Fig 2: APOE-KO canines develop spontaneous pulmonary fibrotic-like pathology. (A) Schematic of the dog APOE genomic structure (GenBank accession NC_051805) and CRISPR/Cas9 sgRNA targeting sites in exon 3. (B) Genome sequencing of sgRNA-targeted regions in the wild-type (APOE+/+) and mutant (APOE-2236/-34+17 bp) dog genomes. //represents concealed nucleic acid sequence; the dash and △ represent deletions; ∧ and + represent insertions. (C) Breeding scheme for generating homozygous APOE-KO dogs through natural mating. (D) Histological evaluation of lung tissues from WT and homozygous AOPE-KO dogs by H&E and Masson’s trichrome staining. Scale bars: 200 μm. (E) Quantitative analysis of the collagen deposition area from panel D. n = 3 (three biological replicates). (F) Immunohistochemical staining of the fibrotic markers α-SMA, collagen 1, and fibronectin in dog lung tissues. Scale bars: 200 μm. (G) Quantification of the area with positive staining for fibrotic markers in panel F. n = 3. (H) Western blot analysis of α-SMA, collagen 1, and fibronectin expression in dog lung tissues. (I) Densitometric quantification of the western blot results from panel H. n = 3. (J) qPCR measurement of ACTA2, COL1A1, and FN1 mRNA levels in dog lung tissues. n = 3. Abbreviations: sgRNA, single-guide RNA; WT, wild-type; HOM, homozygous; H&E, hematoxylin‒eosin; α-SMA, alpha-smooth muscle actin; KO, knockout. The data are presented as mean ± SD. All statistical analyses for this figure were performed using unpaired two-tailed Student’s t test. Statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 3: apoE suppresses TGF-β/Smad signaling through dual receptor engagement of LRP1 and PLAU. (A) Schematic of RNA sequencing performed in MRC-5 cells treated with the TGF-β and apoE protein. (B) Principal component analysis (PCA) plot demonstrating distinct clustering among the three experimental groups (Control, TGF-β, and TGF-β + apoE). (C) KEGG pathway enrichment analysis showing upregulated pathways in the TGF-β vs. control groups (left) and downregulated pathways in the TGF-β + apoE vs. TGF-β groups (right). (D) Heatmap depicting expression changes in TGF-β signaling pathway genes in the control vs. TGF-β (left) and TGF-β vs. TGF-β + apoE (right) groups. (E) Representative western blot (WB) images of collagen 1, PAI1, SMAD2/3 and p-SMAD2/3 levels in TGF-β-stimulated MRC-5 and primary human lung fibroblasts (HLF) cells with or without apoE treatment. (F) Quantification of the WB band intensity shown in panel E. (G) qPCR analysis of COL1A1 and PAI1 mRNA levels in TGF-β-stimulated MRC-5 and HLF cells with or without apoE treatment. (H) Representative immunofluorescence images of p-SMAD2/3 and SMAD4 in MRC-5 cells following TGF-β stimulation with or without apoE treatment. Scale bars: 20 μm. (I) Quantification of the mean fluorescence intensity of p-SMAD2/3 and SMAD4 within the cell nucleus, as shown in panel H. (J) Representative WB images of collagen 1, PAI1, SMAD2/3 and p-SMAD2/3 levels in MRC-5 and HLF cells following the knockdown of LRP1, PLAU, or LRP1 + PLAU with TGF-β and apoE protein treatment. (K) Quantification of the WB band intensity shown in panel J. (L) qPCR assessment of COL1A1 and PAI1 mRNA levels in MRC-5 and HLF cells following the knockdown of LRP1, PLAU, or LRP1 + PLAU with TGF-β and apoE protein treatment. Abbreviations: SCR, Scramble; Si, SiRNA. The mean ± SD is presented for three independent experiments. All statistical analyses for this figure were performed using one-way ANOVA and Tukey’s method. Statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 4: Single-cell transcriptomics reveals fibroblast-specific LRP1 as an important checkpoint for apoE anti-fibrosis. (A) Uniform manifold approximation and projection (UMAP) visualization of 21 transcriptionally distinct cell populations identified in integrated single-cell RNA sequencing datasets (GSE122960, GSE128033, GSE132771, and GSE128169) derived from human lung tissues of control subjects and IPF patients. (B) Heatmap showing the mean expression patterns of key marker genes across the 21 annotated human lung cell types. (C) Stacked bar plot depicting the proportions of fibroblasts and myofibroblasts in control and IPF human lung tissues. (D) Dot plot illustrating the expression levels of APOE receptors (LDLR, LRP1, LRP8, VLDLR) across the 21 human lung cell types. (E) Violin plot showing LDLR expression across 21 human lung cell types in control and IPF tissues. (F) Violin plot showing LRP1 expression across 21 human lung cell types in control and IPF tissues. (G) Representative immunofluorescence images of fibrotic markers (α-SMA, collagen 1, and fibronectin) in MRC-5 cells following TGF-β stimulation with or without apoE treatment. Scale bars: 50 μm. (H) Quantification of the mean fluorescence intensity shown in panel G. (I) Western blot (WB) analysis of fibrotic marker protein levels (α-SMA, collagen 1, and fibronectin) in TGF-β-stimulated MRC-5 cells with or without apoE treatment. (J) Quantification of the WB band intensity shown in panel I. (K) qPCR analysis of ACTA2, COL1A1, and FN1 mRNA levels in TGF-β-stimulated MRC-5 cells with or without apoE treatment. The mean ± SD is presented for three independent experiments. Statistical analyses were performed using one-way ANOVA and Tukey’s method (H-K). Statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 5: RGX-104 attenuates lung fibrosis in a bleomycin-induced mouse model and fibrotic cocktail-stimulated human precision-cut lung slices. (A) Schematic of the experimental design for RGX-104 treatment in the BLM-induced murine pulmonary fibrosis model. (B) Serum apoE levels in BLM-induced mice treated with RGX-104 or corn oil. n = 4 (four biological replicates). (C) Representative immunohistochemistry images of apoE expression in lung tissues from BLM-challenged mice treated with RGX-104 or corn oil. Scale bars: 200 μm. (D) Quantification of the apoE-positive area shown in panel C. n = 4. (E) Western blot (WB) analysis of apoE protein levels in lung tissues from BLM-induced mice treated with RGX-104 or corn oil. (F) Quantification of the WB band intensity shown in panel E. n = 3. (G) qPCR analysis of Apoe mRNA levels in lung tissues from BLM-challenged mice treated with RGX-104 or corn oil. n = 4. (H) Representative Masson’s trichrome-stained lung sections from BLM-challenged mice treated with RGX-104 or corn oil. Scale bars: 1 mm (overview); 100 μm (magnified). (I) Semiquantitative Ashcroft score (left) and collagen area quantification (right) were used to assess fibrosis severity, as shown in panel H. n = 4. (J) Quantitative analysis of hydroxyproline content in BLM-induced mice treated with RGX-104 or corn oil. n = 4. (K) WB analysis of fibrotic markers (α-SMA, collagen 1, and fibronectin) in lung tissues from BLM-challenged mice treated with RGX-104 or corn oil. (L) Quantification of the WB band intensity shown in panel K. n = 3. (M) qPCR analysis of Acta2, Col1a1, and Fn1 mRNA levels in lung tissues from BLM-induced mice treated with RGX-104 or corn oil. n = 4. (N) Representative immunofluorescence images of coimmunostaining for α-SMA (red) and p-Smad3 (green) in BLM-challenged mice treated with RGX-104 or corn oil. α-SMA served as a marker for fibroblasts. Scale bars: 50 μm. (O) Quantification of the p-Smad3 fluorescence intensity of the fibroblasts in panel N. n = 4. (P) Schematic of RGX-104 treatment of cocktail-stimulated human precision-cut lung slices (PCLS). (Q) Representative immunohistochemistry images of apoE expression in cocktail-stimulated human PCLS treated with or without RGX-104. Scale bars: 200 μm. (R) Quantification of the apoE-positive area shown in panel Q. n = 4. (S) Representative immunofluorescence staining of fibrotic markers (α-SMA, collagen 1, and fibronectin) in fibrotic cocktail-stimulated human PCLS treated with or without RGX-104. Scale bars: 50 μm. (T) Quantification of the mean fluorescence intensity shown in panel S. n = 4. (U) WB analysis of fibrotic markers (α-SMA, collagen 1, and fibronectin) in fibrotic cocktail-stimulated human PCLS treated with or without RGX-104. (V) Quantification of the WB band intensity shown in panel U. n = 3. (W) Representative immunofluorescence images of coimmunostaining for α-SMA (red) and p-SMAD3 (green) in cocktail-stimulated human PCLS treated with or without RGX-104. Scale bars: 50 μm. (X) Quantification of the p-SMAD3 fluorescence intensity of the fibroblasts in panel W. n = 4. Abbreviations: BLM, bleomycin; α-SMA, α-smooth muscle actin. The data are presented as mean ± SD. All statistical analyses for this figure were performed using one-way ANOVA and Tukey’s method. Statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant.