lesions (a late finding) in the pulmonary arteries, is a major driver of morbidity and mortality in PH. Available PH therapies consist of vasodilators that do not specifically target lesion formation or expansion and neither prevent progression nor reverse disease. This paucity of curative treatments highlights the need for new drug discovery targeting crucial steps of artery remodeling in PH. The cell dynamics and molecular signals driving neointimal lesion formation have been difficult to elucidate as classic mouse models of PH do not develop neointima. Here, we detail the methods to generate a robust and non-genetic mouse model of PH with medial thickening and neointimal lesion formation in the pulmonary arteries, through chronic exposure to an inflammatory stimulus-house dust mite (HDM). This model rapidly generates human-like pulmonary arterial lesions following a reproducible time course, allowing scrutiny of the cellular and molecular mechanisms controlling each stage of artery remodeling. Further, we outline optimal tissue handling, sectioning, and staining methodologies for detailed quantitative analysis of artery medial thickening and neointimal lesion formation and expansion. Finally, we present a method for staged pharmacologic intervention to identify molecules and pathways required at each step of the pulmonary arterial remodeling process. The advantages of this mouse model of PH over currently available animal models are five-fold. (i) It allows the use of the full range of genetic and single cell tools available in mice to manipulate and study the process of vascular remodeling seen in human disease, including the formation of neointimal lesions in a controlled and cell specific manner. (ii) The vascular lesions develop in a stereotyped manner with predictable timing, allowing for pharmacologic manipulation at discrete stages of vessel remodeling. (iii) It is rapid, with development of PH and vascular remodeling in a timeframe of two to eight weeks. (iv) It uses simple techniques and requires neither surgery, unusual equipment, or extensive personnel training. (v) The staining and quantitation methodologies we present are a significant improvement over those currently in use in the field. We hope that dissemination of this model and the associated detailed methods will speed up the development of novel and more effective PH therapeutics. Graphic abstract: Chronic perivascular inflammation induces medial thickening and neointima formation in pulmonary arteries, following a stereotyped time course, and allowing staged pharmacologic intervention during specific remodeling events, as well as quantitative assessment of vascular changes.

Background: Pulmonary arterial hypertension (PAH) is a fatal disease characterized by profound vascular remodeling in which pulmonary arteries narrow because of medial thickening and occlusion by neointimal lesions, resulting in elevated pulmonary vascular resistance and right heart failure. Therapies targeting the neointima would represent a significant advance in PAH treatment; however, our understanding of the cellular events driving neointima formation, and the molecular pathways that control them, remains limited. Methods: We comprehensively map the stepwise remodeling of pulmonary arteries in a robust, chronic inflammatory mouse model of pulmonary hypertension. This model demonstrates pathological features of the human disease, including increased right ventricular pressures, medial thickening, neointimal lesion formation, elastin breakdown, increased anastomosis within the bronchial circulation, and perivascular inflammation. Using genetic lineage tracing, clonal analysis, multiplexed in situ hybridization, immunostaining, deep confocal imaging, and staged pharmacological inhibition, we define the cell behaviors underlying each stage of vascular remodeling and identify a pathway required for neointima formation. Results: Neointima arises from smooth muscle cells (SMCs) and not endothelium. Medial SMCs proliferate broadly to thicken the media, after which a small number of SMCs are selected to establish the neointima. These neointimal founder cells subsequently undergoing massive clonal expansion to form occlusive neointimal lesions. The normal pulmonary artery SMC population is heterogeneous, and we identify a Notch3-marked minority subset of SMCs as the major neointimal cell of origin. Notch signaling is specifically required for the selection of neointimal founder cells, and Notch inhibition significantly improves pulmonary artery pressure in animals with pulmonary hypertension. Conclusions: This work describes the first nongenetically driven murine model of pulmonary hypertension (PH) that generates robust and diffuse occlusive neointimal lesions across the pulmonary vascular bed and does so in a stereotyped timeframe. We uncover distinct cellular and molecular mechanisms underlying medial thickening and neointima formation and highlight novel transcriptional, behavioral, and pathogenic heterogeneity within pulmonary artery SMCs. In this model, inflammation is sufficient to generate characteristic vascular pathologies and physiological measures of human PAH. We hope that identifying the molecular cues regulating each stage of vascular remodeling will open new avenues for therapeutic advancements in the treatment of PAH.

The Tabula Muris ConsortiumWe have created a compendium of single cell transcriptome data from the model organism Mus musculus comprising more than 100,000 cells from 20 organs and tissues. These data represent a new resource for cell biology, revealing gene expression in poorly characterized cell populations and allowing for direct and controlled comparison of gene expression in cell types shared between tissues, such as T-lymphocytes and endothelial cells from distinct anatomical locations. Two distinct technical approaches were used for most tissues: one approach, microfluidic droplet-based 3’-end counting, enabled the survey of thousands of cells at relatively low coverage, while the other, FACS-based full length transcript analysis, enabled characterization of cell types with high sensitivity and coverage. The cumulative data provide the foundation for an atlas of transcriptomic cell biology.

Progressive tissue fibrosis is a major cause of the morbidity and mortality associated with repeated epithelial injuries and accumulation of myofibroblasts. Successful treatment options are limited by an incomplete understanding of the molecular mechanisms that regulate myofibroblast accumulation. Here, we employed in vivo lineage tracing and real-time gene expression transgenic reporting methods to analyze the early embryonic transcription factor T-box gene 4 (TBX4), and determined that TBX4-lineage mesenchymal progenitors are the predominant source of myofibroblasts in injured adult lung. In a murine model, ablation of TBX4-expressing cells or disruption of TBX4 signaling attenuated lung fibrosis after bleomycin-induced injury. Furthermore, TBX4 regulated hyaluronan synthase 2 production to enable fibroblast invasion of matrix both in murine models and in fibroblasts from patients with severe pulmonary fibrosis. These data identify TBX4 as a mesenchymal transcription factor that drives accumulation of myofibroblasts and the development of lung fibrosis. Targeting TBX4 and downstream factors that regulate fibroblast invasiveness could lead to therapeutic approaches in lung fibrosis.

Most vertebrate organs are composed of epithelium surrounded by support and stromal tissues formed from mesenchyme cells, which are not generally thought to form organized progenitor pools. Here, we use clonal cell labeling with multicolor reporters to characterize individual mesenchymal progenitors in the developing mouse lung. We observe a diversity of mesenchymal progenitor populations with different locations, movements, and lineage boundaries. Airway smooth muscle (ASM) progenitors map exclusively to mesenchyme ahead of budding airways. Progenitors recruited from these tip pools differentiate into ASM around airway stalks; flanking stalk mesenchyme can be induced to form an ASM niche by a lateral bud or by an airway tip plus focal Wnt signal. Thus, mesenchymal progenitors can be organized into localized and carefully controlled domains that rival epithelial progenitor niches in regulatory sophistication.

The regulation of the balance between proliferation and differentiation in the mesenchymal compartment of the lung is largely uncharacterized, unlike its epithelial counterpart. In this study, we determined that miR-142-3p contributes to the proper proliferation of mesenchymal progenitors by controlling the level of WNT signaling. miR-142-3p can physically bind to adenomatous polyposis coli mRNA, functioning to regulate its expression level. In miR-142-3p loss-of-function experiments, proliferation of parabronchial smooth muscle cell progenitors is significantly impaired, leading to premature differentiation. Activation of WNT signaling in the mesenchyme, or Apc loss of function, can both rescue miR-142-3p knockdown. These findings show that in the embryonic lung mesenchyme, the microRNA machinery modulates the level of WNT signaling, adding an extra layer of control in the feedback loop between FGFR2C and β-catenin-mediated WNT signaling.

Development of the pulmonary system is essential for terrestrial life. The molecular pathways that regulate this complex process are beginning to be defined, and such knowledge is critical to our understanding of congenital and acquired lung diseases. A recent workshop was convened by the National Heart, Lung, and Blood Institute to discuss the developmental principles that regulate the formation of the pulmonary system. Emerging evidence suggests that key developmental pathways not only regulate proper formation of the pulmonary system but are also reactivated upon postnatal injury and repair and in the pathogenesis of human lung diseases. Molecular understanding of early lung development has also led to new advances in areas such as generation of lung epithelium from pluripotent stem cells. The workshop was organized into four different topics, including early lung cell fate and morphogenesis, mechanisms of lung cell differentiation, tissue interactions in lung development, and environmental impact on early lung development. Critical points were raised, including the importance of epigenetic regulation of lung gene expression, the dearth of knowledge on important mesenchymal lineages within the lung, and the interaction between the developing pulmonary and cardiovascular system. This manuscript describes the summary of the discussion along with general recommendations to overcome the gaps in knowledge in lung developmental biology.

Some of the most serious diseases involve altered size and structure of the arterial wall. Elucidating how arterial walls are built could aid understanding of these diseases, but little is known about how concentric layers of muscle cells and the outer adventitial layer are assembled and patterned around endothelial tubes. Using histochemical, clonal, and genetic analysis in mice, here we show that the pulmonary artery wall is constructed radially, from the inside out, by two separate but coordinated processes. One is sequential induction of successive cell layers from surrounding mesenchyme. The other is controlled invasion of outer layers by inner layer cells through developmentally regulated cell reorientation and radial migration. We propose that a radial signal gradient controls these processes and provide evidence that PDGF-B and at least one other signal contribute. Modulation of such radial signaling pathways may underlie vessel-specific differences and pathological changes in arterial wall size and structure.

During embryonic development, organs arise along the gut tube as a series of buds in a stereotyped anterior-posterior (A-P) pattern. Using chick-quail chimeras and in vitro tissue recombination, we studied the interactions governing the induction and maintenance of endodermal organ identify focusing on the pancreas. Though several permissive signals in pancreatic development have been previously identified, here we provide evidence that lateral plate mesoderm sends instructive signals to the endoderm, signals that induce expression of the pancreatic genes Pdx1, p48, Nkx6.1, glucagon, and insulin. Moreover, this instructive signal directs cells to form ectopic insulin-positive islet-like clusters in endoderm that would otherwise form more rostral organs. Once generated, endocrine cells no longer require interaction with mesoderm, but nonendocrine cells continue to require permissive signals from the mesoderm. Stimulation of activin, BMP, or retinoic acid signaling is sufficient to induce Pdx1 expression in endoderm anterior to the pancreas. Lateral plate mesoderm appears to pattern the endoderm in a posterior-dominant fashion as first noted in the patterning of the neural tube at the same embryonic stage. These findings argue for a central role of the mesoderm in coordinating the A-P pattern of all three primary germ layers.