Figure 6. Endothelial Wnt ligand secretion reestablishes metabolic zonation in the CCl₄-acutely injured liver.

(A) (Left) Schematic of CCl₄ administration and tissue harvesting using Cldn2-EGFP mice. (Right) Double-immunofluorescence results show physical association of claudin-2/GFP⁺ hepatocytes (arrows) with hepatic endothelial cells (PECAM⁺) throughout the recovery period that follows CCl₄-acute injury. (B) (Top, left) Schematic of the experimental strategy and tissue harvesting. ALT/AST serum levels demonstrate that CCl₄ promotes liver damage in Cdh5-CreERT2 and Cdh5-CreERT2;Wls^f/f mice injected with tamoxifen. (n = 3). p values were determined by one-way ANOVA with Bonferroni’s multiple comparisons test, NS, not significant (p>0.05), ***p<0.001. (Right and bottom) Diagrams indicate the experimental strategy and tissue harvesting. Double-immunofluorescence and quantitative results show progressive expansion and full restoration of Zone 3 (GS⁺, arrows) and Zone 2 (Cyp2e1⁺, arrows) and transient macrophage infiltrates (F4/80⁺, arrows; arrowheads are Kupffer cells) in the liver of Cdh5-CreERT2 mice after acute CCl₄ administration. In contrast, Zone 3 is nearly absent, Zone 2 is significantly smaller, and Zone 1 is expanded, in Cdh5-CreERT2;Wls^f/f livers 5-, 7- and 15 days post-CCl₄. Perivenous macrophage infiltrates are observed in both Cdh5-CreERT2 and Cdh5-CreERT2;Wls^f/f livers 5–7 days post-CCl₄ but not 15 days after CCl₄ administration. p values were determined by two-tailed unpaired Student’s t-test, ***p<0.001, (n = 3). (C) (Left) Schematic of CCl₄ administration and tissue harvesting. (Right) Triple-immunofluorescence results show that CD117 proteins are restricted to the Lyve-1⁺/Lyve-1^LOW sinusoidal endothelium traversing Zones 2/3 in the normal and CCl₄-injected (day 3) adult liver. (D) Schematic of CD117⁺ and Lyve1⁺ HECs isolation. Nonparenchymal liver cells (NPCs) were isolated using a two-step collagenase perfusion method and incubated with CD31-coated Dynabeads. The eluted CD31⁺ (HEC) fraction was incubated with CD117-coated Dynabeads to isolate CD117⁺CD31⁺ (‘pericentral/perivenous’) HECs. The unbound fraction from this step was incubated with Lyve-1-coated Dynabeads to separate Lyve⁺CD31⁺ hepatic sinusoidal cells from other HECs. (E) QRT-PCR results show comparable Lyve1 transcript expression in CD117⁺/CD31⁺ and Lyve1⁺/CD31⁺ isolates from saline-injected (‘control’) or CCl₄-injected (day 3) livers, lower Kit expression in LSECs from injured livers compared to control livers, and higher Wnt2, Wnt9b and Rspo3 expression in LSECs from injured livers compared to control livers. (Three individual livers per condition were used to isolate LSECs.) (A–C) Each image represents 2–4 individual livers. Asterisks: central veins. (B) NS, not significant (p>0.05), ***p<0.001, (n = 3). Scale bars: 50 μm (C), 100 μm (A, B).