Dataset Information

Endothelial Dysfunction in Steatotic Human Donor Livers: A Pilot Study of the Underlying Mechanism During Subnormothermic Machine Perfusion.

ABSTRACT: Background:Steatosis is a major risk factor for primary nonfunction in liver transplantations. Steatotic livers recover poorly from ischemia reperfusion injury, in part due to alterations in the microcirculation, although the exact mechanism is unclear. In this study, we tested if there were any alterations in the shear stress sensing Kruppel-like factor 2 (KLF2) and its likely downstream consequences in the ex vivo perfused human liver endothelium, which would imply perturbations in microcirculatory flow in macrosteatotic livers disrupts laminar flow to evaluate if this is a potential therapeutic target for steatotic livers. Methods:Using a subnormothermic machine perfusion system, 5 macrosteatotic and 4 nonsteatotic human livers were perfused for 3 hours. Flow, resistance, and biochemical profile were monitored. Gene expression levels of nitric oxide synthase 3 (eNOS), KLF2, and thrombomodulin were determined. Nitric oxide (NO) was measured in the perfusion fluid and activation of eNOS was measured with Western blotting. Results:Flow dynamics, injury markers, and bile production were similar in both groups. Kruppel-like factor 2 expression was significantly higher in nonsteatotic livers. Western blotting analyses showed significantly higher levels of activated eNOS in nonsteatotic livers, consistent with an increase in NO production over time. Macrosteatotic livers showed decreased KLF2 upregulation, eNOS activity, and NO production during machine perfusion. Conclusions:These results indicate a perturbed KLF2 sensing in steatotic livers, which aligns with perturbed microcirculatory state. This may indicate endothelial dysfunction and contribute to poor posttransplantation outcomes in fatty livers, and further studies to confirm by evaluation of flow and testing treatments are warranted.

SUBMITTER: Beijert I

PROVIDER: S-EPMC5959347 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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Endothelial Dysfunction in Steatotic Human Donor Livers: A Pilot Study of the Underlying Mechanism During Subnormothermic Machine Perfusion.

Beijert Irene I Mert Safak S Huang Viola V Karimian Negin N Geerts Sharon S Hafiz Ehab O A EOA Markmann James F JF Yeh Heidi H Porte Robert J RJ Uygun Korkut K

Transplantation direct 20180423 5

<h4>Background</h4>Steatosis is a major risk factor for primary nonfunction in liver transplantations. Steatotic livers recover poorly from ischemia reperfusion injury, in part due to alterations in the microcirculation, although the exact mechanism is unclear. In this study, we tested if there were any alterations in the shear stress sensing Kruppel-like factor 2 (KLF2) and its likely downstream consequences in the ex vivo perfused human liver endothelium, which would imply perturbations in mic ...[more]

PMID: 29796416

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Project description:Background For 50 years, static cold storage (SCS) has been the gold standard for solid organ preservation in transplantation. Although logistically convenient, this preservation method presents important constraints in terms of duration and cold ischemia-induced lesions. We aimed to develop a machine perfusion (MP) protocol for recovery of vascularized composite allografts (VCA) after static cold preservation and determine its effects in a rat limb transplantation model.Methods Partial hindlimbs were procured from Lewis rats and subjected to SCS in Histidine-Tryptophan-Ketoglutarate solution for 0, 12, 18, 24, and 48 hours. They were then either transplanted (Txp), subjected to subnormothermic machine perfusion (SNMP) for 3 hours with a modified Steen solution, or to SNMP + Txp. Perfusion parameters were assessed for blood gas and electrolytes measurement, and flow rate and arterial pressures were monitored continuously. Histology was assessed at the end of perfusion. For select SCS durations, graft survival and clinical outcomes after transplantation were compared between groups at 21 days.Results Transplantation of limbs preserved for 0, 12, 18, and 24-hour SCS resulted in similar survival rates at postoperative day 21. Grafts cold-stored for 48 hours presented delayed graft failure (p = 0.0032). SNMP of limbs after 12-hour SCS recovered the vascular resistance, potassium, and lactate levels to values similar to limbs that were not subjected to SCS. However, 18-hour SCS grafts developed significant edema during SNMP recovery. Transplantation of grafts that had undergone a mixed preservation method (12-hour SCS + SNMP + Txp) resulted in better clinical outcomes based on skin clinical scores at day 21 post-transplantation when compared to the SCS + Txp group (p = 0.01613).Conclusion To date, VCA MP is still limited to animal models and no protocols are yet developed for graft recovery. Our study suggests that ex vivo SNMP could help increase the preservation duration and limit cold ischemia-induced injury in VCA transplantation.

Project description:High-quality human hepatocytes form the basis of drug safety and efficacy tests, cell-based therapies, and bridge-to-transplantation devices. Presently the only supply of cells derives from an inadequate pool of suboptimal disqualified donor livers. Here we evaluated whether machine perfusion could ameliorate ischemic injury that many of these livers experience prior to hepatocyte isolation.Non-heparinized female Lewis rat livers were exposed to an hour of warm ischemia (34°C) and then perfused for 3h. Five different perfusion conditions that utilized the cell isolation apparatus were investigated, namely: (1) modified Williams Medium E and (2) Lifor, both with active oxygenation (95%O(2)/5%CO(2)), as well as (3) Lifor passively oxygenated with ambient air (21%O(2)/0.04%CO(2)), all at ambient temperatures (20 ± 2°C). At hypothermic temperatures (5 ± 1°C) and under passive oxygenation were (4) University of Wisconsin solution (UW) and (5) Vasosol. Negative and positive control groups comprised livers that had ischemia (WI) and livers that did not (Fresh) prior to cell isolation, respectively.Fresh livers yielded 32 ± 9 million cells/g liver while an hour of ischemia reduced the cell yield to 1.6 ± 0.6 million cells/g liver. Oxygenated Williams Medium E and Lifor recovered yields of 39 ± 11 and 31 ± 2.3 million cells/g liver, respectively. The passively oxygenated groups produced 15 ± 7 (Lifor), 13 ± 7 (Vasosol), and 10 ± 6 (UW)million cells/g liver. Oxygenated Williams Medium E was most effective at sustaining pH values, avoiding the accumulation of lactate, minimizing edematous weight gain and producing bile during perfusion.Machine perfusion results in a dramatic increase in cell yields from livers that have had up to an hour of warm ischemia, but perfusate choice significantly impacts the extent of recovery. Oxygenated Williams Medium E at room temperature is superior to Lifor, UW and Vasosol, largely facilitated by its high oxygen content and low viscosity.

Dataset Information

Endothelial Dysfunction in Steatotic Human Donor Livers: A Pilot Study of the Underlying Mechanism During Subnormothermic Machine Perfusion.

Publications

Endothelial Dysfunction in Steatotic Human Donor Livers: A Pilot Study of the Underlying Mechanism During Subnormothermic Machine Perfusion.

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