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. 2018 May;18(5):1110-1121.
doi: 10.1111/ajt.14586. Epub 2017 Dec 18.

Heme oxygenase-1 regulates sirtuin-1-autophagy pathway in liver transplantation: From mouse to human

Kojiro Nakamura  1 , Shoichi Kageyama  1 , Shi Yue  1 , Jing Huang  1 , Takehiro Fujii  1 , Bibo Ke  1 , Rebecca A Sosa  2 , Elaine F Reed  2 , Nakul Datta  1 , Ali Zarrinpar  1 , Ronald W Busuttil  1 , Jerzy W Kupiec-Weglinski  1
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"V体育ios版" Heme oxygenase-1 regulates sirtuin-1-autophagy pathway in liver transplantation: From mouse to human

Kojiro Nakamura et al. Am J Transplant. 2018 May.

Abstract

Liver ischemia-reperfusion injury (IRI) represents a major risk factor of early graft dysfunction and a key obstacle to expanding the donor pool in orthotopic liver transplantation (OLT). Although graft autophagy is essential for resistance against hepatic IRI, its significance in clinical OLT remains unknown. Despite recent data identifying heme oxygenase-1 (HO-1) as a putative autophagy inducer, its role in OLT and interactions with sirtuin-1 (SIRT1), a key autophagy regulator, have not been studied. We aimed to examine HO-1-mediated autophagy induction in human OLT and in a murine OLT model with extended (20 hours) cold storage, as well as to analyze the requirement for SIRT1 in autophagy regulation by HO-1. Fifty-one hepatic biopsy specimens from OLT patients were collected under an institutional review board protocol 2 hours after portal reperfusion, followed by Western blot analyses. High HO-1 levels correlated with well-preserved hepatocellular function and enhanced SIRT1/LC3B expression VSports手机版. In mice, HO-1 overexpression by genetically modified HO-1 macrophage therapy was accompanied by decreased OLT damage and increased SIRT1/LC3B expression, whereas adjunctive inhibition of SIRT1 signaling diminished HO-1-mediated hepatoprotection and autophagy induction. Our translational study confirms the clinical relevance of HO-1 cytoprotection and identifies SIRT1-mediated autophagy pathway as a new essential regulator of HO-1 function in IR-stressed OLT. .

Keywords: basic (laboratory) research/science; biopsy; immunobiology; liver disease: immune/inflammatory; liver transplantation/hepatology; organ perfusion and preservation; tissue injury and repair; translational research/science. V体育安卓版.

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Conflict of interest statement

The authors declare that no conflict of interest exists.

Disclosure

The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.

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Figure 1
Figure 1. Adoptive transfer of AdHO-1 BMM ameliorates IR-damage in OLT
Mouse (C57BL/6) livers subjected to 20h of cold storage were transplanted to syngeneic mice. BMM (5×106) transfected with AdHO-1 or Adβ-gal (2.5×109 pfu) or untreated BMM were infused into liver grafts via portal vein immediately prior to reperfusion. Liver grafts and serum samples were analyzed at 6h post-OLT. (A) Representative (n=3) Western blot-assisted detection of HO-1; β-actin expression served as an internal control. (B) Representative H&E staining (n=3; original magnification, ×100). (C) Suzuki histological grading of liver IRI and sALT levels. (n=4-6/group); *p<0.05 vs. OLT: +BMM (AdHO-1). Data shown as mean±SD.
Figure 2
Figure 2. Adoptive transfer of AdHO-1 BMM inhibits pro-inflammatory signature and enhances anti-inflammatory program in OLT
Mouse (C57BL/6) livers subjected to 20h of cold storage and transplanted to syngeneic mice were infused immediately before reperfusion via portal vein with BMM (naïve, AdHO-1 or Ad β-gal). Samples were analyzed at 6h post-OLT. qRT-PCR-assisted detection of mRNA coding for IL10 and TGFβ (A), TNFα, MCP1, CXCL10 (B). Data were normalized to HPRT gene expression (n=4/group) * p<0.05 vs. OLT: +BMM (AdHO-1). Data shown as mean±SD.
Figure 3
Figure 3. Adoptive transfer of AdHO-1 BMM suppresses neutrophil trafficking and mitigates hepatocellular death in OLT
Mouse (C57BL/6) livers subjected to 20h of cold storage and transplanted to syngeneic mice were infused via portal vein with BMM (naïve, AdHO-1 or Ad β-gal). Samples were analyzed at 6h post-OLT. (A) Representative immunohistochemical Ly6G (upper panels) and TUNEL (lower panels) staining. (B) Quantification of Ly6G (left) and TUNEL (right) positive cells/HPF (n=4-6/group). * p<0.05 vs. OLT: +BMM (AdHO-1). Data shown as mean±SD.
Figure 4
Figure 4. Adoptive transfer of AdHO-1 BMM enhances SIRT1/LC3B expression in OLT
Mouse (C57BL/6) livers subjected to 20h of cold storage and transplanted to syngeneic mice were infused via portal vein with BMM (naïve, AdHO-1 or Ad β-gal). Samples were analyzed at 6h post-OLT. (A) Representative (n=3) Western blot-asisted detection of SIRT1 and LC3B; β-actin expression served as an internal control. (B) qRT-PCR-assisted detection of mRNA coding for HO-1 and SIRT1. Data were normalized to HPRT gene expression (n=4/group) * p<0.05 vs. OLT: +BMM (AdHO-1). Data shown as mean±SD. (C) Representative (n=3) immunohistochemical detection of LC3B in OLT at 6h post-reperfusion.
Figure 5
Figure 5. Post-transplant HO-1 expression negatively correlates with hepatocellular damage and positively with SIRT1/LC3B expression in human OLT
(A) Post-transplant liver biopsies (Bx; 2h post-reperfusion) were collected from fifty-one OLT patients. Bx samples were analyzed by Western blots with β-actin normalization. (B) Relationship between post-transplant HO-1 expression and sALT levels at postoperative day 1 (POD1). (C/D) Relationship between post-transplant HO-1 and SIRT1 (C) and LC3B (D) expression. r: Spearman’s correlation coefficient.
Figure 6
Figure 6. Increased post-transplant HO-1 levels correlate with attenuated hepatocellular damage and enhanced SIRT1 – LC3B pathway in human OLT
(A) Bx samples collected from the fifty-one OLT patients were classified into low HO-1 (n=26) and high HO-1 (n=25) groups. (B) Western blot-assisted expression of SIRT1 and LC3B. Data shown in dot plots and bars indicate mean±SEM. # p<0.05 (Mann-Whitney U test). (C) Representative Western-blots assisted detection of HO-1, SIRT1 and LC3B (Case A/B: low HO-1 group, Case C/D: high HO-1 group) (D) sALT levels at postoperative day 1-14 (POD1-14) shown as mean±SEM. Dotted line indicates low HO-1, while the solid line high HO-1. # p<0.05 (Mann-Whitney U test). (E) The cumulative probability of OLT survival (Kaplan-Meier method). Dotted line indicates low HO-1, while the solid line high HO-1 groups (log-rank test). (F) Representative TUNEL and LC3B staining pattern in OLT.
Figure 7
Figure 7. SIRT1 inhibition depresses LC3B expression and abrogates hepatoprotection in AdHO-1 BMM-conditioned IR-OLT
Mouse (C57BL/6) livers subjected to 20h of cold storage were transplanted to syngeneic mice and conditioned with AdHO-1 BMM with or without adjunctive SIRT1 inhibition (EX527, 10mg/kg i.p). Samples were analyzed at 6h post-OLT. (A) Representative H&E staining (original magnification, ×100). (B) sALT levels (left) and Suzuki histological grading of liver IRI (right). *p<0.05 vs. OLT: + AdHO-1 BMM + DMSO (n=3-4/group). Data shown as mean±SD. (C) Representative (n=3) Western blot-asisted detection of LC3B; β-actin expression served as an internal control. (D) Representative (n=3) immunhistochemical detection of LC3B.
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