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Heme Oxygenase-1-Modified Bone Marrow Mesenchymal Stem Cells Combined with Normothermic Machine Perfusion Repairs Bile Duct Injury in a Rat Model of DCD Liver Transplantation via Activation of Peribiliary Glands through the Wnt Pathway.

ABSTRACT: Livers from donors after circulatory death (DCD) are inevitably exposed to a longer warm ischemic period, which might increase the incidence of postoperative bile duct complications. Bone marrow mesenchymal stem cells (BMMSCs) have tissue repair properties. The present study was aimed at exploring the repair effect of heme oxygenase-1- (HO-1-) modified BMMSCs (HO-1/BMMSCs) combined with normothermic machine perfusion (NMP) on bile duct injury after DCD liver transplantation and at revealing the underlying mechanisms. Rat livers were exposed to in situ warm ischemia for 30 min; then, NMP was performed through the portal vein for 4 h with BMMSCs, HO-1/BMMSCs, or neither before implantation. Obvious bile duct histological damage and liver functional damage were observed postoperatively. In the group treated with HO-1/BMMSCs combined with NMP (HBP group), liver functions and bile duct histology were improved; meanwhile, cell apoptosis was reduced and cell proliferation was active. A large number of regenerative cells appeared at the injured site, and the defective bile duct epithelium was restored. Dilatation of peribiliary glands (PBGs), proliferation of PBG cells, high expression of vascular endothelial growth factor (VEGF), and increased proportion of bile duct progenitor cells with stem/progenitor cells biomarkers were observed. Blocking Wnt signaling significantly inhibited the repair effect of HO-1/BMMSCs on bile duct injury. In conclusion, HO-1/BMMSCs combined with NMP were relevant to the activation of biliary progenitor cells in PBGs which repaired bile duct injury in DCD liver transplantation via the Wnt signaling pathway. Proliferation and differentiation of PBG cells were involved in the renewal of the injured biliary epithelium.

SUBMITTER: Tian X

PROVIDER: S-EPMC8275434 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Peribiliary glands (PBG) are a source of stem/progenitor cells organized in a cellular network encircling large bile ducts. Severe cholangiopathy with loss of luminal biliary epithelium has been proposed to activate PBG, resulting in cell proliferation and differentiation to restore biliary epithelial integrity. However, formal evidence for this concept in human livers is lacking. We therefore developed an ex vivo model using precision-cut slices of extrahepatic human bile ducts obtained from discarded donor livers, providing an intact anatomical organization of cell structures, to study spatiotemporal differentiation and migration of PBG cells after severe biliary injury. Postischemic bile duct slices were incubated in oxygenated culture medium for up to a week. At baseline, severe tissue injury was evident with loss of luminal epithelial lining and mural stroma necrosis. In contrast, PBG remained relatively well preserved and different reactions of PBG were noted, including PBG dilatation, cell proliferation, and maturation. Proliferation of PBG cells increased after 24 hours of oxygenated incubation, reaching a peak after 72 hours. Proliferation of PBG cells was paralleled by a reduction in PBG apoptosis and differentiation from a primitive and pluripotent (homeobox protein Nanog+/ sex-determining region Y-box 9+) to a mature (cystic fibrosis transmembrane conductance regulator+/secretin receptor+) and activated phenotype (increased expression of hypoxia-inducible factor 1 alpha, glucose transporter 1, and vascular endothelial growth factor A). Migration of proliferating PBG cells in our ex vivo model was unorganized, but resulted in generation of epithelial monolayers at stromal surfaces. Conclusion: Human PBG contain biliary progenitor cells and are able to respond to bile duct epithelial loss with proliferation, differentiation, and maturation to restore epithelial integrity. The ex vivo spatiotemporal behavior of human PBG cells provides evidence for a pivotal role of PBG in biliary regeneration after severe injury.

Project description:Normothermic machine perfusion (NMP) provides clinicians an opportunity to assess marginal livers before transplantation. However, objective criteria and point-of-care (POC) biomarkers to predict risk and guide decision making are lacking. In this investigation, we characterized trends in POC biomarkers during NMP and compared primate donation after circulatory death (DCD) livers with short and prolonged warm ischemic injury. Following asystole, livers were subjected to either 5 minutes (DCD-5min, n = 4) or 45 minutes (DCD-45min, n = 4) of warm ischemia time. Livers were flushed with heparinized UW solution, and preserved in cold storage before NMP. During flow-controlled NMP, circulating perfusate and tissue biopsies were collected at 0, 2, 4, 6, and 8 hours for analysis. DCD-45min livers had greater terminal portal vein pressure (8.5 vs. 13.3 mm Hg, P = 0.027) and terminal portal vein resistance (16.3 vs. 32.4 Wood units, P = 0.005). During perfusion, DCD-45min livers had equivalent terminal lactate clearance (93% vs. 96%, P = 0.344), greater terminal alanine aminotransferase (163 vs. 883 U/L, P = 0.002), and greater terminal perfusate gamma glutamyltransferase (GGT) (5.0 vs. 31.7 U/L, P = 0.002). DCD-45min livers had higher circulating levels of flavin mononucleotide (FMN) at hours 2 and 4 of perfusion (136 vs. 250 ng/mL, P = 0.029; and 158 vs. 293 ng/mL, P = 0.003; respectively). DCD-5min livers produced more bile and demonstrated progressive decline in bile lactate dehydrogenase, whereas DCD-45min livers did not. On blinded histologic evaluation, DCD-45min livers demonstrated greater injury and necrosis at late stages of perfusion, indicative of nonviability. Conclusion: Objective criteria are needed to define graft viability during NMP. Perfusate lactate clearance does not discriminate between viable and nonviable livers during NMP. Perfusate GGT and FMN may represent POC biomarkers predictive of liver injury during NMP.

Project description:Background: A major limitation in expanding the use of donation after circulatory death (DCD) livers in transplantation is the increased risk of graft failure secondary to ischemic cholangiopathy. Warm ischemia causes thrombosis and injury to the peribiliary vascular plexus (PVP), which is supplied by branches of the hepatic artery, causing higher rates of biliary complications in DCD allografts. Aims/Objectives: We aimed to recondition discarded DCD livers with tissue plasminogen activator (tPA) while on normothermic machine perfusion (NMP) to improve PVP blood flow and reduce biliary injury. Methods: Five discarded DCD human livers underwent 12 h of NMP. Plasminogen was circulated in the base perfusate prior to initiation of perfusion and 1 mg/kg of tPA was administered through the hepatic artery at T = 0.5 h. Two livers were split prior to perfusion (S1, S2), with tPA administered in one lobe, while the other served as a control. The remaining three whole livers (W1-W3) were compared to seven DCD control liver perfusions (C1-C7) with similar hepatocellular and biliary viability criteria. D-dimer levels were measured at T = 1 h to verify efficacy of tPA. Lactate, total bile production, bile pH, and difference in biliary injury scores before and after perfusion were compared between tPA and non-tPA groups using unpaired, Mann-Whitney tests. Results: Average weight-adjusted D-dimer levels were higher in tPA livers in the split and whole-liver model, verifying drug function. There were no differences in perfusion hepatic artery resistance, portal vein resistance, and arterial lactate between tPA livers and non-tPA livers in both the split and whole-liver model. However, when comparing biliary injury between hepatocellular and biliary non-viable whole livers, tPA livers had significantly lower PVP injury scores (0.67 vs. 2.0) and mural stroma (MS) injury scores (1.3 vs. 2.7). Conclusion: This study demonstrates that administration of tPA into DCD livers during NMP can reduce PVP and MS injury. Further studies are necessary to assess the effect of tPA administration on long term biliary complications.

Project description:BackgroundHuman albumin/dextran (HA-D), bovine-gelatin (BG), and packed red blood cells plus plasma have been used in European and North-American clinical trials of normothermic ex situ liver perfusion (NEsLP). We compared the effects of these perfusates in a porcine model during NEsLP and after transplantation.MethodsPorcine livers were retrieved 30 minutes after circulatory death. After 5 hours of NEsLP, grafts were transplanted. Three groups (n = 6) were assessed (HA-D vs BG vs whole blood [WB]). One group of static cold storage (SCS) was evaluated for comparison with the perfusion groups. Hemodynamic variables, liver and endothelial injury, and function were assessed during NEsLP and posttransplantation.ResultsHepatic artery flow was higher since the beginning of NEsLP in the HA-D group (HA-D, 238 ± 90 mL/min vs BG, 97 ± 33 mL/min vs WB, 148 ± 49 mL/min; P = 0.01). Hyaluronic acid was lower in the HA-D at the end of perfusion (HA-D, 16.28 ± 7.59 ng/μL vs BG, 76.05 ± 15.30 ng/μL vs WB, 114 ± 46 ng/μL; P < 0.001). After transplant, aspartate aminotransferase was decreased in the HA-D group when compared with the rest of the groups (HA-D, 444 ± 226 IU/L vs BG, 1033 ± 694 IU/L vs WB, 616 ± 444 IU/L vs SCS, 2235 ± 1878 IU/L). At 5 hours after transplant, lactate was lower in the HA-D group (HA-D, 3.88 ± 1.49 mmol/L vs BG, 7.79 ± 2.68 mmol/L vs WB, 8.16 ± 3.86 mmol/L vs SCS, 9.06 ± 3.54 mmol/L; P = 0.04). International Normalized Ratio was improved in HA-D group compared to the rest of the groups (HA-D, 1.23 ± 0.30 vs BG, 1.63 ± 0.20 vs WB, 1.50 ± 0.31 vs SCS, 1.97 ± 1.55; P = 0.03) after transplantation. In contrast, BG displayed lower aspartate aminotransferase levels during NEsLP (HA-D, 183 ± 53 IU/L vs BG, 142 ± 52 IU/L vs WB, 285 ± 74 IU/L; P = 0.01) and less cleaved-caspase-3 staining (HA-D, 2.05 ± 0.73% vs BG, 0.95 ± 1.14% vs WB, 1.74 ± 0.54% vs SCS, 7.95 ± 2.38%) compared with the other groups. On the other hand, the bile from the WB showed higher pH (HA-D, 7.54 ± 0.11 vs BG, 7.34 ± 0.37 vs WB, 7.59 ± 0.18) and lower glucose levels (HA-D, 0.38 ± 0.75 mmol/L vs BG, 1.42 ± 1.75 mmol/L vs WB, 0 ± 0 mmol/L) by the end of perfusion.ConclusionsOverall HA-D displayed more physiologic conditions during NEsLP that were reflected in less graft injury and improved liver function and survival after transplantation. Optimization of the perfusates based on the beneficial effects found with these different solutions would potentially improve further the outcomes through the use of NEsLP in marginal grafts.

Project description:BackgroundNormothermic machine perfusion (NMP) of liver grafts donated after circulatory death (DCD) has shown promise in large animal and clinical trials. Following procurement, initial flush with a cold preservation solution is the standard of care. There is concern that initial cooling followed by warming may exacerbate liver injury, and the optimal initial flush temperature has yet to be identified. We hypothesize that avoidance of the initial cold flush will yield better quality liver grafts.MethodsTwenty-four anaesthetized pigs were withdrawn from mechanical ventilation and allowed to arrest. After 60-minutes of warm ischemia to simulate a DCD procurement, livers were flushed with histidine-tryptophan-ketoglutarate (HTK) at 4°C, 25°C or 35°C (n = 4 per group). For comparison, an adenosine-lidocaine crystalloid solution (AD), shown to have benefit at warm temperatures in heart perfusions, was also used (n = 4 per group). During 12-hours of NMP, adenosine triphosphate (ATP), lactate, transaminase levels, and histological injury were determined. Bile production and hemodynamics were monitored continuously.ResultsATP levels recovered substantially following 1-hour of NMP reaching pre-ischemic levels by the end of NMP with no difference between groups. There was no difference in peak aspartate aminotransferase (AST) or in lactate dehydrogenase (LDH). Portal vein resistance was lowest in the 4°C group reaching significance after 2 hours (0.13 CI -0.01,0.277, p = 0.025). Lactate levels recovered promptly with no difference between groups. Comparison to AD groups showed no statistical difference in the abovementioned parameters. On electron microscopy the HTK4°C group had the least edema with mean cell thickness of 2.92μm (p = 0.41) while also having the least sinusoidal dilatation with a mean diameter of 5.36μm (p = 0.04). For AD, the 25°C group had the lowest mean cell thickness at 3.14μm (p = 0.09).ConclusionsAvoidance of the initial cold flush failed to demonstrate added benefit over standard 4°C HTK in this DCD model of liver perfusion.

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Heme Oxygenase-1-Modified Bone Marrow Mesenchymal Stem Cells Combined with Normothermic Machine Perfusion Repairs Bile Duct Injury in a Rat Model of DCD Liver Transplantation via Activation of Peribiliary Glands through the Wnt Pathway.

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