Oval Cells Contribute to Fibrogenesis of Marginal Liver Grafts under Stepwise Regulation of Aldose Reductase and Notch Signaling

Abstract

Background and Aims: Expanded donor criteria poses increased risk for late phase complications such as fibrosis that lead to graft dysfunction in liver transplantation. There remains a need to elucidate the precise mechanisms of post-transplant liver damage in order to improve the long-term outcomes of marginal liver grafts. In this study, we aimed to examine the role of oval cells in fibrogenic development of marginal liver grafts and explore the underlying mechanisms. Methods: Using an orthotopic rat liver transplantation model and human post-transplant liver biopsy tissues, the dynamics of oval cells in marginal liver grafts was evaluated by the platform integrating immuno-labeling techniques and ultrastructure examination. Underlying mechanisms were further explored in oval cells and an Aldose reductase (AR) knockout mouse model simulating marginal graft injury. Results: We demonstrated that activation of aldose reductase initiated oval cell proliferation in small-for-size fatty grafts during ductular reaction at the early phase after transplantation. These proliferative oval cells subsequently showed prevailing biliary differentiation and exhibited features of mesenchymal transition including dynamically co-expressing epithelial and mesenchymal markers, developing microstructures for extra-cellular matrix degradation (podosomes) or cell migration (filopodia and blebs), and acquiring the capacity in collagen production. Mechanistic studies further indicated that transition of oval cell-derived biliary cells toward mesenchymal phenotype ensued fibrogenesis in marginal grafts under the regulation of notch signaling pathway. Conclusions: Oval cell activation and their subsequent lineage commitment contribute to post-transplant fibrogenesis of small-for-size fatty liver grafts. Interventions targeting oval cell dynamics may serve as potential strategies to refine current clinical management.

Keywords: aldose reductase; hepatic bipotent cells; notch signaling.; small-for-size fatty graft injury.

Figure 1

Hepatic progenitor cells (oval cells) proliferated in small-for-size fatty grafts and exhibited dominant biliary differentiation. (A) Suppressed hepatocyte regeneration in small-for-size fatty grafts (Ki-67 staining, arrows) post-transplantation (n=6). (B) Statistical analysis of Ki-67 positive hepatocytes under microscopic examination. (C) Enhanced expression of Ov6 (hepatic progenitor cell marker, arrows) in the proliferative ductules of small-for-size fatty grafts (n=6). (D) Integrated intensity of Ov6 expression. (E) Increased proliferation of oval cells (Ov6 staining, arrows) in human small-for-size fatty graft. (F) Co-localization of Ov6 and E-cadherin (day 4, day 7) and development of defined lumina (arrowhead) on day 7 in the ductules of rat small-for-size fatty grafts. * p<0.05; ** p<0.01.

Figure 2

Aldose reductase (AR) regulated oval cell proliferation. (A) Over-expression of aldose reductase in proliferative biliary cells of rat or human small-for-size fatty grafts (the arrows indicated the biliary cells). (B) Effect of AR plasmid (solid line) and AR inhibitor Zopolrestat (dashed line) on the growth of oval cells. (mean ± SD, n=3, p<0.05) (C) Effect of AR plasmid and Zopolrestat on the expression of cell cycle related genes. (D) Zopolrestat blocked cell cycle of oval cells at G1/S. (E) In AR wild type mouse model simulating small-for-size fatty graft injury, activated oval cells (Trop2, green color) co-expressed Ep-CAM (magenta color) (arrows) (n=6). In contrast, fewer oval cells were observed in AR knockout mouse model.

Figure 3

Proliferative biliary cells in small-for-size fatty graft dynamically co-expressed CK19 and Vimentin. (A) Co-localization of epithelial (CK19) and mesenchymal (Vimentin) markers in reactive ductules (arrowheads) of post-transplant small-for-size fatty grafts. (B) Co-localization of CK19 and Vimentin in small-for-size fatty graft revealed by 3D reconstruction (z-stacking under confocal microscopy). (C) Dynamic expression of CK19 (decreased, pink arrows) and Vimentin (increased, yellow arrows) in the proliferative ductules of small-for-size fatty grafts (serial sections). (D) Dynamic co-localization of CK19 and Vimentin in the proliferative ductules (arrowheads) of human small-for-size fatty graft at 6 and 11 months post-transplantation.

Figure 4

Transitional biliary cells in small-for-size fatty graft showed features of collagen remodeling and migration. (A) Podosome (arrowhead) with increased lysosome-like structures (magnified image, arrow) in the cytoplasm of transitional biliary cells (day 14). (B) Accumulation of podosome-associated protein Cortactin (magenta color) was accompanied with concurrent augmentation of matrix-degrading protease Mmp14 (magenta color) in biliary cells (arrowheads). (C) Filopodia protrusions (arrowheads) extended from the basal or lateral sides of transitional biliary cells and contacted adjacent extracellular matrix (asterisk). Microfilaments (magnified image, arrow) parallel to cell membrane were identified in the cytosol near protrusions. (D) Co-localization of Arpc2 (a protein for filopodia formation, arrowheads) and Mmp9 in biliary tubules (bright field, circled by dashed line) in small-for-size fatty graft (day 14). (E) In the leading edge of transitional cells (pink color), membrane blebs formed (magnified image, arrow) and contacted with passing non-biliary cells (magnified image, asterisks). (F) Discontinuity of base membrane (Laminin staining, arrowheads) surrounding biliary cells in post-transplant human small-for-size fatty graft (6 month).

Figure 5

Biliary cells in small-for-size fatty graft showed collagen-producing capacity. Prominent expression of Pro-collagen I alpha 1 mRNA was identified in the biliary cells of small-for-size fatty grafts (A, upper panels and lower left panel; green color, CK19; red color, pro-collagen I α 1) (In situ hybridization, day 14). In contrast, signals of procollagen I α 1 were not detectable in the whole normal grafts (A, lower right panel). Masson's trichrome staining revealed collagen deposition (B, upper panel, arrows) surrounding the proliferative ductules of small-for-size fatty grafts (outlined by dashed lines, day 14).

Figure 6

Notch signaling was activated in small-for-size fatty graft. (A) Increased mRNA level of Notch 2 in small-for-size fatty graft (rat, mean ± SD, n=6). * p<0.05. (B) Over-expression of Notch 2 (pink arrows, DAB) in biliary cells of small-for-size fatty graft (rat) on day 7, day 14 post-transplantation in contrast to the whole normal graft (rat). (C) Up-regulation of Hes-1 (pink arrows, DAB) in biliary cells of small-for-size fatty graft was accompanied with an increase of Jag-1 (yellow arrows, Emerald) in adjacent stromal cells (biliary cells were circled inside dashed lines). (D) Increased Notch 2 expression (DAB, arrows) in biliary cells of post-transplant human small-for-size fatty graft (6 months) compared with that of the whole normal graft. Abbreviation: DAB, 3,3'-Diaminobenzidine.

Figure 7

Notch signaling modulated mesenchymal transition and fibrogenesis of biliary-differentiated oval cells. (A) Oval cells formed acinus structures inside the matrigel after 4 days of induction. (B) Acinus polarity and biliary differentiation were revealed by immunofluorescent staining. (C) Augmented mRNA levels of Notch 2 and effector Hes-1 in biliary-differentiated oval cells after addition of TGFβ-1. (D) mRNA levels of epithelial (CK7, CK19, E-cadherin), mesenchymal (SMA) and fibrogenic (Collagen I alpha 1, Fibronectin) markers in biliary-differentiated oval cells with or without the presence of TGFβ-1. (E) Addition of γ-secretase inhibitor dibenzazepine (DBZ, 5 nM) mitigated the alteration of epithelial/mesenchymal markers and fibrogenic markers induced by TGFβ-1. (F) Representative epithelial/mesenchymal markers attenuated by DBZ in the presence of TGFβ-1(western blot). Data represent the mean ± SD (n=3) and are representative of at least 2 independent experiments in duplicate. * p<0.05, ** p<0.01.