Figure 1.
Partial hepatectomy triggers hepatic accumulation of Fn14(+) cells in WT mice.
Healthy adult wild type (WT) mice (n = 80) underwent PH. Expression of Fn14 was evaluated at various time points by (A) qRT-PCR analysis, (B) Representative western blot, quantification of protein levels for Fn14 normalized to β-actin and (C) Immunohistochemistry (magnification 20×). To assure reagent specificity, similar analysis was done in mice that were genetically-deficient in Fn14(Fn14 KO). Mean +/− SEM results from all mice (n = 4–5/time point) are graphed relative to baseline, pre-PH (time 0), * p<0.05.
Figure 2.
Post- PH growth of Fn14(+) cells is Fn14-dependent and requires TWEAK.
(A) Liver sections from WT mice were double-stained for Fn14 (brown) and Ki67 (green), a proliferation marker. Representative image of liver from 6 h after PH is displayed. Arrow indicates double positive cells. Mean +/− SEM numbers of positive cells in 10 random 40× fields/section in 5 mice/group/time point. (B) qRT-PCR analysis of TWEAK mRNA expression in whole liver RNA from WT and Fn14 KO mice at various time points after PH; * p<0.05 vs WT. (C) Fn14 immunohistochemistry and morphometry analysis in WT mice, WT mice treated with Anti-TWEAK antibodies, TWEAK KO mice, and Fn14 KO mice at baseline (0 h) and 48 h after PH. * p<0.05 vs time 0, # p 0.05 vs WT. Representative images are displayed (magnification 20×).
Figure 3.
Deletion of Fn14 inhibits post- PH proliferation of hepatocytes and cholangiocytes.
Fn14 KO mice and wild type (WT) mice underwent PH. Proliferative activity was evaluated by (A) BrdU incorporation and (B) Ki67 immunostaining. Representative images are displayed (magnification 20×). Arrows indicate Brdu(+) or Ki67(+) cells (red-hepatocyte, black-ductular cells). Labeled hepatocytes/ductular cells were counted in 10 randomly-selected 20× magnification fields/section. Mean +/− SEM results from all mice (n = 3–4/group/time point) are graphed relative to baseline, pre-PH (time 0) in WT group. * p<0.05 vs time 0, # p<0.05 vs WT. Representative images of 48 hours post-PH liver are displayed (magnification 20×).
Figure 4.
Deletion of Fn14 impairs liver regeneration and survival after PH.
Healthy adult wild type (WT) and Fn14 KO mice underwent PH. (A) Liver regeneration ratios (assessed by dividing liver weight at sacrifice by the estimated original liver weight) were compared between WT and Fn14 KO mice at various time points post-PH. (B) Serum bilirubin levels were measured using a Total Bilirubin Test Kit (BIOTRON Diagnostics). (C) Percentage of infarct areas per high-powered field (HPF) were also quantified at in 50 randomly-chosen fields/section. Mean +/− SEM results from 3–4 mice/group/time point are shown. * p<0.01 vs WT. (D) Survival rates were compared between WT and Fn14 KO mice at various time points post-PH. The P values for survival curve are calculated using the log rank test.
Figure 5.
Deletion of TWEAK and inhibition TWEAK activity with anti-TWEAK antibody inhibits proliferation of hepatocytes and cholangiocytes.
TWEAK KO mice, WT mice and WT mice that were treated with anti-TWEAK antibodies underwent PH. Ki67 labeled hepatocytes or ductular cells were counted in 10 randomly-selected 20× magnification fields/section. Mean +/− SEM results from all mice (n = 3–4/time point) are graphed relative to baseline, pre-PH (time 0) in WT group. * p<0.05 vs time 0, # p 0.05 vs WT. Representative images of 48 hours post-PH liver are displayed (magnification 20×).
Figure 6.
Inhibiting TWEAK/Fn14 signaling attenuates the induction of mitogens associated with liver regeneration.
QRT-PCR analysis of whole liver expression of TNFα, IL6 and HGF mRNA in (A) WT and Fn14 KO mice (B) WT, TWEAK KO and WT mice treated with Anti-TWEAK antibodies after PH. Mean +/− SEM results from all mice (n = 3–4/time point) are graphed relative to baseline, pre-PH (time 0) in WT group. * p<0.05 vs time 0, # p<0.05 vs WT.
Figure 7.
Deletion of Fn14 inhibits accumulation of progenitor cells after PH.
Fn14 KO mice and wild type (WT) mice underwent PH. Expression of progenitor markers was evaluated at various time points by (A) qRT-PCR analysis, (B) Immunohistochemistry and (C) Western blot. Mean +/− SEM results from all mice (n = 5/time point) are graphed relative to baseline, pre-PH (time 0) in WT group, * p<0.05 vs time 0, # p<0.05 vs WT. Representative images of 48 hours post-PH liver are displayed (magnification 40×).
Figure 8.
Liver Fn14(+) cells co-localize with progenitor cells.
(A) Serial section staining for Fn14 and progenitor markers (AFP and LGR5) in WT mice 48 hours after PH (magnification 40×). (B) Confocal images of Fn14 and CK18 co-staining in WT mice liver 24 hours after PH (magnification 40×). Yellow indicates co-localization of these two markers. (C) Double immunofluorescence for Fn14 and LGR5 in WT mice liver 48 hours after PH (magnification 20×). Yellow indicates co-localization of these two markers. (C) Confocal images of Fn14 and LGR5 co-stained primary hepatocytes isolated from WT mice 24 hours after PH (magnification 63×).
Figure 9.
TWEAK is required for optimal liver regeneration after PH.
TWEAK KO mice, WT mice and WT mice that were treated with anti-TWEAK antibodies underwent PH. Expression of progenitor markers was evaluated at various time points by (A) qRT-PCR analysis and (B) Immunohistochemistry. Mean +/− SEM results from all mice (n = 5/time point) are graphed relative to baseline, pre-PH (time 0) in WT group, * p<0.05 vs time 0, # p<0.05 vs WT. Representative images of 48 hours post-PH liver are displayed (magnification 20×).