Biology subjects

Lingampally, A.

Publications and source records attributed to Lingampally, A..

6 recordsLinked to original sources

bioRxiv2024

Exploring the Antifibrotic Potential of the heparan sulfate mimetic OTR4120: Insights from Preclinical Models

RationaleIdiopathic pulmonary fibrosis (IPF) is a debilitating lung disease characterized by excessive deposition of extracellular matrix (ECM), resulting in lung function impairment. Heparan sulfate mimetics (HSm) have been suggested to have potential antifibrotic effects by regulating ECM. This study aims to investigate the impact of a specific HSm named OTR4120 on fibrotic processes in ex vivo, in vitro and in vivo models. MethodsHuman Precision Cut Lung Slices (hPCLS) treated with a fibrotic cocktail alone or with OTR4120 were evaluated using second-harmonic imaging microscopy (SHIM) to assess collagen deposition. Human embryonic fibroblast WI-38 cell line and primary fibroblasts obtained from human donors were differentiated into myofibroblasts (MYF) using TGF-{beta}1 and treated with OTR4120 or a control vehicle. Gene expression analysis for MYF markers was performed using quantitative PCR (qPCR). Protein expression of MYF markers was evaluated using immunofluorescence techniques. Bulk-RNA sequencing analysis on WI-38 cells cultured under different experimental conditions was conducted. Finally, the therapeutic effects of OTR4120 on a bleomycin-induced fibrosis mouse model were investigated. ResultsSHIM analysis on OTR4120-treated hPCLS showed a decrease in collagen deposition. OTR4120 treatment of primary fibroblasts and WI-38 cells exposed to TGF-{beta}1 significantly reduced the expression of MYF markers. Bulk-RNA sequencing analysis on OTR4120-treated WI-38 cells showed significant impacts on fibrosis-related processes. Therapeutic application of OTR4120 in vivo to bleomycin-induced fibrosis mice resulted in enhanced fibrosis resolution. ConclusionOTR4120 has potential therapeutic benefits as an antifibrotic agent in the context of lung fibrosis. Further investigations are necessary to understand the precise mechanism through which OTR4120 exerts its antifibrotic effects.

cell biology↗

bioRxiv2024

Lineage-tracing of Acta2+ cells in aged mice during lung fibrosis formation and resolution supports the lipofibroblasts to myofibroblast reversible switch

Idiopathic pulmonary fibrosis (IPF) develops mostly in old man and is characterized by the irreversible accumulation of excessive extracellular matrix components by activated myofibroblasts (aMYFs) leading to lung failure. Following bleomycin administration in young mice, fibrosis formation associated with efficient resolution takes place, the later limiting the clinical relevance of this model for IPF. In young mice, we previously reported that aMYFs captured during fibrosis formation differentiate towards a lipofibroblast (LIF)-like phenotype during resolution. In this study, we used aged mice in combination with bleomycin administration to trigger enhanced fibrosis formation and delayed resolution, as a more relevant model for IPF and examined the heterogeneity and fate of aMYFs at different time points. Alveolosphere assay were carried out to compare the alveolar resident mesenchymal niche activity for AT2 stem cells in young versus old mice. Lineage tracing of the Acta2+ aMYFs in old mice exposed to bleomycin followed by scRNAseq of the lineage-traced cells isolated during fibrosis formation and resolution was performed to delineate the heterogeneity of aMYFs during fibrosis formation and their fate during resolution. Data mining of human mesenchymal cells from IPF and control datasets were also performed to decipher the heterogeneity of aMYFs and investigate differentiation trajectories during fibrosis formation. Our results show that alveolar resident mesenchymal cells from old mice display decreased supporting activity for AT2 stem cells. We report that aMYFs consist of four subclusters displaying unique pro-alveologenic versus pro-fibrotic profiles. Alveolar fibroblasts displaying a high LIF-like signature largely constitute both the origin and fate of aMYFs during fibrosis formation and resolution, respectively. The heterogeneity of aMYFs is conserved in humans and a significant proportion of human aMYFs displays a high LIF signature. In conclusion, our data indicate that the cellular and molecular bases of aMYFs formation and differentiation towards the LIF phenotype are conserved between young and old mice. Importantly, our work identifies a subcluster of aMYFs that is potentially relevant for future management of IPF.

pathology↗

bioRxiv2023

Lung injury shifts pulmonary capillary endothelial cells towards regeneration-associated Lrg1+ subpopulations with delayed dynamics in aged mice

Aging increases the risk of developing fibrotic diseases by hampering tissue regeneration after injury. Using longitudinal single-cell RNA-seq and spatial transcriptomics, here we compare the transcriptome of bleomycin-induced fibrotic lungs of young and aged mice, at 3 time points corresponding to the peak of fibrosis, regeneration and resolution. We find that lung injury shifts the transcriptomic profiles of three pulmonary capillary endothelial cells (PCEC) subpopulations. The associated signatures are linked to pro-angiogenic signaling with strong Lrg1 expression and do not progress similarly throughout the resolution process between young and old animals. Moreover, part of this set of resolution-associated markers is also detected in PCEC from samples of patients with idiopathic pulmonary fibrosis. Finally, we find that aging also alters the transcriptome of PCEC which display typical pro-fibrotic and pro-inflammatory features. We propose that age-associated alterations in specific PCEC subpopulations may interfere with the process of lung progenitor differentiation, thus contributing to the persistent fibrotic process typical of human pathology.

pathology↗

bioRxiv2022

Fgfr2b signaling is essential for the maintenance of the alveolar epithelial type 2 lineage during lung homeostasis in mice

Fibroblast growth factor receptor 2b (Fgfr2b) signaling is essential throughout lung development to form the alveolar epithelial lineage. However, its role in alveolar epithelial type 2 cells (AT2s) homeostasis was recently considered dispensable. SftpcCreERT2; tdTomatoflox/flox mice were used to delete Fgfr2b expression in cells belonging to the AT2 lineage, which contains mature AT2s and a novel SftpcLow lineage-traced population called "injury activated alveolar progenitors" or IAAPs. Upon continuous tamoxifen exposure for either one or two weeks to delete Fgfr2b, a shrinking of the AT2s is observed. Mature AT2s exit the cell cycle, undergo apoptosis and fail to form alveolospheres in vitro. However, the lung morphometry appears normal, suggesting the involvement of compensatory mechanisms. In mutant lungs, IAAPs which escaped Fgfr2b deletion expand, display enhanced alveolosphere formation in vitro and increase drastically their AT2 signature suggesting differentiation towards mature AT2s. Interestingly, a significant increase in AT2s and decrease in IAPPs occurs after a one-week tamoxifen exposure followed by an eight-week chase period. While mature AT2s partially recover their alveolosphere formation capabilities, the IAAPs no longer display this property. Single-cell RNA seq analysis confirms that AT2s and IAAPs represent stable and distinct cell populations and recapitulate some of their characteristics observed in vivo. Our results underscore the essential role played by Fgfr2b signaling in the maintenance of the AT2 lineage in the adult lung and suggest that the IAAPs could represent a new population of AT2 progenitors.

developmental biology↗

bioRxiv2022

Characterization of alveolar epithelial lineage heterogeneity during the late pseudoglandular stage of mouse lung development

The specification, characterization, and fate of alveolar type 1 and type 2 (AT1 and 2) progenitors during embryonic lung development remains mostly elusive. In this paper, we build upon our previously published work on the regulation of airway epithelial progenitors by fibroblast growth factor receptor 2b (Fgfr2b) signalling during early (E12.5) and mid (E14.5) pseudoglandular lung development. Here, we looked at the regulation by Fgfr2b signalling on alveolar progenitors during late pseudoglandular/early canalicular (E14.5-E16.5) development. Using a dominant negative mouse model to conditionally inhibit Fgfr2b ligands at E16.5, we used gene array analyses to characterize a set of potential direct targets of Fgfr2b signalling. By mining published single-cell RNA sequence (scRNAseq) datasets, we showed that these Fgfr2b signature genes narrow on a discreet subset of AT2 cells at E17.5 and in adult lungs. Furthermore, we demonstrated that Fgfr2b signalling is lost in AT2 cells in their transition to AT1 cells during repair after injury. We also used CreERT2-based mouse models to conditionally knock-out the Fgfr2b gene in AT2 and in AT1 progenitors, as well as lineage label these cells. We found, using immunofluorescence, that in wildtype controls AT1 progenitors labeled at E14.5-E15.5 contribute a significant proportion to AT2 cells at E18.5; while AT2 progenitors labeled at the same time contribute significantly to the AT1 lineage. We show, using immunofluorescence and FACS-based analysis, that knocking out of Fgfr2b at E14.5-E15.5 in AT2 progenitors leads to an increase in lineage-labeled AT1 cells at E18.5; while the reverse is true in AT1 progenitors. Furthermore, we demonstrate that increased Fgfr signalling in AT2 progenitors reduces their contribution to the AT1 pool. Taken together, our results suggest that a significant proportion of AT2 and AT1 progenitors are cross-lineage committed during late pseudoglandular development, and that lineage commitment is regulated in part by Fgfr2b signalling. We have characterized a set of direct Fgfr2b targets at E16.5 which are likely involved in alveolar lineage formation. These signature genes concentrate on a subpopulation of AT2 cells later in development, and are downregulated in AT2 cells transitioning to the AT1 lineage during repair after injury in adults. Our findings highlight the extensive heterogeneity of alveolar cells by elucidating the role of Fgfr2b signalling in these cells during early alveolar lineage formation, as well as during repair after injury.

developmental biology↗

bioRxiv2020

Identification a novel subset of alveolar type 2 cells expanding following pneumonectomy and enriched in PD-L1

Alveolar type 2 (AT2) cells are heterogeneous cells; where specialized AT2 subpopulations within this lineage exhibit stem cell properties. However, the existence of quiescent, immature cells within the AT2 lineage, which get activated during lung regeneration, is unknown. SftpcCreERT2/+; tdTomatoflox/flox mice were used for the labelling of AT2 cells and labeled subpopulations were analyzed by flow cytometry, qPCR, ATAC-seq, gene arrays, pneumonectomy, and culture of precision-cut lung slides. Human lungs from donor and IPF were also analyzed. In mice, we detected two distinct AT2 subpopulations with low tdTomato level (TomLow) and high tdTomato level (TomHigh). TomLow express lower level of AT2 differentiation markers, Fgfr2b and Etv5, while TomHigh, as bona fide mature AT2 cells, show higher level of Sftpc, Sftpb, Sftpa1, Fgfr2b, and Etv5. ATAC-seq analysis indicates that TomLow and TomHigh constitute two distinct cell populations with specific silencing of Sftpc, Rosa26 and cell cycle gene loci in TomLow. Upon pneumonectomy, TomLow but not TomHigh cells proliferate and upregulate the expression of Fgfr2b, Etv5, Sftpc, Ccnd1 and Ccnd2 compared to sham. TomLow cells overexpress PD-L1, an immune inhibitory membrane receptor ligand, which is used by flow cytometry to differentially isolate these two sub-populations. In the human lung, PD-L1 and HTII-280 antibodies are used by flow cytometry to differentially sort mature AT2 (HTII-280-high, PD-L1-low) as well as an additional subpopulation of epithelial cells characterized by HTII-280-Low and PD-L1-high. We have identified a novel population of AT2 quiescent immature progenitor cells in mouse that proliferate upon pneumonectomy and provided evidence for the existence of such cells in human. Significance of the workThe characterization and mechanism of the proliferation of a novel and relevant pool of AT2 progenitor cells for the repair/regeneration process after injury are critical to improving respiratory function in patients with lung disease.

cell biology↗