Liver repair and regeneration after ischemia-reperfusion injury is associated with prolonged fibrosis

Abstract

Liver recovery after hepatic ischemia-reperfusion (I/R) injury is characterized by clearance of dead tissue and its replacement with functional liver parenchyma. Previous reports have observed fibrosis after liver I/R. To determine whether liver fibrosis after I/R was a pathologic consequence of the injury response, we assessed the development of liver fibrosis after I/R and its impact on subsequent insult. A murine model of partial I/R was used to induce liver injury and study the reparative response. During liver remodeling after I/R, expression of the profibrotic genes increased in the ischemic liver. Histologically, α-smooth muscle actin (α-SMA)-positive hepatic stellate cells (HSCs)/myofibroblasts increased, and collagen deposition was enhanced along the injured site. Selective staining experiments showed that HSCs, not portal fibroblasts, were the major source of myofibroblasts. During liver repair after I/R, liver fibrosis was readily observed at the interface between necrotic tissue and regenerating liver in association with HSCs/myofibroblasts. The number of HSCs/myofibroblasts decreasing shortly after the full resolution of necrotic injury and restoration are normal liver architecture. However, liver fibrosis persisted for several more weeks before gradually resolving. Resolution of liver fibrosis was accompanied by upregulated expression of matrix metalloproteinase-13. After resolution of fibrosis, the administration of CCl4 did not result in exacerbated liver injury, suggesting that I/R injury does not predispose the liver to future fibrotic insults. The data suggest that liver fibrosis is a component of tissue repair after I/R, is caused by myofibroblasts derived from HSC, and does not increase susceptibility of the liver to subsequent hepatic injury. NEW & NOTEWORTHY This study is the first to assess pathology of liver fibrosis during the reparative process after ischemia-reperfusion (I/R) injury. Here we show that profibrotic gene expression increased in the liver after I/R, and collagen accumulation produced by hepatic stellate cells (HSCs)/myofibroblasts enhanced at the interface between necrotic tissue and regenerating liver. Liver fibrosis gradually resolved concomitant with decreasing activation of HSC and upregulating matrix metalloproteinase-13. After resolution of fibrosis, the liver was not more susceptible to subsequent hepatic injury.

Keywords: hepatic stellate cells; liver fibrosis; liver regeneration; liver repair.

Fig. 1.

Development of liver fibrosis after ischemia-reperfusion (I/R) injury. A: liver sections from mice undergoing 45-min ischemia followed by reperfusion were analyzed. Sirius red staining of liver sections shows increased collagen deposition 4 days after I/R injury and maintaining a similar level of fibrosis up to 2 wk after I/R. Hematoxylin-eosin staining shows significant liver injury 1 day after reperfusion with repair and resolution of injury to nearly normal liver architecture by 1 wk after reperfusion. Original magnification is ×100. B: liver sections from mice undergoing 90-min ischemia followed by reperfusion were analyzed. Sirius red staining of liver sections shows collagen deposition within 4 days after I/R injury and increasing fibrosis during the time of liver repair (up to 2 wk after I/R). Hematoxylin-eosin staining shows massive liver injury 1 day after I/R with repair and resolution of the injury to nearly normal liver architecture by 2 wk after reperfusion. There was much more liver injury and liver fibrosis in mice undergoing 90 min of ischemia compared with those undergoing 45 min of ischemia. Original magnification is ×100. Quantification of necrosis area and fibrosis area shows an inverse relationship between necrotic area and extent of liver fibrosis. Data are means ± SE with n = 3 per group. *P < 0.05 compared with control.

Fig. 2.

Profibrotic gene expression after ischemia-reperfusion (I/R) injury. Liver mRNA expression of α-smooth muscle actin (α-SMA), collagen-1α1, and tissue inhibitor of metalloproteinase-1 (TIMP-1) after I/R injury were measured by qPCR. Expression of these genes increased during liver repair and returned to near baseline expression by 2 wk after reperfusion. Data are means ± SE with n = 4 per group. *P < 0.05 compared with control. #P < 0.05 compared with control and 4 days.

Fig. 3.

Expansion of myofibroblasts is restricted to areas of liver necrosis. The location of myofibroblasts and their proximity to areas of liver fibrosis were determined by immunohistochemistry for α-smooth muscle actin (α-SMA) and Sirius red staining in serial sections at 1 wk after ischemia-reperfusion (I/R). A: myofibroblasts expanded at the border between viable liver parenchyma and necrotic areas and also in the periportal regions within necrotic areas of the liver. Collagen deposition was observed in the same areas. Original magnification is ×400. B: virtually no α-SMA-positive cells were observed around bile ducts in nonnecrotic areas of liver, whereas collagen deposition was still detected in these areas. Original magnification is ×400. C: fibrosis was observed where the necrotic tissue was replaced with reconstructed liver parenchyma at 2 wk after I/R. Original magnification is ×200.

Fig. 4.

Origin of hepatic myofibroblasts after ischemia-reperfusion (I/R) injury. To determine the nature of myofibroblasts after I/R injury, serial sections of liver obtained 1 wk after I/R were stained by immunohistochemistry for α-smooth muscle actin (α-SMA) (myofibroblasts), desmin (hepatic stellate cells), and Thy-1 (portal fibroblasts). There is clear colocalization of α-SMA and desmin but no colocalization of α-SMA and Thy-1. Original magnification is ×630.

Fig. 5.

Resolution of liver fibrosis after ischemia-reperfusion (I/R). A: collagen deposition was determined by Sirius red staining. Liver fibrosis peaked 2 wk after reperfusion and was nearly resolved 8 wk after reperfusion. Fibrosis area was measured as a percentage of total area. Data are means ± SE with n = 3 per group. Original magnification is ×100. *P < 0.05 compared with 2 wk. B: myofibroblasts were identified by immunohistochemical staining for α-smooth muscle actin (α-SMA). The number of myofibroblasts decreased before the regression of fibrosis. α-SMA-positive area was measured as a percentage of total area. Data are means ± SE with n = 3 per group. Original magnification is ×400. *P < 0.05 compared with 1 wk. C: expression of matrix metalloproteinases (MMPs) was assessed in lysates of postischemic liver tissue by Western blot. The expression of MMP-2 and MMP-9 peaked 1 wk after I/R and then returned to baseline levels. MMP-13 expression decreased 1 wk after I/R and then was increased at 2, 4, and 8 wk after I/R. Figure is representative of 3 independent experiments.

Fig. 6.

Impact of fibrosis on liver repair after ischemia-reperfusion (I/R). Mice were injected intraperitoneally with vehicle or 0.75 mg/kg gliotoxin 48, 72, and 96 h after reperfusion. A: stellate cells in the ischemic liver were assessed 4 days after I/R by desmin staining. Desmin-positive area was measured as a percentage of total area. Gliotoxin significantly reduced desmin-positive area. Data are means ± SE with n = 4 per group. *P < 0.05 compared with vehicle group. B: liver recovery, assessed by area of liver necrosis, was determined 7 days after I/R by hematoxylin and eosin staining. Gliotoxin significantly increased necrosis area. Data are means ± SE with n = 6–7 per group. *P < 0.05 compared with vehicle group.

Fig. 7.

Prior ischemia-reperfusion (I/R) injury and fibrosis do not predispose the liver to injury from subsequent insults. A–C: mice subjected to I/R injury were allowed to partially recover (2 wk) before being injected intraperitoneally with CCl4 twice weekly for 2 wk. Tissues and samples were analyzed at 3 wk and 4 wk after I/R. B: liver injury was measured by serum alanine aminotransferase (ALT), and expression of inflammatory and profibrotic genes was assessed by measuring liver TNF-α and α-smooth muscle actin (α-SMA) mRNA expression by qPCR. Data are means ± SE with n = 4 per group. *P < 0.05 compared with sham group. C: liver histology was assessed by hematoxylin-eosin staining. Original magnification is ×100. D–F: mice subjected to I/R injury were allowed to recover fully (8 wk) before being injected intraperitoneally with CCl4 twice weekly for 2 wk. Tissues and samples were analyzed at 9 wk and 10 wk after I/R. E: serum ALT and liver TNF-α and α-SMA mRNA expression were measured as described above. Data are means ± SE with n = 4 per group. *P < 0.05 compared with sham group. F: liver histology was assessed by H and E staining. Original magnification is ×100. There were no differences in the development of liver injury between mice undergoing previous sham or I/R injury.