Failure of fibrotic liver regeneration in mice is linked to a severe fibrogenic response driven by hepatic progenitor cell activation

Abstract

Failure of fibrotic liver to regenerate after resection limits therapeutic options and increases demand for liver transplantation, representing a significant clinical problem. The mechanism underlying regenerative failure in fibrosis is poorly understood. Seventy percent partial hepatectomy (PHx) was performed in C57Bl/6 mice with or without carbon tetrachloride (CCl4)-induced liver fibrosis. Liver function and regeneration was monitored at 1 to 14 days thereafter by assessing liver mass, alanine aminotransferase (ALT), mRNA expression, and histology. Progenitor (oval) cell mitogen tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and TWEAK-neutralizing antibody were used to manipulate progenitor cell proliferation in vivo. In fibrotic liver, hepatocytes failed to replicate efficiently after PHx. Fibrotic livers showed late (day 5) peak of serum ALT (3542 ± 355 IU/L compared to 93 ± 65 IU/L in nonfibrotic livers), which coincided with progenitor cell expansion, increase in profibrogenic gene expression and de novo collagen deposition. In fibrotic mice, inhibition of progenitor activation using TWEAK-neutralizing antibody after PHx resulted in strongly down-regulated profibrogenic mRNA, reduced serum ALT levels and improved regeneration. Failure of hepatocyte-mediated regeneration in fibrotic liver triggers activation of the progenitor (oval) cell compartment and a severe fibrogenic response. Inhibition of progenitor cell proliferation using anti-TWEAK antibody prevents fibrogenic response and augments fibrotic liver regeneration. Targeting the fibrogenic progenitor response represents a promising strategy to improve hepatectomy outcomes in patients with liver fibrosis.

Figure 1

Impaired liver regeneration in fibrotic mice, as measured by survival rate, body weight, and restoration of liver volume after PHx. A: Robust liver fibrosis was induced by chronic CCl₄ injections for 6 weeks, leading to a fourfold increase in total hepatic collagen content (μg per liver) in mice receiving CCl₄ via oral gavage. ∗P < 0.001 versus normal mice receiving mineral oil only. B: Connective tissue staining (Sirius red) showing histological signs of early bridging fibrosis. Scale bar = 200 μm. Original magnification: ×100 (left panel); ×200 (right panel). C: Reduced survival in mice with pre-established liver fibrosis after 70% PHx. Survival rate was calculated using the Kaplan-Meier method and was 90.8% in normal mice (137 of 144) compared to 68.9% in fibrotic mice (70 of 86. P < 0.05 versus normal mice. D: Loss of body weight after hepatectomy is more significant in fibrotic mice after PHx. P < 0.05 versus normal mice. Each mouse was weighed every day until day 10 post-PHx, results represent percent change in individual mouse body weight versus body weight before hepatectomy (means ± SEM). E: Restoration of liver volume is compromised in fibrotic livers. Regeneration ratio was assessed by dividing each regenerated liver weight at sacrifice by the estimated remnant liver weight, as described in Materials and Methods. Data are expressed as means ± SEM (6 to 12 mice per group), as described in Materials and Methods. ^∗∗P < 0.0001 compared to normal mice at corresponding time points.

Figure 2

Repressed hepatocyte replication and increased hepatocyte cell death in fibrotic mice after PHx. A: Marked increase in serum transaminases at late stages (4 to 6 days) after PHx suggests massive hepatocyte death in mice with pre-established fibrosis. Serum ALT levels were monitored over time in normal and fibrotic mice after PHx. Means ± SEM (n = 6 to 12 mice per group). ^∗P < 0.01 at day 1 and ^∗∗P < 0.0001 at day 5 versus normal mice. B: Quantification of Ki-67⁺ nuclei of hepatocytes after PHx. Hepatocytes were identified by typical morphological appearance and Ki-67⁺ nuclei were counted in 10 high-power fields in at least five individual mice per time point. Each bar represents means ± SEM of the number of positive hepatocytes and high-power field. ^∗P < 0.05, ^∗∗P < 0.0001 versus normal mice. C: H&E staining of liver samples at selected time points after hepatectomy. Arrow indicates eosinophilic area of vanishing dead hepatocytes in periportal areas. Original magnification: ×200 (left and middle); ×400 (right). Scale bar = 200 μm. Left image, day 0; middle and right images, day 5. D: TUNEL staining notably increased at day 5 after PHx in fibrotic livers. and localize within and at the interface areas of hepatocytic death (arrows), suggesting apoptotic cell death mechanism. E: Hepatocytes in fibrotic mice fail to replicate after PHx. Representative images from liver sections stained with proliferation marker Ki-67 show low replicative activity in hepatocytes in fibrotic (upper row) versus nonfibrotic livers (lower row) at day 2 after hepatectomy. Arrows indicate positive Ki-67 staining in hepatocyte nuclei. Scale bar = 100 μm.

Figure 3

Massive activation of hepatic progenitor (oval) cell compartment in late stages of regeneration in fibrotic livers. A: Expansion of hepatic progenitor (oval) cells in portal areas starting from day 5 after PHx in fibrotic liver. Representative images from liver sections stained for oval cell marker A6 at selected time points in normal and fibrotic mice after hepatectomy. Upper panel: Normal liver without hepatectomy at days 0, 5, and 10 after PHx. Lower panel: Fibrotic liver at 0, 5, and 10 days after hepatectomy. Arrow indicates positive staining. Original magnification, ×100. Scale bar = 200 μm. B: Quantification of A6⁺ oval cells numbers after PHx in normal (open bars) versus fibrotic (closed bars). Ten randomly chosen portal vein areas were assessed in sections from five individual mice (means ± SEM of A6⁺ cell counts per high-power fields). ^∗P < 0.001, ^∗∗P < 0.0001 versus livers without hepatectomy. C: Serum TGFβ1 levels after PHx differ significantly in normal and fibrotic mice and increase dramatically at day 5 in fibrotic mice. Serum TGFβ1 was determined using an eBioscience Human/Mouse TGF beta1 ELISA kit , as described in Materials and Methods. D: A6⁺ progenitor (oval) cells actively proliferate in fibrotic liver after hepatectomy. Representative image is shown (5 days post-PHx, fibrotic) of double-immunofluorescence staining of liver samples for progenitor cell and proliferation markers that frequently colocalize (arrows). PCNA, green, nuclear proliferation marker; A6, red, cytoplasmic oval cell marker; DAPI, blue, nuclei. Original magnification, ×200. Scale bar = 50 μm.

Figure 4

PHx elicits delayed, additive profibrogenic response in fibrotic mice. A: Connective tissue staining (Sirius Red) demonstrates reactive collagen deposition around duct-like proliferations (arrows). Representative liver section of fibrotic mice 10 days after PHx. Oval cell marker A6 staining labels these ductular structures of the same area. B: Dramatic increase in profibrogenic expression which peaks 5 days after PHx in regenerating fibrotic liver (black bars) but not in normal liver (white bars). Liver samples were collected at selected time points after hepatectomy, and hepatic mRNA levels of procollagen α1(I) (collagen), α-SMA, TIMP-1, TGFβ1, and TGFβ2, and integrin β6 were analyzed by real-time PCR. Results represent means ± SEM expressed as fold change over normal mouse controls from 4 to 6 mice per group. ^∗P < 0.05 versus fibrotic livers before hepatectomy; ^∗∗P < 0.05 versus normal liver at corresponding time points.

Figure 5

Manipulation of hepatic progenitor expansion during regeneration modulates fibrogenic response and fibrotic liver regeneration. Experimental manipulation of the hepatic progenitor (oval) cell proliferative response post-PHx was effectively achieved in both directions: robust increase in A6⁺ progenitor cells in animals receiving TWEAK-Fc injections (TWEAK, 75 μg per mouse, n = 10), as well as dramatic inhibition of the oval cell response in mice receiving neutralizing anti-TWEAK antibody (α-TWEAK, 75 μg per mouse, n = 6) compared to mice injected with irrelevant isotype IgG (P1.17, 75 μg per mouse, n = 8). A: Representative images from liver sections stained with oval cell marker A6 IHC in fibrotic mice at day 5 after PHx. Connective tissue (Sirius Red) staining (B) and proliferation marker (Ki-67) IHC (C) of livers from fibrotic mice treated with isotype control antibody (Iso), TWEAK-Fc (TWEAK), or anti-TWEAK antibody (anti-TWEAK) 5 days after 70% PHx. Note increased proliferation predominantly of hepatocytes in anti-TWEAK group and mostly within ductular structures in TWEAK-treated mice (quantified in Table 3). C: Representative images shown. Original magnification, ×200. D: Manipulation of oval cell proliferation modulates profibrogenic response associated with fibrotic liver regeneration. Significant decrease in profibrogenic mRNA expression 5 days after PHx in mice receiving TWEAK-neutralizing antibody (anti-TWEAK, 75 μg per mouse). Hepatic mRNA level of TGFβ1, COL1A1 (procollagen type 1), α-SMA, TIMP-1, TGFβ2, and integrin β6 were analyzed by real-time RT-PCR. Each bar represents means ± SEM expressed as fold change over normal controls (n = 6 to 8 per group). ^∗P < 0.05, ^∗∗P < 0.01 compared to isotype control group (P1.17).