The importance of detailed epigenomic profiling of different cell types within organs.

The human body consists of hundreds of kinds of cells specified from a single genome overlaid with cell type-specific epigenetic information. Comprehensively profiling the body's distinct epigenetic landscapes will allow researchers to verify cell types used in regenerative medicine and to determine the epigenetic effects of disease, environmental exposures and genetic variation. Key marks/factors that should be investigated include regions of nucleosome-free DNA accessible to regulatory factors, histone marks defining active enhancers and promoters, DNA methylation levels, regulatory RNAs, and factors controlling the three-dimensional conformation of the genome. Here we use the lung to illustrate the importance of investigating an organ's purified cell epigenomes, and outline the challenges and promise of realizing a comprehensive catalog of primary cell epigenomes

Transcriptomic profiling of primary alveolar epithelial cell differentiation in human and rat

AbstractCell-type specific gene regulation is a key to gaining a full understanding of how the distinct phenotypes of differentiated cells are achieved and maintained. Here we examined how changes in transcriptional activation during alveolar epithelial cell (AEC) differentiation determine phenotype. We performed transcriptomic profiling using in vitro differentiation of human and rat primary AEC. This model recapitulates in vitro an in vivo process in which AEC transition from alveolar type 2 (AT2) cells to alveolar type 1 (AT1) cells during normal maintenance and regeneration following lung injury. Here we describe in detail the quality control, preprocessing, and normalization of microarray data presented within the associated study (Marconett et al., 2013). We also include R code for reproducibility of the referenced data and easily accessible processed data tables

SMAD3 prevents binding of NKX2.1 and FOXA1 to the SpB promoter through its MH1 and MH2 domains

Mechanisms of gene repression by transforming growth factor-beta (TGF-beta) are not well understood. TGF-beta represses transcription of pulmonary surfactant protein-B gene in lung epithelial cells. Repression is mediated by SMAD3 through interactions with NKX2.1 and FOXA1, two key transcription factors that are positive regulators of SpB transcription. In this study, we found that SMAD3 interacts through its MAD domains, MH1 and MH2 with NKX2.1 and FOXA1 proteins. The sites of interaction on NKX2.1 are located within the NH2 and COOH domains, known to be involved in transactivation function. In comparison, weaker interaction of FOXA1 winged helix, and the NH2-terminal domains was documented with SMAD3. Both in vitro studies and in vivo ChIP assays show that interaction of SMAD3 MH1 and MH2 domains with NKX2.1 and FOXA1 results in reduced binding of NKX2.1 and FOXA1 to their cognate DNA-binding sites, and diminished promoter occupancy within the SpB promoter. Thus, these studies reveal for the first time a mechanism of TGF-beta-induced SpB gene repression that involves interactions between specific SMAD3 domains and the corresponding functional sites on NKX2.1 and FOXA1 transcription factors

Translocation of PEGylated quantum dots across rat alveolar epithelial cell monolayers

Farnoosh Fazlollahi1,8, Arnold Sipos1,2, Yong Ho Kim1,2, Sarah F Hamm-Alvarez6, Zea Borok1–3, Kwang-Jin Kim1,2,5–7, Edward D Crandall1,2,4,8 1Will Rogers Institute Pulmonary Research Center, 2Department of Medicine, 3Department of Biochemistry and Molecular Biology, 4Department of Pathology, 5Department of Physiology and Biophysics, 6Department of Pharmacology and Pharmaceutical Sciences, 7Department of Biomedical Engineering, 8Mork Family Department of Chemical Engineering and Materials Science, University of Southern California, Los Angeles, CA, USA Background: In this study, primary rat alveolar epithelial cell monolayers (RAECM) were used to investigate transalveolar epithelial quantum dot trafficking rates and underlying transport mechanisms. Methods: Trafficking rates of quantum dots (PEGylated CdSe/ZnS, core size 5.3 nm, hydrodynamic size 25 nm) in the apical-to-basolateral direction across RAECM were determined. Changes in bioelectric properties (ie, transmonolayer resistance and equivalent active ion transport rate) of RAECM in the presence or absence of quantum dots were measured. Involvement of endocytic pathways in quantum dot trafficking across RAECM was assessed using specific inhibitors (eg, methyl-ß-cyclodextrin, chlorpromazine, and dynasore for caveolin-, clathrin-, and dynamin-mediated endocytosis, respectively). The effects of lowering tight junctional resistance on quantum dot trafficking were determined by depleting Ca2+ in apical and basolateral bathing fluids of RAECM using 2 mM EGTA. Effects of temperature on quantum dot trafficking were studied by lowering temperature from 37°C to 4°C. Results: Apical exposure of RAECM to quantum dots did not elicit changes in transmonolayer resistance or ion transport rate for up to 24 hours; quantum dot trafficking rates were not surface charge-dependent; methyl-ß-cyclodextrin, chlorpromazine, and dynasore did not decrease quantum dot trafficking rates; lowering of temperature decreased transmonolayer resistance by approximately 90% with a concomitant increase in quantum dot trafficking by about 80%; and 24 hours of treatment of RAECM with EGTA decreased transmonolayer resistance by about 95%, with increased quantum dot trafficking of up to approximately 130%. Conclusion: These data indicate that quantum dots do not injure RAECM and that quantum dot trafficking does not appear to take place via endocytic pathways involving caveolin, clathrin, or dynamin. We conclude that quantum dot translocation across RAECM takes place via both transcellular and paracellular pathways and, based on comparison with our prior studies, interactions of nanoparticles with RAECM are strongly dependent on nanoparticle composition and surface properties. Keywords: alveolar epithelial barrier, transport, paracellular pathways, endocytosi

Tissue-dependent consequences of Apc inactivation on proliferation and differentiation of ciliated cell progenitors via Wnt and Notch signaling

The molecular signals that control decisions regarding progenitor/stem cell proliferation versus differentiation are not fully understood. Differentiation of motile cilia from progenitor/stem cells may offer a simple tractable model to investigate this process. Wnt and Notch represent two key signaling pathways in progenitor/stem cell behavior in a number of tissues. Adenomatous Polyposis Coli, Apc is a negative regulator of the Wnt pathway and a well known multifunctional protein. Using the cre-LoxP system we inactivated the Apc locus via Foxj1-cre, which is expressed in cells committed to ciliated cell lineage. We then characterized the consequent phenotype in two select tissues that bear motile cilia, the lung and the testis. In the lung, Apc deletion induced β-catenin accumulation and Jag1 expression in ciliated cells and by lateral induction, triggered Notch signaling in adjacent Clara cells. In the bronchiolar epithelium, absence of Apc blocked the differentiation of a subpopulation of cells committed to the ciliogenesis program. In the human pulmonary adenocarcinoma cells, Apc over-expression inhibited Jag1 expression and promoted motile ciliogenic gene expression program including Foxj1, revealing the potential mechanism. In the testis, Apc inactivation induced β-catenin accumulation in the spermatogonia, but silenced Notch signaling and depleted spermatogonial stem cells, associated with reduced proliferation, resulting in male infertility. In sum, the present comparative analysis reveals the tissue-dependent consequences of Apc inactivation on proliferation and differentiation of ciliated cell progenitors by coordinating Wnt and Notch signaling

Efficient generation and transcriptomic profiling of human iPSC-derived pulmonary neuroendocrine cells

Expansion of pulmonary neuroendocrine cells (PNECs) is a pathological feature of many human lung diseases. Human PNECs are inherently difficult to study due to their rarity (\u3c1% of total lung cells) and a lack of established protocols for their isolation. We used induced pluripotent stem cells (iPSCs) to generate induced PNECs (iPNECs), which express core PNEC markers, including ROBO receptors, and secrete major neuropeptides, recapitulating known functions of primary PNECs. Furthermore, we demonstrate that differentiation efficiency is increased in the presence of an air-liquid interface and inhibition of Notch signaling. Single-cell RNA sequencing (scRNA-seq) revealed a PNEC-associated gene expression profile that is concordant between iPNECs and human fetal PNECs. In addition, pseudotime analysis of scRNA-seq results suggests a basal cell origin of human iPNECs. In conclusion, our model has the potential to provide an unlimited source of human iPNECs to explore PNEC pathophysiology associated with several lung diseases

Platelet CLEC-2 protects against lung injury via effects of its ligand podoplanin on inflammatory alveolar macrophages in the mouse

There is no therapeutic intervention proven to prevent acute respiratory distress syndrome (ARDS). Novel mechanistic insights into the pathophysiology of ARDS are therefore required. Platelets are implicated in regulating many of the pathogenic processes that occur during ARDS; however, the mechanisms remain elusive. The platelet receptor CLEC-2 has been shown to regulate vascular integrity at sites of acute inflammation. Therefore the purpose of this study was to establish the role of CLEC-2 and its ligand podoplanin in a mouse model of ARDS. Platelet-specific CLEC-2-deficient, as well as alveolar epithelial type I cell (AECI)-specific or hematopoietic-specific podoplanin deficient, mice were established using cre-loxP strategies. Combining these with intratracheal (IT) instillations of lipopolysaccharide (LPS), we demonstrate that arterial oxygen saturation decline in response to IT-LPS in platelet-specific CLEC-2-deficient mice is significantly augmented. An increase in bronchoalveolar lavage (BAL) neutrophils and protein was also observed 48 h post-IT-LPS, with significant increases in pro-inflammatory chemokines detected in BAL of platelet-specific CLEC-2-deficient animals. Deletion of podoplanin from hematopoietic cells but not AECIs also reduces lung function and increases pro-inflammatory chemokine expression following IT-LPS. Furthermore, we demonstrate that following IT-LPS, platelets are present in BAL in aggregates with neutrophils, which allows for CLEC-2 interaction with podoplanin expressed on BAL inflammatory alveolar macrophages. Taken together, these data suggest that the platelet CLEC-2-podoplanin signaling axis regulates the severity of lung inflammation in mice and is a possible novel target for therapeutic intervention in patients at risk of developing ARDS. </jats:p

Stem Cells, Cell Therapies, and Bioengineering in Lung Biology and Disease 2021

The 9th biennial conference titled “Stem Cells, Cell Therapies, and Bioengineering in Lung Biology and Diseases” was hosted virtually, due to the ongoing COVID-19 pandemic, in collaboration with the University of Vermont Larner College of Medicine, the National Heart, Lung, and Blood Institute, the Alpha-1 Foundation, the Cystic Fibrosis Foundation, and the International Society for Cell & Gene Therapy. The event was held from July 12th through 15th, 2021 with a pre-conference workshop held on July 9th. As in previous years, the objectives remained to review and discuss the status of active research areas involving stem cells (SCs), cellular therapeutics, and bioengineering as they relate to the human lung. Topics included 1) technological advancements in the in situ analysis of lung tissues, 2) new insights into stem cell signaling and plasticity in lung remodeling and regeneration, 3) the impact of extracellular matrix in stem cell regulation and airway engineering in lung regeneration, 4) differentiating and delivering stem cell therapeutics to the lung, 5) regeneration in response to viral infection, and 6) ethical development of cell-based treatments for lung diseases. This selection of topics represents some of the most dynamic and current research areas in lung biology