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Three-dimensional analysis of ductular reactions and their correlation with liver regeneration and fibrosis

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Abstract

The liver has multiple regeneration modes, including hepatocellular hypertrophy and self-renewal of hepatocytes. When hepatocyte proliferation is impaired, hepatic progenitor cells may proliferate through ductular reaction (DR), differentiate into hepatocytes, and contribute to fibrosis. However, the three-dimensional spatial relationship between DR and regenerating hepatocytes and dynamic changes in DR associated with fibrosis remain poorly understood. Here, we performed three-dimensional (3D) imaging of cleared 42 liver explants with chronic and acute liver diseases and 4 normal livers to visualize DR. In chronic hepatic liver diseases, such as viral hepatitis, steatohepatitis, autoimmune hepatitis, and cryptogenic cirrhosis, the total length and number of branches of DR showed a significant positive correlation. We studied the spatial relationship between DR and GS-expressing cells using glutamine synthetase (GS) and cytokeratin 19 (CK19) as markers of liver regeneration and DR, respectively. The percentage of CK19-positive cells that co-expressed GS was less than 10% in chronic liver diseases. In contrast, nearly one-third of CK19-positive cells co-expressed GS in acute liver diseases, and chronic cholestatic liver diseases, including primary biliary cholangitis and primary sclerosing cholangitis, showed no co-expression. We also found that DR was longer and had more branching in livers with progressive fibrosis compared to those with regressive fibrosis. Our results suggest that DR displays varying degrees of spatial complexity and contribution to liver regeneration. DR may serve as hepatobiliary junctions that maintain continuity between hepatocytes and bile ducts rather than hepatocyte regeneration in chronic liver diseases.

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Data Availability

All data are available from the corresponding author upon reasonable requests.

Abbreviations

DR:

Ductular reaction

3D:

Three-dimensional

2D:

Two-dimensional

GS:

Glutamine synthetase

CK:

Cytokeratin

DMSO:

Dimethyl sulfoxide

PBS:

Phosphate-buffered saline

DBE:

Dibenzyl ether

PSC:

Primary sclerosing cholangitis

PBC:

Primary biliary cholangitis

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Funding

This study was supported by the Department of Pathology at the John Hopkins University School of Medicine.

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Authors and Affiliations

  1. Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    Tadashi Yoshizawa, Jae W. Lee, Seung-Mo Hong, DongJun Jung, Denis Wirtz, Ralph H. Hruban, Laura D. Wood & Kiyoko Oshima

  2. Department of Pathology and Bioscience, Hirosaki University Graduate School of Medicine, Hirosaki Aomori, Japan

    Tadashi Yoshizawa

  3. Department of Pathology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea

    Seung-Mo Hong

  4. Department of Medicine, Graduate School, University of Ulsan, Seoul, Republic of Korea

    DongJun Jung

  5. Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    Michaël Noë, Ralph H. Hruban & Laura D. Wood

  6. Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD, USA

    Wojciech Zbijewski

  7. Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD, USA

    Ashley Kiemen, Pei-Hsun Wu & Denis Wirtz

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  1. Tadashi Yoshizawa
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Contributions

TY and KO designed the study. TY, SH, and DJ performed experiments and data analysis. MN, WZ, AK, PW, and DW provided technical support. JWL, RHH, LDW, and KO performed data analysis and interpreted data. TY and KO developed methods and wrote the manuscript. All authors read and approved the manuscript.

Corresponding author

Correspondence to Kiyoko Oshima.

Ethics declarations

Studies were conducted in accordance with the 1996 Declaration of Helsinki and approved by the institutional review board (IRB) at the Johns Hopkins Hospital.

Conflict of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Supplementary Information

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428_2023_3641_MOESM1_ESM.tif

Supplementary file1 Supplementary Figure 1 |Quantification of unit volume. The nodule diameter (R) was measured (A) and the volume was set as shown in (B) (R/2 × R/4 × R/8). Scale bar represents 100 μm (TIF 9840 KB)

Supplementary file2 Supplementary Movie 1 | 3D surface rendering image of bile ducts in control liver. Bile ducts are highlighted by CK19, which is shown in green. (MP4 113931 KB)

Supplementary file3 Supplementary Movie 2 | 3D surface rendering image of DR and GS in chronic hepatic liver diseases. DR is highlighted by CK19. CK19 and GS are shown in green and red, respectively. Cells co-expressing CK19 and GS are shown in silver. (MP4 212073 KB)

Supplementary file4 Supplementary Movie 3 | 3D surface rendering image of DR and GS in acute liver diseases. DR is highlighted by CK19. CK19 and GS are shown in green and red, respectively. Cells co-expressing CK19 and GS are shown in silver. (MP4 362346 KB)

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Yoshizawa, T., Lee, J.W., Hong, SM. et al. Three-dimensional analysis of ductular reactions and their correlation with liver regeneration and fibrosis. Virchows Arch (2023). https://doi.org/10.1007/s00428-023-03641-3

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