Journal: bioRxiv

Article Title: Liver Lymphatic Dysfunction as a Driver of Fibrosis and Cirrhosis Progression

doi: 10.1101/2025.01.11.632552

Figure Lengend Snippet: (A) Representative immunofluorescence (IF) images of proliferating cell nuclear antigen (PCNA; green), LYVE1 (red) and DAPI in sham, 1-week, 2-week, 4-week BDL mouse livers. PCNA-positive nuclei with DAPI in LYVE 1 positive LVs (arrows) are proliferating lymphatic endothelial cells (LyECs). (B) Quantification of PCNA-positive LyECs in livers from sham(n=5), 1-week(n=5), 2-week(n=5), and 4-week(n=7) BDL mice. *p< 0.05, **p< 0.01, and ***p< 0.001. Scale bars: 100 μm. PV, portal vein.

Article Snippet: Human liver LyECs suspension with basal medium (Cat#cAP-03, Angio-Proteomie) was prepared at a concentration of 1.5×10 5 cells/ml.

Techniques: Immunofluorescence

Journal: bioRxiv

Article Title: Liver Lymphatic Dysfunction as a Driver of Fibrosis and Cirrhosis Progression

doi: 10.1101/2025.01.11.632552

Figure Lengend Snippet: (A) Schematic of liver LyECs isolation and its RNA sequencing in mice. RNA sequencing was performed in primary mouse liver LyECs isolated from IL7-promoter-driven GFP knock-in mice 2 weeks(n=3) and 4 weeks(n=3) after BDL or sham surgery (n=3). (B) Heatmap (left) and Venn diagram (right) showing genes differentially expressed in livers from BDL2w and BDL4w compared to Sham mice (fold change > 1.5; P ≤ 0.01). (C) Ingenuity Pathway Analysis (IPA) showing canonical signaling pathways altered in mouse liver LyECs from BDL2w(n=3) and BDL4w(n=3) mice. (D) Top potential upstream regulators in liver LyECs from BDL4w mice. (E) Heatmaps displaying TGF-β response genes differentially expressed in sham (n=3), BDL2w (n=3) and BDL4w (n=3). (F) The list of the Gene Set Enrichment Analysis(GSEA) signaling pathways altered in human liver LyECs (n=9) in cirrhotic patients from different etiologies compared to normal human subject (n=6). GSE136103. (G) The IPA biological functions altered in mouse liver LyECs from BDL2w(n=3) and BDL4w(n=3) mice. The vertical line indicates a Benjamini-Hochberg correction at −log, equating to P = 0.05.

Article Snippet: Human liver LyECs suspension with basal medium (Cat#cAP-03, Angio-Proteomie) was prepared at a concentration of 1.5×10 5 cells/ml.

Techniques: Isolation, RNA Sequencing Assay, Knock-In

Journal: bioRxiv

Article Title: Liver Lymphatic Dysfunction as a Driver of Fibrosis and Cirrhosis Progression

doi: 10.1101/2025.01.11.632552

Figure Lengend Snippet: (A) TGF-β1 expression in livers from sham (n=6), 1-week(n=6), 2-week(n=5), and 4-week(n=7) BDL mice. (B) Representative immunofluorescence (IF) images of phospho-SMAD2 (green), LYVE1 (a LV marker; red) and DAPI in sham and 4-week BDL mouse livers. The arrows indicate Phospho-SMAD2-positive nuclei with DAPI in LYVE 1 positive lymphatic endothelial cells (LyECs). (right graph) Phospho-SMAD2-positive LVs number in livers from sham(n=5), 1-week(n=5), 2-week(n=5), and 4-week(n=7) BDL mice. (C) Representative IF images of phospho-SMAD2 (green), PDPN (red) with DAPI in healthy control livers(n=8) and PSC patients further classified into compensated cirrhosis (CC, n=7) and decompensated cirrhosis (DC, n=6). Quantification of phospho-SMAD2-positive LVs numbers in livers from healthy control (n=8) and PSC patients (CC vs. DC)(right panel). (D) A correlation between phospho-SMAD2-positive LV number and total LV number in PSC livers (n=13). (E) Representative images of tube formation assay to assess lymphangiogenesis and their quantification (F). Human primary liver LyECs incubated with or without VEGF-C (100ng/ml), hTGF-β1 (1ng/ml) and TGFβ inhibitor (LY364947) for 4 hours. Five images were taken per well and analyzed by the angiogenesis analyzer macro of ImageJ software. Representative data from three independent experiments are shown. (G) BrdU cell proliferation assay in human primary liver LyECs incubated with or without VEGF-C (100 ng/mL), hTGF-β1 (1ng/ml) and TGF-β inhibitor (LY364947) for 4 hours. Representative data from three independent experiments are shown. (H) Schematic of TGF-β inhibitor injection in mice. DMSO (control) or TGF-β inhibitor (LY364947, 0.02mg/mouse) was injected subcutaneously 2 weeks after BDL surgery followed by four more injections with a three-day interval between each administration until 4 weeks. (I) Representative immunofluorescence (IF) images and quantification (J, K) of LVs in livers from 4-week BDL mice treated with control-DMSO (n=8) or TGF-β inhibitor (n=8). LYVE-1 (a LV marker; red) and DAPI (blue). *P < 0.05, **P < 0.01, and ***P < 0.001. Scale bars: 100 μm.

Article Snippet: Human liver LyECs suspension with basal medium (Cat#cAP-03, Angio-Proteomie) was prepared at a concentration of 1.5×10 5 cells/ml.

Techniques: Expressing, Immunofluorescence, Marker, Control, Tube Formation Assay, Incubation, Software, BrdU Cell Proliferation Assay, Injection

Journal: bioRxiv

Article Title: Liver Lymphatic Dysfunction as a Driver of Fibrosis and Cirrhosis Progression

doi: 10.1101/2025.01.11.632552

Figure Lengend Snippet: (A) Phospho-SMAD2/3 levels in human primary liver LyECs treated with human TGF-β1(hTGFβ1, 1ng/ml) at indicated time. (B) Quantification of PAI-1 mRNA expression in human primary liver LyECs treated with human recombinant TGF1 or control-DMSO for 24 hours. Representative data from three independent experiments. *p< 0.05.

Article Snippet: Human liver LyECs suspension with basal medium (Cat#cAP-03, Angio-Proteomie) was prepared at a concentration of 1.5×10 5 cells/ml.

Techniques: Expressing, Recombinant, Control