iPSC-Derived Airway Epithelial Cells: Progress, Promise, and Challenges.

Induced pluripotent stem cells (iPSCs) generated from somatic cell sources are pluripotent and capable of indefinite expansion in vitro. They provide an unlimited source of cells that can be differentiated into lung progenitor cells for potential clinical use in pulmonary regenerative medicine. This review gives a comprehensive overview of recent progress toward the use of iPSCs to generate proximal and distal airway epithelial cells and mix lung organoids. Furthermore, their potential applications and future challenges for the field are discussed, with a focus on the technological hurdles that must be cleared before stem cell therapeutics can be used for clinical treatment.

Lung adenocarcinoma-related TNF-α-dependent inflammation upregulates MHC-II on alveolar type II cells through CXCR-2 to contribute to Treg expansion.

MHC-II on alveolar type-II (AT-II) cells is associated with immune tolerance in an inflammatory microenvironment. Recently, we found TNF-α upregulated MHC-II in AT-II in vitro. In this study, we explored whether TNF-α-mediated inflammation upregulates MHC-II on AT-II cells to trigger Treg expansion in inflammation-driven lung adenocarcinoma (IDLA). Using urethane-induced mice IDLA model, we found that IDLA cells mainly arise from AT-II cells, which are the major source of MHC-II. Blocking urethane-induced inflammation by TNF-α neutralization inhibited tumorigenesis and reversed MHC-II upregulation on tumor cells of AT-II cellular origin in IDLA. MHC-II-dependent AT-II cells were isolated from IDLA-induced Treg expansion. In human LA samples, we found high expression of MHC-II in tumor cells of AT-II cellular origin, which was correlated with increased Foxp3+ T cells infiltration as well as CXCR-2 expression. CXCR-2 and MHC-II colocalization was observed in inflamed lung tissue and IDLA cells of AT-II cellular origin. Furthermore, at the pro-IDLA inflammatory stage, TNF-α-neutralization or CXCR-2 deficiency inhibited the upregulation of MHC-II on AT-II cells in inflamed lung tissue. Thus, tumor cells of AT-II cellular origin contribute to Treg expansion in an MHC-II-dependent manner in TNF-α-mediated IDLA. At the pro-tumor inflammatory stage, TNF-α-dependent lung inflammation plays an important role in MHC-II upregulation on AT-II cells.

Differential effects of Nintedanib and Pirfenidone on lung alveolar epithelial cell function in ex vivo murine and human lung tissue cultures of pulmonary fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a fatal interstitial lung disease. Repetitive injury and reprogramming of the lung epithelium are thought to be critical drivers of disease progression, contributing to fibroblast activation, extracellular matrix remodeling, and subsequently loss of lung architecture and function. To date, Pirfenidone and Nintedanib are the only approved drugs known to decelerate disease progression, however, if and how these drugs affect lung epithelial cell function, remains largely unexplored. METHODS: We treated murine and human 3D ex vivo lung tissue cultures (3D-LTCs; generated from precision cut lung slices (PCLS)) as well as primary murine alveolar epithelial type II (pmATII) cells with Pirfenidone or Nintedanib. Murine 3D-LTCs or pmATII cells were derived from the bleomycin model of fibrosis. Early fibrotic changes were induced in human 3D-LTCs by a mixture of profibrotic factors. Epithelial and mesenchymal cell function was determined by qPCR, Western blotting, Immunofluorescent staining, and ELISA. RESULTS: Low µM concentrations of Nintedanib (1 µM) and mM concentrations of Pirfenidone (2.5 mM) reduced fibrotic gene expression including Collagen 1a1 and Fibronectin in murine and human 3D-LTCs as well as pmATII cells. Notably, Nintedanib stabilized expression of distal lung epithelial cell markers, especially Surfactant Protein C in pmATII cells as well as in murine and human 3D-LTCs. CONCLUSIONS: Pirfenidone and Nintedanib exhibit distinct effects on murine and human epithelial cells, which might contribute to their anti-fibrotic action. Human 3D-LTCs represent a valuable tool to assess anti-fibrotic mechanisms of potential drugs for the treatment of IPF patients.

Angiotensin II Brings More Questions Than Answers.

The approval of synthetic human angiotensin II (Giapreza, LaJolla Pharmaceuticals) by the FDA in December 2017 provides clinicians with a new tool in the treatment of distributive shock. Angiotensin II (ATII) was approved based on the results of the ATHOS-3 trial. In this trial, patients who received angiotensin II were more likely to achieve a mean arterial pressure of 75 mmHg or an increase in mean arterial pressure of 10 mmHg above that seen in patients who received a placebo. However, the results of ATHOS-3 also highlighted important concerns about thrombotic and infectious complications associated with ATII. Given that the cost of medication acquisition is approximately $1,500 per vial, practitioners must also decide how to implement ATII into practice in the most cost-effective manner. This commentary examines the current controversies surrounding both the safety and efficacy of ATII.

Stretch-induced apoptosis in rat alveolar epithelial cells is mediated by the intrinsic mitochondrial pathway.

BACKGROUND: Alveolar type II (ATII) cells in the lung are exposed to mechanical stretch during breathing and mechanical ventilation. Increased mechanical stretch contributes to lung injury by induction of apoptosis and necrosis in ATII cells. AIM OF THE STUDY: In this study, we investigated the intrinsic and the extrinsic apoptosis pathways, and their involvement in our model of stretch-induced apoptosis. MATERIAL AND METHODS: ATII cells were stretched on elastic membranes for 24 h and apoptosis was determined at different time points. Several factors of the intrinsic and the extrinsic pathway were investigated by FACS, ELISA, and Western blot. We also investigated the inhibition of reactive oxygen species (ROS) expression and cytochrome C release and their influence on induction of apoptosis by stretching. RESULTS: In comparison to static cells, ROS generation and cytochrome C release were increased in stretched cells while at the same time, mitochondrial membrane potential was reduced. Both inhibition of ROS generation by tocopherol and inhibition of cytochrome C release by cyclosporine A led to reduction of stretch-induced apoptosis. An increase of caspase 8 and 9 was observed in stretched cells compared to static cells. In contrast, factors of the extrinsic pathway such as TNF-alpha, TRAIL, TNFRI, Fas, FasL, and FADD were not different to static cells at all time points or where not detectable. CONCLUSION: Together, these findings suggest predominance of the intrinsic pathway in stretch-induced apoptosis.

Inhibition of Wnt/ß-catenin signaling suppresses bleomycin-induced pulmonary fibrosis by attenuating the expression of TGF-ß1 and FGF-2.

Pulmonary fibrosis is a progressive lung disorder of unknown etiology, which is characterized by alterations in alveolar epithelium function, fibroblast activation, and increased extracellular matrix deposition. Recent studies have demonstrated that PF is associated with uncontrolled production of cytokines after lung injury. In the present study, we found that transforming growth factor-ß1 (TGF-ß1) and fibroblast growth factor 2 (FGF-2) were both upregulated in bleomycin-induced fibrotic lung tissue and primary murine alveolar epithelial Type II (ATII) cells treated with bleomycin. Furthermore, we discovered that TGF-ß1 could induce the differentiation of lung resident mesenchymal stem cells (LR-MSCs) into fibroblasts, which may play an essential role in PF. LR-MSCs incubated with FGF-2 showed modest alterations in the expression of α-SMA and Vimentin. Moreover, in our study, we found that Wnt/ß-catenin signaling was activated both in vitro and in vivo as a result of bleomycin treatment. Interestingly, we also found that suppression of the Wnt/ß-catenin signaling could significantly attenuate bleomycin-induced PF accompanied with decreased expression of TGF-ß1 and FGF-2 in vitro and in vivo. These results support that controlling the aberrant expression of TGF-ß1 and FGF-2 via inhibition of Wnt/ß-catenin signaling could serve as a potential therapeutic strategy for PF.

Role of Wnt/ß-Catenin Signaling in Epithelial Differentiation of Lung Resident Mesenchymal Stem Cells.

Accumulating evidence has demonstrated that stem cells have the ability to repair the lung tissue injuries following either injection of cultured cells or bone marrow transplantation. As a result, increasing attention has focused on the lung resident mesenchymal stem cells (LR-MSCs) for repairing damaged lung tissues. Meanwhile, some studies have revealed that Wnt/ß-catenin signaling plays an important role in the epithelial differentiation of mesenchymal stem cells (MSCs). In the current study, our aim was to explore the roles of Wnt/ß-catenin signaling on cell proliferation and epithelial differentiation of LR-MSCs. We have successfully isolated the stem cell antigen (Sca)-1(+) CD45(-) CD31(-) cells which were proposed to be LR-MSCs by magnetic-activated cell sorting (MACS). Furthermore, we demonstrated the expression of epithelial markers on LR-MSCs following indirect co-culture of these cells with alveolar epithelial type II (ATII) cells, confirming the epithelial phenotype of LR-MSCs following co-culture. In order to clarify the regulatory mechanisms of Wnt/ß-catenin signaling in epithelial differentiation of LR-MSCs, we measured the protein levels of several important members involved in Wnt/ß-catenin signaling in the presence or absence of some canonical activators and inhibitors of the ß-catenin pathways. In conclusion, our study demonstrated that Wnt/ß-catenin signaling may be an essential mechanism underlying the regulation of epithelial differentiation of LR-MSCs.

The C-terminus of the long AKAP13 isoform (AKAP-Lbc) is critical for development of compensatory cardiac hypertrophy.

The objective of this study was to determine the role of A-Kinase Anchoring Protein (AKAP)-Lbc in the development of heart failure, by investigating AKAP-Lbc-protein kinase D1 (PKD1) signaling in vivo in cardiac hypertrophy. Using a gene-trap mouse expressing a truncated version of AKAP-Lbc (due to disruption of the endogenous AKAP-Lbc gene), that abolishes PKD1 interaction with AKAP-Lbc (AKAP-Lbc-ΔPKD), we studied two mouse models of pathological hypertrophy: i) angiotensin (AT-II) and phenylephrine (PE) infusion and ii) transverse aortic constriction (TAC)-induced pressure overload. Our results indicate that AKAP-Lbc-ΔPKD mice exhibit an accelerated progression to cardiac dysfunction in response to AT-II/PE treatment and TAC. AKAP-Lbc-ΔPKD mice display attenuated compensatory cardiac hypertrophy, increased collagen deposition and apoptosis, compared to wild-type (WT) control littermates. Mechanistically, reduced levels of PKD1 activation are observed in AKAP-Lbc-ΔPKD mice compared to WT mice, resulting in diminished phosphorylation of histone deacetylase 5 (HDAC5) and decreased hypertrophic gene expression. This is consistent with a reduced compensatory hypertrophy phenotype leading to progression of heart failure in AKAP-Lbc-ΔPKD mice. Overall, our data demonstrates a critical in vivo role for AKAP-Lbc-PKD1 signaling in the development of compensatory hypertrophy to enhance cardiac performance in response to TAC-induced pressure overload and neurohumoral stimulation by AT-II/PE treatment.

Idiopathic Pulmonary Fibrosis Caused by Damaged Mitochondria and Imbalanced Protein Homeostasis in Alveolar Epithelial Type II Cell.

Alveolar epithelial Type II (ATII) cells are closely associated with early events of Idiopathic pulmonary fibrosis (IPF). Proteostasis dysfunction, endoplasmic reticulum (ER) stress, and mitochondrial dysfunction are known causes of decreased proliferation of alveolar epithelial cells and the secretion of pro-fibrotic mediators. Here, a large body of evidence is systematized and a cascade relationship between protein homeostasis, endoplasmic reticulum stress, mitochondrial dysfunction, and fibrotropic cytokines is proposed, providing a theoretical basis for ATII cells dysfunction as a possible pathophysiological initiating event for idiopathic pulmonary fibrosis.

PAI-1 Regulation of p53 Expression and Senescence in Type II Alveolar Epithelial Cells.

Cellular senescence contributes importantly to aging and aging-related diseases, including idiopathic pulmonary fibrosis (IPF). Alveolar epithelial type II (ATII) cells are progenitors of alveolar epithelium, and ATII cell senescence is evident in IPF. Previous studies from this lab have shown that increased expression of plasminogen activator inhibitor 1 (PAI-1), a serine protease inhibitor, promotes ATII cell senescence through inducing p53, a master cell cycle repressor, and activating p53-p21-pRb cell cycle repression pathway. In this study, we further show that PAI-1 binds to proteasome components and inhibits proteasome activity and p53 degradation in human lung epithelial A549 cells and primary mouse ATII cells. This is associated with a senescence phenotype of these cells, manifested as increased p53 and p21 expression, decreased phosphorylated retinoblastoma protein (pRb), and increased senescence-associated beta-galactose (SA-ß-gal) activity. Moreover, we find that, although overexpression of wild-type PAI-1 (wtPAI-1) or a secretion-deficient, mature form of PAI-1 (sdPAI-1) alone induces ATII cell senescence (increases SA-ß-gal activity), only wtPAI-1 induces p53, suggesting that the premature form of PAI-1 is required for the interaction with the proteasome. In summary, our data indicate that PAI-1 can bind to proteasome components and thus inhibit proteasome activity and p53 degradation in ATII cells. As p53 is a master cell cycle repressor and PAI-1 expression is increased in many senescent cells, the results from this study will have a significant impact not only on ATII cell senescence/lung fibrosis but also on the senescence of other types of cells in different diseases.

HUB genes transcriptionally regulate lipid metabolism in alveolar type II cells under LPS stimulation.

Objective: Alveolar type II (ATII) cells produce pulmonary surfactant (PS) essential for maintaining lung function. The aberration or depletion of PS can cause alveolar collapse, a hallmark of acute respiratory distress syndrome (ARDS). However, the intricacies underlying these changes remain unclear. This study aimed to elucidate the mechanisms underlying PS perturbations in ATII cells using transcriptional RNA-seq, offering insights into the pathogenesis of ARDS. Methods: ATII cells were identified using immunofluorescence targeting surface-active protein C. We used 24-h lipopolysaccharide (LPS)-induced ATII cells as an ARDS cell model. The efficacy of the injury model was gauged by detecting the presence of tumour necrosis factor-α and interleukin-6. RNA-seq analysis was performed to investigate the dynamics of PS deviation in unaltered and LPS-exposed ATII cells. Results: Whole-transcriptome sequencing revealed that LPS-stimulated ATII cells showed significantly increased transcription of genes, including Lss, Nsdhl, Hmgcs1, Mvd, Cyp51, Idi1, Acss2, Insig1, and Hsd17b7, which play key roles in regulating cholesterol biosynthesis. We further verified gene levels using real-time quantitative PCR, and the results showed that the mRNA expression of these genes increased, which was consistent with the RNA-seq results. Conclusion: Our study revealed pivotal transcriptional shifts in ATII cells after LPS exposure, particularly in nine key lipid and cholesterol metabolism genes. This altered expression might disrupt the lipid balance, ultimately affecting PS function. This finding deepens our understanding of the aetiology of ARDS and may lead to new therapeutic directions.

Short telomeres in alveolar type II cells associate with lung fibrosis in post COVID-19 patients with cancer.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the coronavirus disease 2019 (COVID-19) pandemic. The severity of COVID-19 increases with each decade of life, a phenomenon that suggest that organismal aging contributes to the fatality of the disease. In this regard, we and others have previously shown that COVID-19 severity correlates with shorter telomeres, a molecular determinant of aging, in patient's leukocytes. Lung injury is a predominant feature of acute SARS-CoV-2 infection that can further progress to lung fibrosis in post-COVID-19 patients. Short or dysfunctional telomeres in Alveolar type II (ATII) cells are sufficient to induce pulmonary fibrosis in mouse and humans. Here, we analyze telomere length and the histopathology of lung biopsies from a cohort of alive post-COVID-19 patients and a cohort of age-matched controls with lung cancer. We found loss of ATII cellularity and shorter telomeres in ATII cells concomitant with a marked increase in fibrotic lung parenchyma remodeling in post- COVID-19 patients compared to controls. These findings reveal a link between presence of short telomeres in ATII cells and long-term lung fibrosis sequel in Post-COVID-19 patients.

Regeneration or Repair? The Role of Alveolar Epithelial Cells in the Pathogenesis of Idiopathic Pulmonary Fibrosis (IPF).

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive interstitial lung disease (ILD) with unknown etiology in which gradual fibrotic scarring of the lungs leads to usual interstitial pneumonia (UIP) and, ultimately, to death. IPF affects three million people worldwide, and the only currently available treatments include the antifibrotic drugs nintedanib and pirfenidone, which effectively reduce fibrosis progression are, unfortunately, not effective in curing the disease. In recent years, the paradigm of IPF pathogenesis has shifted from a fibroblast-driven disease to an epithelium-driven disease, wherein, upon recurrent microinjuries, dysfunctional alveolar type II epithelial cells (ATII) are not only unable to sustain physiological lung regeneration but also promote aberrant epithelial-mesenchymal crosstalk. This creates a drift towards fibrosis rather than regeneration. In the context of this review article, we discuss the most relevant mechanisms involved in IPF pathogenesis with a specific focus on the role of dysfunctional ATII cells in promoting disease progression. In particular, we summarize the main causes of ATII cell dysfunction, such as aging, environmental factors, and genetic determinants. Next, we describe the known mechanisms of physiological lung regeneration by drawing a parallel between embryonic lung development and the known pathways involved in ATII-driven alveolar re-epithelization after injury. Finally, we review the most relevant interventional clinical trials performed in the last 20 years with the aim of underlining the urgency of developing new therapies against IPF that are not only aimed at reducing disease progression by hampering ECM deposition but also boost the physiological processes of ATII-driven alveolar regeneration.

Abnormal respiratory progenitors in fibrotic lung injury.

Recent advances in single-cell RNA sequencing (scRNA-seq) and epithelium lineage labeling have yielded identification of multiple abnormal epithelial progenitor populations during alveolar type 2 (ATII) cell differentiation into alveolar type 1 (ATI) cells during regenerative lung post-fibrotic injury. These abnormal cells include basaloid/basal-like cells, ATII transition cells, and persistent epithelial progenitors (PEPs). These cells occurred and accumulated during the regeneration of distal airway and alveoli in response to both chronic and acute pulmonary injury. Among the alveolar epithelial progenitors, PEPs express a distinct Krt8+ phenotype that is rarely found in intact alveoli. However, post-injury, the Krt8+ phenotype is seen in dysplastic epithelial cells. Fully understanding the characteristics and functions of these newly found, injury-induced abnormal behavioral epithelial progenitors and the signaling pathways regulating their phenotype could potentially point the way to unique therapeutic targets for fibrosing lung diseases. This review summarizes recent advances in understanding these epithelial progenitors as they relate to uncovering regenerative mechanisms.

Characterization of circRNA-miRNA-mRNA networks regulating oxygen utilization in type II alveolar epithelial cells of Tibetan pigs.

Understanding the signaling pathway regulatory mechanisms in type II alveolar epithelial (ATII) cells, the progenitor cells responsible for proliferating and regenerating type I alveolar epithelial (ATI) and ATII cells, in Tibetan pigs is beneficial for exploring methods of preventing and repairing cellular damage during hypoxia. We simulated a hypoxic environment (2% O2) for culture ATII cells of Tibetan pigs and Landrace pigs, with cells cultured under normoxic conditions (21% O2) as a control group, and performed integrated analysis of circular RNA (circRNA)-microRNA (miRNA)-messenger RNA (mRNA) regulatory axes by whole-transcriptome sequencing. Functional enrichment analysis indicated that the source genes of the differential expressed circRNAs (DEcircRNAs) were primarily involved in cell proliferation, cellular processes, and cell killing. A series of DEcircRNAs were derived from inhibitors of apoptosis proteins and led to a key autonomous effect as modulators of cell repair in Tibetan pigs under hypoxia. The significant higher expression of COL5A1 in TL groups may inhibited apoptosis of ATII cells in Tibetan pigs under lower oxygen concentration, and may lead their better survive in the hypoxia environment. In addition, a competing endogenous RNA (ceRNA) network of functional interactions was constructed that included novel_circ_000898-ssc-miR-199a-5p-CAV1 and novel_circ_000898-ssc-miR-378-BMP2, based on the node genes ssc-miR-199a-5p and ssc-miR-378, which may regulate multiple miRNAs and mRNAs that mediate endoplasmic reticulum (ER) stress-induced apoptosis and inflammation and attenuate hypoxia-induced injury in ATII cells under hypoxic conditions. These results broaden our knowledge of circRNAs, miRNAs, and mRNAs associated with hypoxia and provide new insights into the hypoxic response of ATII cells in Tibetan pigs.

Alternative splicing signature of alveolar type II epithelial cells of Tibetan pigs under hypoxia-induced.

Alternative splicing (AS) allows the generation of multiple transcript variants from a single gene and affects biological processes by generating protein diversity in organisms. In total, 41,642 AS events corresponding to 9,924 genes were identified, and SE is the most abundant alternatively spliced type. The analysis of functional categories demonstrates that alternatively spliced differentially expressed genes (DEGs) were enriched in the MAPK signaling pathway and hypoxia-inducible factor 1 (HIF-1) signaling pathway. Proteoglycans in cancer between the normoxic (21% O2, TN and LN) and hypoxic (2% O2, TL and LL) groups, such as SLC2A1, HK1, HK2, ENO3, and PFKFB3, have the potential to rapidly proliferate alveolar type II epithelial (ATII) cells by increasing the intracellular levels of glucose and quickly divert to anabolic pathways by glycolysis intermediates under hypoxia. ACADL, EHHADH, and CPT1A undergo one or two AS types with different frequencies in ATII cells between TN and TL groups (excluding alternatively spliced DEGs shared between normoxic and hypoxic groups), and a constant supply of lipids might be obtained either from the circulation or de novo synthesis for better growth of ATII cells under hypoxia condition. MCM7 and MCM3 undergo different AS types between LN and LL groups (excluding alternatively spliced DEGs shared between normoxic and hypoxic groups), which may bind to the amino-terminal PER-SIM-ARNT domain and the carboxyl terminus of HIF-1α to maintain their stability. Overall, AS and expression levels of candidate mRNAs between Tibetan pigs and Landrace pigs revealed by RNA-seq suggest their potential involvement in the ATII cells grown under hypoxia conditions.

Ssc-miR-141 Attenuates Hypoxia-Induced Alveolar Type II Epithelial Cell Injury in Tibetan Pigs by Targeting PDCD4.

The Tibetan pig is an endemic economic animal in the plateau region of China, and has a unique adaptation mechanism to the plateau hypoxic environment. Research into microRNAs (miRNAs) involved in the mechanism underlying hypoxia adaptation of Tibetan pig is very limited. Therefore, we isolated alveolar type II epithelial (ATII) cells from the lungs of the Tibetan pig, cultured them in normoxia/hypoxia (21% O2; 2% O2) for 48 h, and performed high-throughput sequencing analysis. We identified a hypoxic stress-related ssc-miR-141 and predicted its target genes. The target genes of ssc-miR-141 were mainly enriched in mitogen-activated protein kinase (MAPK), autophagy-animal, and Ras signaling pathways. Further, we confirmed that PDCD4 may serve as the target gene of ssc-miR-141. Real-time quantitative polymerase chain reaction (RT-qPCR) analysis was performed to confirm the expression levels of ssc-miR-141 and PDCD4, and a dual-luciferase gene reporter system was used to verify the targeted linkage of ssc-miR-141 to PDCD4. The results showed that the expression level of ssc-miR-141 in the hypoxia group was higher than that in the normoxia group, while the expression level of PDCD4 tended to show the opposite trend and significantly decreased under hypoxia. These findings suggest that ssc-miR-141 is associated with hypoxia adaptation and provide a new insight into the role of miRNAs from ATII cells of Tibetan pig in hypoxia adaptation.

Transcriptome and Metabolome Integration Provides New Insights Into the Regulatory Networks of Tibetan Pig Alveolar Type II Epithelial Cells in Response to Hypoxia.

Tibetan pigs show a widespread distribution in plateau environments and exhibit striking physiological and phenotypic differences from others pigs for adaptation to hypoxic conditions. However, the regulation of mRNAs and metabolites as well as their functions in the alveolar type II epithelial (ATII) cells of Tibetan pigs remain undefined. Herein, we carried out integrated metabolomic and transcriptomic profiling of ATII cells between Tibetan pigs and Landrace pigs across environments with different oxygen levels to delineate their signature pathways. We observed that the differentially accumulated metabolites (DAMs) and differentially expressed genes (DEGs) profiles displayed marked synergy of hypoxia-related signature pathways in either Tibetan pigs or Landrace pigs. A total of 1,470 DEGs shared between normoxic (TN, ATII cells of Tibetan pigs were cultured under 21% O2; LN, ATII cells of Landrace pigs were cultured under 21% O2) and hypoxic (TL, ATII cells of Tibetan pigs were cultured under 2% O2; LL, ATII cells of Landrace pigs were cultured under 2% O2) groups and 240 DAMs were identified. Functional enrichment assessment indicated that the hypoxia-related genes and metabolites were primarily involved in glycolysis and aldosterone synthesis and secretion. We subsequently constructed an interaction network of mRNAs and metabolites related to hypoxia, such as guanosine-3', 5'-cyclic monophosphate, Gly-Tyr, and phenylacetylglycine. These results indicated that mitogen-activated protein kinase (MAPK) signaling, aldosterone synthesis and secretion, and differences in the regulation of MCM and adenosine may play vital roles in the better adaptation of Tibetan pigs to hypoxic environments relative to Landrace pigs. This work provides a new perspective and enhances our understanding of mRNAs and metabolites that are activated in response to hypoxia in the ATII cells of Tibetan pigs.

RNA-seq analysis of the human surfactant air-liquid interface culture reveals alveolar type II cell-like transcriptome.

Understanding pulmonary diseases requires robust culture models that are reproducible, sustainable in long-term culture, physiologically relevant, and suitable for assessment of therapeutic interventions. Primary human lung cells are physiologically relevant but cannot be cultured in vitro long term and, although engineered organoids are an attractive choice, they do not phenotypically recapitulate the lung parenchyma; overall, these models do not allow for the generation of reliable disease models. Recently, we described a new cell culture platform based on H441 cells that are grown at the air-liquid interface to produce the SALI culture model, for studying and correcting the rare interstitial lung disease surfactant protein B (SPB) deficiency. Here, we report the characterization of the effects of SALI culture conditions on the transcriptional profile of the constituent H441 cells. We further analyze the transcriptomics of the model in the context of surfactant metabolism and the disease phenotype through SFTPB knockout SALI cultures. By comparing the gene expression profile of SALI cultures with that of human lung parenchyma obtained via single-cell RNA sequencing, we found that SALI cultures are remarkably similar to human alveolar type II cells, implying clinical relevance of the SALI culture platform as a non-diseased human lung alveolar cell model.

Targeting the lung epithelium after intravenous delivery by directed evolution of underexplored sites on the AAV capsid.

Advances in adeno-associated virus (AAV) engineering have provided exciting new tools for research and potential solutions for gene therapy. However, the lung has not received the same tailored engineering as other major targets of debilitating genetic disorders. To address this, here we engineered the surface-exposed residues AA452-458 of AAV9 capsid proteins at the three-fold axis of symmetry and employed a Cre-transgenic-based screening platform to identify AAV capsids targeted to the lung after intravenous delivery in mice. Using a custom image processing pipeline to quantify transgene expression across whole tissue images, we found that one engineered variant, AAV9.452sub.LUNG1, displays dramatically improved transgene expression in lung tissue after systemic delivery in mice. This improved transduction extends to alveolar epithelial type II cells, expanding the toolbox for gene therapy research for diseases specific to the lung.