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Knockout of secretin receptor reduces biliary damage and liver fibrosis in Mdr2−/− mice by diminishing senescence of cholangiocytes

Laboratory Investigation (2018) | Download Citation

Abstract

Secretin receptor (SR), only expressed by cholangiocytes, plays a key role in the regulation of biliary damage and liver fibrosis. The aim of this study was to determine the effects of genetic depletion of SR in Mdr2−/− mice on intrahepatic biliary mass, liver fibrosis, senescence, and angiogenesis. 12 wk SR−/−, Mdr2−/−, and SR−/−/Mdr2−/− mice with corresponding wild-type mice were used for the in vivo studies. Immunohistochemistry or immunofluorescence was performed in liver sections for (i) biliary expression of SR; (ii) hematoxylin and eosin; (iii) intrahepatic biliary mass by CK-19; (iv) fibrosis by Col1a1 and α-SMA; (v) senescence by SA-β-gal and p16; and (vi) angiogenesis by VEGF-A and CD31. Secretin (Sct) and TGF-β1 levels were measured in serum and cholangiocyte supernatant by ELISA. In total liver, isolated cholangiocytes or HSCs, we evaluated the expression of fibrosis markers (FN-1 and Col1a1); senescence markers (p16 and CCL2); microRNA 125b and angiogenesis markers (VEGF-A, VEGFR-2, CD31, and vWF) by immunoblots and/or qPCR. In vitro, we measured the paracrine effect of cholangiocyte supernatant on the expression of senescent and fibrosis markers in human hepatic stellate cells (HHSteCs). The increased level of ductular reaction, fibrosis, and angiogenesis in Mdr2−/− mice was reduced in SR−/−/Mdr2−/− mice. Enhanced senescence levels in cholangiocytes from Mdr2−/− mice were reversed to normal in SR−/−/Mdr2−/− mice. However, senescence was decreased in HSCs from Mdr2−/− mice but returned to normal values in SR−/−/Mdr2−/− mice. In vitro treatment of HHSteCs with supernatant from cholangiocyte lacking SR (containing lower biliary levels of Sct-dependent TGF-β1) have decreased fibrotic reaction and increased cellular senescence. Sct-induced TGF-β1 secretion was mediated by microRNA 125b. Our data suggest that differential modulation of angiogenesis-dependent senescence of cholangiocytes and HSCs may be important for the treatment of liver fibrosis in cholangiopathies.

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Acknowledgements

This work was supported partly by the Dr. Nicholas C. Hightower Centennial Chair of Gastroenterology from Scott & White, a VA Research Career Scientist Award, a VA Merit award to GA (5I01BX000574), a VA Merit Award (5I01BX002192) to SG, a VA Merit Award (1I01BX001724) to FM from the United States and a VA Merit Award (1I01BX003031) to HF, Department of Veterans Affairs Biomedical Laboratory Research and Development Service, by University of Rome “La Sapienza” to PO, and the NIH grants DK058411, DK110035, DK076898, DK108959, and DK062975 to GA, FM, and SG. CW is supported by grants from American Diabetes Association (1–17-IBS-145) and the NIH grant DK095862. The content is the responsibility of the author(s) alone and does not necessarily reflect the views or policies of the Department of Veterans Affairs or the United States Government.

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Affiliations

  1. Department of Medical Physiology, Texas A&M University College of Medicine, Temple, TX, 76504, USA

    • Tianhao Zhou
    • , Nan Wu
    • , Julie Venter
    • , Thao K Giang
    • , Heather Francis
    • , Konstantina Kyritsi
    • , Shannon Glaser
    •  & Gianfranco Alpini
  2. Research, Central Texas Veterans Health Care System, Temple, TX, 76504, USA

    • Fanyin Meng
    • , Heather Francis
    • , Shannon Glaser
    •  & Gianfranco Alpini
  3. Baylor Scott & White Digestive Disease Research Center, Baylor Scott & White Health, Temple, TX, 76504, USA

    • Fanyin Meng
    • , Heather Francis
    • , Shannon Glaser
    •  & Gianfranco Alpini
  4. Academic Research Integration, Baylor Scott & White Healthcare, Temple, TX, 76504, USA

    • Fanyin Meng
  5. Department of Nutrition and Food Science, Texas A&M University, College Station, TX, 77840, USA

    • Chaodong Wu
  6. Department of Anatomical, Histological, Forensic Medicine and Orthopaedics Sciences, Sapienza, Rome, Italy

    • Antonio Franchitto
    • , Paolo Onori
    • , Romina Mancinelli
    •  & Eugenio Gaudio
  7. Eleonora Lorillard Spencer Cenci Foundation, Rome, Italy

    • Antonio Franchitto
  8. Department of Medicine, Gastroenterology, Sapienza, Rome, Italy

    • Domenico Alvaro
  9. Clinic of Gastroenterology and Hepatology, Università Politecnica delle Marche, Ospedali Riuniti - University Hospital, Ancona, Italy

    • Marco Marzioni

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The authors declare that they have no conflict of interest.

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Correspondence to Shannon Glaser or Gianfranco Alpini.

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DOI

https://doi.org/10.1038/s41374-018-0093-9