Inhaled Angiopoietin‐Like 4 Antisense Oligonucleotide Therapy for Lung Injury and Fibrosis

Figures

• 

  Figure 1. In vitro validation of ANGPTL4‐ASO knockdown efficacy in human and mouse cell lines. A,B) Dose‐dependent suppression of ANGPTL4 expression in human HaCaT keratinocytes A) and mouse AML12 hepatocytes B) under normoxic and hypoxic conditions. Cells were treated with increasing concentrations (1–100 000 nM) of two ANGPTL4‐ASOs. Gene expression was normalized to TBP (human) or 18S (mouse) and presented as fold change relative to normoxia. C,D) Dose‐response curves and IC50 determinations for human ANGPTL4‐ASOs in HaCaT cells C) and mouse Angptl4‐ASOs in AML12 cells D) under hypoxic conditions. IC50 values: human ANGPTL4‐ASO1 (76.05 nM), ANGPTL4‐ASO2 (854 nM); mouse Angptl4‐ASO1 (8.47 nM), Angptl4‐ASO2 (54.97 nM). Error bars represent Standard Deviation (SD). IC50 values were calculated using non‐linear regression analysis.

• 

  Figure 2. Angptl4‐ASO reduces lung inflammation and tissue damage in PAO1 infection. A) Experimental timeline of the P. aeruginosa (PAO1) lung infection mouse model. D0: PAO1 and/or Angptl4‐ASO inhalation; D2: sacrifice. B) Immunofluorescence staining of ANGPTL4 (red) in mouse lung sections. Nuclei were counterstained with DAPI (blue). Ctrl and ASO‐only groups show minimal ANGPTL4 expression in bronchial epithelium and walls. PAO1 group exhibits significant ANGPTL4 expression in bronchial epithelium, surrounding areas, lung interstitium, and alveoli. PAO1‐ASO group shows significant downregulation compared to PAO1 group (scale bar: 20 µm). C) Hematoxylin and eosin (H&E) staining of mouse lung sections. Control and ASO‐only treated lungs (a‐d) show no inflammatory exudation or cellular infiltration. PAO1 treated lungs (e, f) display severely damaged lung alveolar structure and extensive cellular infiltration (yellow arrows indicates neutrophils; white arrows indicates macrophages). PAO1‐ASO treated lungs (g, h) exhibit notable recovery from tissue injury, preserved alveolar structure, and reduced immune cell infiltration (Black scale bar: 100 µm; Red scale bar: 10 µm). D) Quantification of…

• 

  Figure 3. Angptl4‐ASO treatment reduces lung inflammation in PR8 influenza infection. A) Experimental timeline of the Influenza virus PR8 lung infection mouse model. D0: PR8 and/or Angptl4‐ASO inhalation; D7: sacrifice. B) Immunofluorescence staining of ANGPTL4 (red) in mouse lung sections. Nuclei were counterstained with DAPI (blue). Ctrl and ASO‐only groups show minimal ANGPTL4 expression in bronchial epithelium and walls. PR8 group exhibits significant ANGPTL4 expression in bronchial epithelium, surrounding areas, lung interstitium, and alveoli. PR8‐ASO group shows significant downregulation compared to PR8 group, with only minimal expression in bronchial epithelium and walls (White arrows indicate sites of ANGPTL4 expression; scale bar: 20 µm). C) Representative H&E staining of mouse lung sections. Ctrl and ASO‐only treated lungs (a‐d) show no inflammatory exudation or cellular infiltration. PR8 treated lungs (e, f) display severely damaged lung alveolar structure and extensive cellular infiltration. PR8‐ASO treated lungs (g, h) exhibit notable relief of inflammatory response, preserved alveolar structure, and reduced cellular infiltration (Black scale bar: 100 µm; Red scale bar: 10 µm).

• 

  Figure 4. Transcriptome analysis reveals molecular activities of Angptl4‐ASO on day 2 post‐PAO1 infection. A) Principal component analysis (PCA) plot showing the clustering of lung transcriptomes across treatment groups (n = 3 per group). B) Heatmap illustrating DEGs in between treatment groups. Each row represents a gene, and each column a sample. Red indicates higher expression levels and blue indicates lower expression levels. C) Heatmap of the differential gene expression of cytokines and chemokines between Angptl4‐ASO treated and untreated mice infected with PAO1. The genes are grouped based on their molecular families. Purple indicates higher expression while green indicates lower expression in PAO1‐ASO relative to PAO1 mice. D) Enrichment plots of gene sets related to fibroblast growth factor (FGF) production and fibroblast activation. E) Heatmap of the differential expression of genes related to fibroblast activation and gap junction between Angptl4‐ASO treated and untreated mice infected with PAO1. Purple indicates higher expression while green indicates lower expression in PAO1‐ASO relative to PAO1 mice.

• 

  Figure 5. Angptl4‐ASO restores glycoprotein expression in infected lung tissues. Mass spectrometry imaging of lung sections showing nuclei (blue), α‐2,6 sialic acid (green), N‐glycan (red), and α‐1,6 fucosyltransferase (yellow) in Ctrl and Angptl4‐ASO‐only groups (top two panels). Robust expression of these glycoproteins is observed under both conditions, indicating intact pulmonary epithelial barriers. Reduced glycoprotein expression in day 2 post‐PAO1 infection A) or day 7 post‐PR8 infection B) (3rd panels), reflecting damage to the epithelial barrier. In contrast, the bottom panels demonstrate that Angptl4‐ASO treatment restores these glycoproteins to levels comparable to those in healthy tissue, suggesting that Angptl4‐ASO strengthens lung barrier function through glycoprotein regulation (scale bar: 50 µm).

• 

  Figure 6. ANGPTL4 expression in human pulmonary fibrosis and treatment response in a mouse model. A) Representative images comparing normal and fibrotic human lung tissue using H&E staining (top), Masson's trichrome staining (middle), and immunofluorescence for ANGPTL4 (bottom). Normal lung tissue displays typical alveolar architecture, while fibrotic tissue shows structural disruption and elevated ANGPTL4 expression (scale bar: 500 µm). B) Experimental timeline and dosing regimen for the pulmonary fibrosis model. Schematic illustration of the experimental design for the BLM‐induced pulmonary fibrosis model. Mice received three intratracheal doses of BLM to induce lung injury and fibrosis at Days 0, 7, and 14, followed by inhaled administration of Angptl4‐ASO or PBS at days 21 and 28. Lung tissues and BALF were collected at days 21 and 35 for histological, biochemical, and molecular analyses. C) Relative Angptl4 mRNA levels in mouse lung tissue across groups at day 35 post‐BLM treatment. Both ASO monotherapy and combination therapy (BLM‐ASO) significantly reduced expression compared to their respective controls. Data are shown as mean ± SD (n = 5 per group). Statistical comparisons were…