The molecular mechanisms by which endothelial cells (ECs) regulate pulmonary vascularization and contribute to alveolar epithelial cell development during lung morphogenesis remain unknown. We tested the hypothesis that delta-like 4 (DLL4), an EC Notch ligand, is critical for alveolarization by combining lung mapping and functional studies in human tissue and DLL4-haploinsufficient mice (Dll4+/lacz). DLL4 expressed in a PECAM-restricted manner in capillaries, arteries, and the alveolar septum from the canalicular to alveolar stage in mice and humans. Dll4 haploinsufficiency resulted in exuberant, nondirectional vascular patterning at E17.5 and P6, followed by smaller capillaries and fewer intermediate blood vessels at P14. Vascular defects coincided with polarization of lung EC expression toward JAG1-NICD-HES1 signature and decreased tip cell-like (Car4) markers. Dll4+/lacZ mice had impaired terminal bronchiole development at the canalicular stage and impaired alveolarization upon lung maturity. We discovered that alveolar type I cell (Aqp5) markers progressively decreased in Dll4+/lacZ mice after birth. Moreover, in human lung EC, DLL4 deficiency programmed a hypersprouting angiogenic phenotype cell autonomously. In conclusion, DLL4 is expressed from the canalicular to alveolar stage in mice and humans, and Dll4 haploinsufficiency programs dysmorphic microvascularization, impairing alveolarization. Our study reveals an obligate role for DLL4-regulated angiogenesis in distal lung morphogenesis.
Sheng Xia, Heather L. Menden, Nick Townley, Sherry M. Mabry, Jeffrey Johnston, Michael F. Nyp, Daniel P. Heruth, Thomas Korfhagen, Venkatesh Sampath
Idiopathic pulmonary fibrosis is a progressive fibrotic lung disease. We previously identified fibrogenic mesenchymal progenitor cells (MPCs) in the lungs of IPF patients that serve as an obligatory driver of progressive fibrosis. Recent single cell RNA sequencing work revealed that IPF MPCs with the highest transcriptomic network entropy differ the most from control MPCs; and that CD44 was a marker of these IPF MPCs. We hypothesize that IPF MPCs with high CD44 (CD44hi) expression will display enhanced fibrogenicity. We demonstrate that CD44 expressing MPCs are present at the periphery of the IPF fibroblastic focus, placing them in regions of active fibrogenesis. In a humanized mouse xenograft model, CD44hi IPF MPCs are more fibrogenic than CD44lo IPF MPCs and knock-down of CD44 diminishes their fibrogenicity. CD44hi IPF MPCs display increased expression of pluripotency markers and self-renewal compared to CD44lo IPF MPCs; properties potentiated by IL-8. The mechanism involves the accumulation of CD44 within the nucleus where it associates with the chromatin modulator protein BRG1 and the Zeb1 transcription factor. This CD44/BRG1/Zeb1 nuclear protein complex targets the Sox2 gene promoting its upregulation and self-renewal. Our data implicates CD44 interaction with the epigenetic modulator protein Brg1 in conveying IPF MPCs with cell-autonomous fibrogenicity.
Libang Yang, Hong Xia, Karen A. Smith, Adam Gilbertsen, Daniel Beisang, Jonathan Kuo, Peter B. Bitterman, Craig A. Henke
High mortality in acute lung injury (ALI) results from sustained proinflammatory signaling by alveolar receptors, such as TNF-α receptor type 1 (TNFR1). Factors that determine the sustained signaling are not known. Unexpectedly, optical imaging of live alveoli revealed a major TNF-α–induced surge of alveolar TNFR1 due to a Ca2+-dependent mechanism that decreased the cortical actin fence. Mouse mortality due to inhaled LPS was associated with cofilin activation, actin loss, and the TNFR1 surge. The constitutively active form of the GTPase, Rac1 (V12Rac1), given intranasally (i.n.) as a noncovalent construct with a cell-permeable peptide, enhanced alveolar filamentous actin (F-actin) and blocked the TNFR1 surge. V12Rac1 also protected against ALI-induced mortality resulting from i.n. instillation of LPS or of Pseudomonas aeruginosa. We propose a potentially new therapeutic paradigm in which actin enhancement by exogenous Rac1 strengthens the alveolar actin fence, protecting against proinflammatory receptor hyperexpression, and therefore blocking ALI.
Galina A. Gusarova, Shonit R. Das, Mohammad N. Islam, Kristin Westphalen, Guangchun Jin, Igor O. Shmarakov, Li Li, Sunita Bhattacharya, Jahar Bhattacharya
Compromised regenerative capacity of lung epithelial cells can lead to cellular senescence, which may precipitate fibrosis. While increased markers of senescence have been reported in idiopathic pulmonary fibrosis (IPF), the origin and identity of these senescent cells remain unclear, and tools to characterize context-specific cellular senescence in human lung are lacking. We observed that the senescent marker p16 is predominantly localized to bronchiolized epithelial structures in scarred regions of IPF and systemic sclerosis associated interstitial lung disease ILD (SSc-ILD) lung tissue, overlapping with the basal epithelial markers Keratin 5 and Keratin 17. Using in vitro models, we derived transcriptional signatures of senescence programming specific to different types of lung epithelial cells, and interrogated these signatures in a single-cell RNA-seq data set derived from control, IPF, and SSc-ILD lung tissue. We identified a population of basal epithelial cells defined by, and enriched for, markers of cellular senescence, and identified candidate markers specific to senescent basal epithelial cells in ILD that can enable future functional studies. Notably, gene expression of these cells significantly overlaps with terminally differentiating cells in stratified epithelia, where it is driven by p53 activation as part of the senescence program.
Daryle J. DePianto, Jason A. Vander Heiden, Katrina B. Morshead, Kai-Hui Sun, Zora Modrusan, Grace Teng, Paul J. Wolters, Joseph R. Arron
IL-13–induced goblet cell metaplasia contributes to airway remodeling and pathological mucus hypersecretion in asthma. miRNAs are potent modulators of cellular responses, but their role in mucus regulation is largely unexplored. We hypothesized that airway epithelial miRNAs play roles in IL-13–induced mucus regulation. miR-141 is highly expressed in human and mouse airway epithelium, is altered in bronchial brushings from asthmatic subjects at baseline, and is induced shortly after airway allergen exposure. We established a CRISPR/Cas9-based protocol to target miR-141 in primary human bronchial epithelial cells that were differentiated at air-liquid-interface, and goblet cell hyperplasia was induced by IL-13 stimulation. miR-141 disruption resulted in decreased goblet cell frequency, intracellular MUC5AC, and total secreted mucus. These effects correlated with a reduction in a goblet cell gene expression signature and enrichment of a basal cell gene expression signature defined by single cell RNA sequencing. Furthermore, intranasal administration of a sequence-specific mmu-miR-141-3p inhibitor in mice decreased Aspergillus-induced secreted mucus and mucus-producing cells in the lung and reduced airway hyperresponsiveness without affecting cellular inflammation. In conclusion, we have identified a miRNA that regulates pathological airway mucus production and is amenable to therapeutic manipulation through an inhaled route.
Sana Siddiqui, Kristina Johansson, Alex Joo, Luke R. Bonser, Kyung Duk Koh, Olivier Le Tonqueze, Samaneh Bolourchi, Rodriel A. Bautista, Lorna Zlock, Theodore L. Roth, Alexander Marson, Nirav R. Bhakta, K. Mark Ansel, Walter E. Finkbeiner, David J. Erle, Prescott G. Woodruff
ORM1-like 3 (ORMDL3) has strong genetic linkage to childhood onset asthma. To determine whether ORMDL3 selective expression in airway smooth muscle (ASM) influences ASM function we used cre/lox techniques to generate transgenic mice (hORMDL3Myh11eGFP-cre) which express human ORMDL3 selectively in smooth muscle cells. In vitro studies of ASM cells isolated from the bronchi of hORMDL3Myh11eGFP-cre mice demonstrated that they developed hypertrophy (quantitated by FACS and image analysis), hyperplasia (assessed by BrdU incorporation), and expressed increased levels of tropomysin proteins TPM1 and TPM4. siRNA knockdown of TPM1 or TPM4 demonstrated their importance to ORMDL3 mediated ASM proliferation but not hypertrophy. In addition, ASM derived from hORMDL3Myh11eGFP-cre mice had increased contractility to histamine in vitro which was associated with increased levels of intracellular Ca2+, increased cell surface membrane Orai1 Ca2+ channels which mediate influx of Ca2+ into the cytoplasm, and increased expression of ASM contractile genes Serca2b and Sm22. In vivo studies of hORMDL3Myh11eGFP-cre mice demonstrated that they had a spontaneous increase in ASM and AHR. ORMDL3 expression in ASM thus induces changes in ASM (hypertrophy, hyperplasia, increased contractility) which may explain the contribution of ORMDL3 to the development of AHR in childhood onset asthma which is highly linked to ORMDL3 on chromosome 17q12-21.
Alexa K. Pham, Marina Miller, Peter Rosenthal, Sudipta Das, Ning Weng, Sunghoon Jang, Richard C. Kurten, Jana Badrani, Taylor A. Doherty, Brian G. Oliver, David H. Broide
Complexity of lung microenvironment and changes in cellular composition during disease make it exceptionally hard to understand molecular mechanisms driving development of chronic lung diseases. Although recent advances in cell-type resolved approaches hold great promise for studying complex diseases, their implementation relies on local access to fresh tissue, as traditional tissue storage methods do not allow viable cell isolation. To overcome these hurdles, we developed a versatile workflow that allows storage of lung tissue with high viability, permits thorough sample quality check before cell isolation, and befits sequencing-based profiling. We demonstrate that cryopreservation enables isolation of multiple cell types from both healthy and diseased lungs. Basal cells from cryopreserved airways retain their differentiation ability, indicating that cellular identity is not altered by cryopreservation. Importantly, using RNA sequencing and EPIC Array, we show that gene expression and DNA methylation signatures are preserved upon cryopreservation, emphasizing the suitability of our workflow for -omics profiling of lung cells. Moreover, we obtained high-quality single-cell RNA sequencing data of cells from cryopreserved human lung, demonstrating that cryopreservation empowers single-cell approaches. Overall, thanks to its simplicity, our workflow is well-suited for prospective tissue collection by academic collaborators and biobanks, opening worldwide access to viable human tissue.
Maria Llamazares Prada, Elisa Espinet, Vedrana Mijošek, Uwe Schwartz, Pavlo Lutsik, Raluca Tamas, Mandy Richter, Annika Behrendt, Stephanie T. Pohl, Naja P. Benz, Thomas Muley, Arne Warth, Claus Peter Heußel, Hauke Winter, Jonathan J. M. Landry, Felix J.F. Herth, Tinne C.J. Mertens, Harry Karmouty-Quintana, Ina Koch, Vladimir Benes, Jan O. Korbel, Sebastian M. Waszak, Andreas Trumpp, David M. Wyatt, Heiko F. Stahl, Christoph Plass, Renata Z. Jurkowska
Ventilation throughout life is dependent upon the formation of pulmonary alveoli which create an extensive surface area wherein the close apposition of respiratory epithelium and endothelial cells of the pulmonary microvascular enables efficient gas exchange. Morphogenesis of the alveoli initiates at late gestation in humans and the early postnatal period in the mouse. Alveolar septation are directed by complex signaling interactions among multiple cell types. Herein, we demonstrate that the expression of insulin-like growth factor 1 receptor (Igf1r) by a subset of pulmonary fibroblasts is required for normal alveologenesis in mice. Postnatal deletion of Igf1r caused alveolar simplification, disrupting alveolar elastin networks and extracellular matrix without altering myofibroblast differentiation or proliferation. Loss of Igf1r impaired contractile properties of lung myofibroblasts, inhibited myosin light chain (MLC) phosphorylation and mechanotransductive nuclear YAP activity. Activation of p-AKT, p-MLC and nuclear YAP in myofibroblasts was dependent on Igf1r. Pharmacologic activation of AKT enhanced MLC phosphorylation, increased YAP activation and ameliorated alveolar simplification in vivo. IGF1R controls mechanosignaling in myofibroblasts required for lung alveologenesis.
Hua He, John Snowball, Fei Sun, Cheng-Lun Na, Jeffrey A. Whitsett
Excess macrophages and smooth muscle cells (SMCs) characterize many cardiovascular diseases, but crosstalk between these cell types is poorly defined. Pulmonary hypertension (PH) is a lethal disease in which lung arteriole SMCs proliferate and migrate, coating the normally unmuscularized distal arteriole. We hypothesized that increased macrophage platelet-derived growth factor (PDGF)-B induces pathological SMC burden in PH. Our results indicate that clodronate attenuates hypoxia-induced macrophage accumulation, distal muscularization, PH and right ventricle hypertrophy (RVH). With hypoxia exposure, macrophage Pdgfb mRNA is upregulated in mice, and LysM Cre mice carrying floxed alleles for hypoxia-inducible factor 1a, 2a, or Pdgfb have reduced macrophage Pdgfb and are protected against distal muscularization and PH. Conversely, LysM Cre, von-Hippel Lindau(flox/flox) mice have increased macrophage Hifa and Pdgfb and develop distal muscularization, PH and RVH in normoxia. Similarly, Pdgfb is upregulated in macrophages from human idiopathic or systemic sclerosis-induced pulmonary arterial hypertension patients, and macrophage-conditioned medium from these patients increases SMC proliferation and migration via PDGF-B. Finally, in mice, orotracheal administration of nanoparticles loaded with Pdgfb siRNA specifically reduces lung macrophage Pdgfb and prevents hypoxia-induced distal muscularization, PH and RVH. Thus, macrophage-derived PDGF-B is critical for pathological SMC expansion in PH, and nanoparticle-mediated inhibition of lung macrophage PDGF-B has profound implications as an interventional strategy for PH.
Aglaia Ntokou, Jui M. Dave, Amy C. Kauffman, Maor Sauler, Changwan Ryu, John Hwa, Erica L. Herzog, Inderjit Singh, W. Mark Saltzman, Daniel M. Greif
Myofibroblasts are the major cellular source of collagen, and their accumulation – via differentiation from fibroblasts and resistance to apoptosis – is a hallmark of tissue fibrosis. Clearance of myofibroblasts by de-differentiation and restoration of apoptosis sensitivity has the potential to reverse fibrosis. Prostaglandin E2 (PGE2) and mitogens such as FGF2 have each been shown to de-differentiate myofibroblasts, but the resultant cellular phenotypes have neither been comprehensively characterized nor compared. Here we show that PGE2 elicited de-differentiation of human lung myofibroblasts via cAMP/PKA while FGF2 utilized MEK/ERK. The two mediators yielded transitional cells with distinct transcriptomes, with FGF2 promoting but PGE2 inhibiting proliferation and survival. The gene expression pattern in fibroblasts isolated from the lungs of mice undergoing resolution of experimental fibrosis resembled that of myofibroblasts treated with PGE2 in vitro. We conclude that myofibroblast de-differentiation can proceed via distinct programs exemplified by treatment with PGE2 and FGF2, with that occurring in vivo most closely resembling the former.
Sean M. Fortier, Loka R. Penke, Dana M. King, Tho X. Pham, Giovanni Ligresti, Marc Peters-Golden
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