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. 2018 Apr 23;4(5):e345.
doi: 10.1097/TXD.0000000000000779. eCollection 2018 May.

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

Irene Beijert  1   2 , Safak Mert  1 , Viola Huang  1 , Negin Karimian  1 , Sharon Geerts  1 , Ehab O A Hafiz  1   3 , James F Markmann  4 , Heidi Yeh  4 , Robert J Porte  2 , Korkut Uygun  1
Affiliations

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

Irene Beijert et al. Transplant Direct. .

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.

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Conflict of interest statement

K.U. is inventor on pending patents relevant to this study (WO/2011/002926; WO/2011/35223) and has a provisional patent application relevant to this study (Massachusetts General Hospital 22743). K.U. has a financial interest in Organ Solutions, a company focused on developing organ preservation technology. K.U.'s interests are managed by the Massachusetts General Hospital and Partners HealthCare in accordance with their conflict of interest policies.

Figures

FIGURE 1
FIGURE 1
Schematic representation of perfusion system. The perfusion system incorporated 2 independent circulations: an arterial and a portal, each with their own pump, oxygenator using carbogen gas (95% O2, 5% CO2), bubble trap, pressure, and flow meter.
FIGURE 2
FIGURE 2
Representative images of lean (A) and macrosteatotic (B) livers at 20× objective magnification. Liver tissue samples were collected at the beginning and the end of perfusion for hemotoxylin and eosin staining scoring for macrosteatosis. Representative images are obtained from 1 lean (panel A) and 1 macrosteatotic (panel B) liver showing postperfusion state. Large fat droplets are prevailing in panel B (black arrow), whereas sinusoids can be barely visible (white arrow). No significant fibrosis has been detected in any of the samples.
FIGURE 3
FIGURE 3
Resistance in the PV (A) and the HA (B) calculated as quotient of pressure and flow. Hepatic artery and PV resistance decreased gradually throughout perfusion in both nonsteatotic and steatotic livers. However, no statistically significant differences were observed between the 2 groups (data are presented as median ± IQR).
FIGURE 4
FIGURE 4
Gene expression analyses. SNMP induces upregulation of KLF2 in both steatotic and nonsteatotic livers although significantly more in the nonsteatotic livers (A). eNOS is not significantly more expressed at the end of SNMP in both steatotic and nonsteatotic livers (B). Thrombomodulin is not significantly more expressed at the end of SNMP in both steatotic and nonsteatotic livers (C). Fold change is calculated from T=0 for each liver (data are presented as median ± IQR).
FIGURE 5
FIGURE 5
NO measurements. Absolute NO concentration increased in the nonsteatotic livers throughout SNMP but did not change in steatotic livers (A). Relative NO production calculated as fold change from T = 0 for each liver (B). Relative NO production fold change increased in the nonsteatotic livers but did not change in steatotic livers (data are presented as median ± IQR).
FIGURE 6
FIGURE 6
Normalized levels of eNOS phosphorylation. Levels of eNOS phosphorylation are significantly higher in non-steatotic livers compared with steatotic livers (data are presented as median ± IQR).
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