Biology subjects

Gokey, J. J.

Publications and source records attributed to Gokey, J. J..

10 recordsLinked to original sources

bioRxiv2026

Integrin α3β1 is required for alveolar-capillary barrier formation through regulation of epithelial differentiation

Laminin-binding integrins regulate critical morphogenic processes, but their specific roles in lung development are unknown. In this study, we combined an in vivo model of epithelial 3 subunit deletion in the murine lung with a multiomics approach to investigate the role of the epithelial 3{beta}1 integrin during murine lung development. We demonstrate that 3{beta}1 integrin is dispensable for airway branching morphogenesis due to delayed epithelial expression. Instead, in vivo deletion of 3{beta}1 integrin impaired alveolar epithelial cell differentiation and disrupted alveolar-capillary barrier formation, functionally limiting gas exchange. A discontinuous alveolar BM contained abnormal collagen IV and laminin networks and widened the gap between epithelial and endothelial cells in the alveolar-capillary barrier niche. Mechanistically, mice deficient in epithelial 3{beta}1 integrin possessed an intracellular trafficking and secretion defect for BM components. Thus, integrin 3{beta}1 orchestrates the temporal maturation of alveolar epithelial cells required for organization of the alveolar-capillary barrier.

developmental biology↗

bioRxiv2026

IPF distal lung epithelial cells acquire a DNA methylation signature consistent with activation of basal cell transcriptional programs

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic interstitial lung disease associated with failed alveolar epithelial repair with an expansion of aberrant airway-like epithelium in the alveolar space leading to lung function decline usually resulting in death within 3-5 years of diagnosis. While single-cell and spatial transcriptomic approaches have been used to characterize disease-emergent cell populations, less is known about the regulation of transcriptional programs that drive failed alveolar epithelial cell repair in IPF. DNA methylation is a fundamental layer of gene regulation that stabilizes differentiated cell identity; however, changes in methylation in the epithelial compartment in IPF have not been studied. To identify novel epigenetic mediators of epithelial cell dysfunction, we performed high-resolution DNA methylation profiling of purified distal lung epithelial cells from 10 age-matched control and 14 IPF lungs using Oxford Nanopore Technologies (ONT) whole-genome, long-read sequencing. We identified widespread methylome remodeling in the IPF lung epithelium, with 84% of differentially methylated regions (DMRs) hypomethylated. DMRs were largely found outside of promoters, with 88% outside of 3 kb from the transcription start site (TSS), consistent with altered distal regulatory element activity. DMRs were enriched for transcription factor (TF) binding site motifs and gene associations consistent with dysregulation of polycomb repressive complex 2 (PRC2) and increases in p63 activity. DNA methylation at DMRs associated with p63 target genes, including KRT5, and genes implicated in failed epithelial repair, including MUC5B, MMP7, and S100A2 inversely correlated with gene expression. Consistent with the observed dysregulation of the IPF epithelial methylome, an experimental co-culture model of alveolar type II epithelial cell (AT2s) to basal-like epithelial cell transdifferentiation revealed widespread hypomethylation. Further, sites associated with NKX2.1 and FOXA1/2 binding, TFs involved in alveolar fate maintenance, were hypermethylated, suggesting loss of epigenetic regulation of alveolar identity. Together, these data implicate DNA methylation in the failed alveolar epithelial repair processes in IPF, potentially providing future therapeutic strategies by identifying putative regulatory elements associated with aberrant transcriptional programs.

molecular biology↗

bioRxiv2025

Loss of Regulator of telomere elongation helicase (Rtel1) impairs alveolar epithelial cell progenitor function but does not exacerbate experimental fibrosis

Inherited loss-of-function mutations in the replication-associated helicase Regulator of telomere elongation helicase (RTEL1) are among the most frequent monogenic causes of familial pulmonary fibrosis (FPF), but the specific functional role of RTEL1 in lung development, homeostasis, and injury-repair has not been determined. Using single-cell RNA-sequencing to define the cellular diversity and molecular programs following intratracheal bleomycin injury in mice, we observed that Rtel1 was most highly expressed in the lung epithelium, and its expression was reduced in PF epithelial cells compared to nonfibrotic controls. While global deletion of Rtel1 leads to embryonic lethality, constitutive deletion of Rtel1 in the lung epithelium (Rtel{Delta}epi) led to normal lung structure and architecture. Rtel1{Delta}epi mice exhibited accelerated telomere attrition and impaired AEC progenitor potential ex-vivo. Despite decreased AEC progenitor function, no differences in lung fibrosis were observed in Rtel{Delta}epi mice compared to controls following single-dose or repetitive intratracheal bleomycin. Together, these data implicate RTEL1 as a regulator of AEC progenitor function but this level of progenitor dysfunction is not alone sufficient to cause or exacerbate experimental lung fibrosis.

cell biology↗

bioRxiv2025

Sustained Yap/Taz activation promotes aberrant alveolar epithelial cell differentiation and drives persistent fibrotic remodeling

YAP/TAZ signaling is required for initiation of lung alveolar repair, yet previous studies in idiopathic pulmonary fibrosis (IPF) predicted increased YAP/TAZ signaling in alveolar epithelial cells (AECs). We investigated whether persistent YAP/TAZ AEC signaling contributes to failed epithelial repair and persistent fibrotic remodeling. In IPF lungs, we identified increased YAP+/TAZ+ AECs and increased expression of YAP/TAZ transcriptional targets compared to donor control lungs. In human lung organoids, pharmacological YAP/TAZ activation resulted in phenotype shifts of AECs into aberrant transitional states. In mice with Yap/Taz activation (YTactive) resulting from deletion of Hippo-kinases Stk3/4 in alveolar-type 2 (AT2) cells, resulted in persistent fibrotic remodeling at 28- and 56-days post-bleomycin injury. Gene promoter activity associated with transitional cell markers (Krt19, Hopx, and Runx2) was increased in YTactive AT2 cells. Immunofluorescent staining showed a loss of AT2 associated Cebpa and increased Krt19 in YTactive lineage traced AT2 cells 28 days post-injury. Inhibition of Yap/Taz using Verteporfin resulted in improved lung repair in YTactive mouse lungs, including increased Cebpa and decreased Krt19+ transitional cells. These findings demonstrate sustained Yap/Taz activation drives abnormal alveolar repair and persistent fibrotic remodeling. Blocking aberrant persistent Yap/Taz activity promotes adaptive repair and has potential as a therapeutic strategy for PF.

molecular biology↗

bioRxiv2023

Image-based spatial transcriptomics identifies molecular niche dysregulation associated with distal lung remodeling in pulmonary fibrosis

The human lung is structurally complex, with a diversity of specialized epithelial, stromal and immune cells playing specific functional roles in anatomically distinct locations, and large-scale changes in the structure and cellular makeup of this distal lung is a hallmark of pulmonary fibrosis (PF) and other progressive chronic lung diseases. Single-cell transcriptomic studies have revealed numerous disease-emergent/enriched cell types/states in PF lungs, but the spatial contexts wherein these cells contribute to disease pathogenesis has remained uncertain. Using sub-cellular resolution image-based spatial transcriptomics, we analyzed the gene expression of more than 1 million cells from 19 unique lungs. Through complementary cell-based and innovative cell-agnostic analyses, we characterized the localization of PF-emergent cell-types, established the cellular and molecular basis of classical PF histopathologic disease features, and identified a diversity of distinct molecularly-defined spatial niches in control and PF lungs. Using machine-learning and trajectory analysis methods to segment and rank airspaces on a gradient from normal to most severely remodeled, we identified a sequence of compositional and molecular changes that associate with progressive distal lung pathology, beginning with alveolar epithelial dysregulation and culminating with changes in macrophage polarization. Together, these results provide a unique, spatially-resolved characterization of the cellular and molecular programs of PF and control lungs, provide new insights into the heterogeneous pathobiology of PF, and establish analytical approaches which should be broadly applicable to other imaging-based spatial transcriptomic studies.

genomics↗

bioRxiv2023

Hypoxia-inducible factor-2 (HIF2) regulates alveolar regeneration after repetitive injury

Idiopathic Pulmonary Fibrosis (IPF) is a progressive and often fatal chronic respiratory disease thought to result from repetitive injury and failed repair of the lung alveoli, and recent studies have identified a number of disease-emergent intermediate/transitional cell states in the IPF lung supporting this concept. In this study, we found that persistent activation of hypoxia-inducible factor (HIF)-signaling in airway-derived, repair-associated cell types/states is a hallmark of dysfunctional epithelial repair in the IPF lung epithelium and experimental models of recurrent lung epithelial injury. Disrupting Hif-signaling attenuated experimental lung fibrosis, reduced mucous-secretory cell polarization, and promoted functional alveolar regeneration following repetitive injury. Mouse and human organoid studies demonstrated that small-molecule-based HIF2 inhibition promoted alveolar epithelial cell proliferation and maturation while preventing the emergence of maladaptive intermediate/transitional states analogous to those in IPF. Together, these studies indicate that targeted HIF2-inhibition represents a novel and effective therapeutic strategy to promote functional lung regeneration, and could be readily translated into human studies of IPF and other chronic interstitial lung diseases with disease modifying effect. One sentence summaryInhibiting hypoxia-inducible-factor 2 (HIF2) promotes functional lung alveolar epithelial repair following recurrent injury.

cell biology↗

bioRxiv2023

Epithelial Yap/Taz are required for functional alveolar regeneration following acute lung injury

A hallmark of idiopathic pulmonary fibrosis (IPF) and other interstitial lung diseases is dysregulated repair of the alveolar epithelium. The Hippo pathway effector transcription factors YAP and TAZ have been implicated as essential for type 1 and type 2 alveolar epithelial cell (AT1 and AT2) differentiation in the developing lung, yet aberrant activation of YAP/TAZ is a prominent feature of the dysregulated alveolar epithelium in IPF. In these studies, we sought to define the functional role of YAP/TAZ activity during alveolar regeneration. We demonstrate that Yap and Taz are normally activated in AT2 cells shortly after injury, and deletion of Yap/Taz in AT2 cells led to pathologic alveolar remodeling, failure of AT2 to AT1 cell differentiation, increased collagen deposition, exaggerated neutrophilic inflammation, and increased mortality following injury induced by a single dose of bleomycin. Loss of Yap/Taz activity prior to a LPS injury prevented AT1 cell regeneration, led to intra-alveolar collagen deposition, and resulted in persistent innate inflammation. Together these findings establish that AT2 cell Yap/Taz activity is essential for functional alveolar epithelial repair and prevention of fibrotic remodeling.

cell biology↗

bioRxiv2023

Repetitive Sulfur Dioxide Exposure in Mice Models Post-Deployment Respiratory Syndrome

Soldiers deployed to Iraq and Afghanistan have a higher prevalence of respiratory symptoms than non-deployed military personnel and some have been shown to have a constellation of findings on lung biopsy termed post-deployment respiratory syndrome (PDRS). Since many of the deployers in this cohort reported exposure to sulfur dioxide (SO2), we developed a model of repetitive exposure to SO2 in mice that phenocopies many aspects of PDRS, including adaptive immune activation, airway wall remodeling, and pulmonary vascular disease (PVD). Although abnormalities in small airways were not sufficient to alter lung mechanics, PVD was associated with the development of pulmonary hypertension and reduced exercise tolerance in SO2 exposed mice. Further, we used pharmacologic and genetic approaches to demonstrate a critical role for oxidative stress and isolevuglandins in mediating PVD in this model. In summary, our results indicate that repetitive SO2 exposure recapitulates many aspects of PDRS and that oxidative stress may mediate PVD in this model, which may be helpful for future mechanistic studies examining the relationship between inhaled irritants, PVD, and PDRS.

pathology↗

bioRxiv2023

Myeloid Cell-Derived IL1β Contributes to Pulmonary Vascular Remodeling in Heart Failure with Preserved Ejection Fraction

BackgroundPulmonary hypertension (PH) in heart failure with preserved ejection fraction (HFpEF) is a common and highly morbid syndrome, but mechanisms driving PH-HFpEF are not well understood. We sought to determine whether a well-accepted murine model of HFpEF also displays features of PH in HFpEF, and we sought to identify pathways that might drive early remodeling of the pulmonary vasculature in HFpEF. MethodsEight week old male and female C57/BL6J mice were given either L-NAME and high fat diet (HFD) or control water/diet for 2,5, and 12 weeks. Bulk RNA sequencing and single cell RNA sequencing was performed to identify early and cell-specific pathways that might regulate pulmonary vascular remodeling in PH-HFpEF. Finally, clodronate liposome and IL1{beta} antibody treatments were utilized to deplete macrophages or IL1{beta}, respectively, to assess their impact on pulmonary vascular remodeling in HFpEF. ResultsMice given L-NAME/HFD developed PH, small vessel muscularization, and right heart dysfunction after 2 weeks of treatment. Inflammation-related gene ontologies were over-represented in bulk RNA sequencing analysis of whole lungs, with an increase in CD68+ cells in both murine and human PH-HFpEF lungs. Cytokine profiling of mouse lung and plasma showed an increase in IL1{beta}, which was confirmed in plasma from patients with HFpEF. Single cell sequencing of mouse lungs also showed an increase in M1-like, pro-inflammatory populations of Ccr2+ monocytes and macrophages, and transcript expression of IL1{beta} was primarily restricted to myeloid-type cells. Finally, clodronate liposome treatment prevented the development of PH in L-NAME/HFD treated mice, and IL1{beta} antibody treatment also attenuated PH in L-NAME/HFD treated mice. ConclusionsOur study demonstrated that a well-accepted model of HFpEF recapitulates features of pulmonary vascular remodeling commonly seen in patients with HFpEF, and we identified myeloid cell derived IL1{beta} as an important contributor to PH in HFpEF.

cell biology↗

bioRxiv2021

Single-cell transcriptomic assessment of cellular phenotype stability in human precision-cut lung slices

Precision-cut lung slices (PCLS) are increasingly utilized for ex vivo disease modeling, but a high-resolution characterization of cellular phenotype stability in PCLS has not been reported. Comparing the single-cell transcriptomic profile of human PCLS after five days of culture to freshly isolated human lung tissue, we found striking changes in endothelial cell and alveolar epithelial cell programs, reflecting both injury and pathways activated in static culture, while immune cell frequencies and programs remained largely intact and similar to the native lung. These cellular dynamics should be considered when utilizing PCLS as a model of the human lung.

cell biology↗