Veerle Heedfeld

Multifactorial biological modulation of warm ischemia reperfusion injury in liver transplantation from non-heart-beating donors eliminates primary nonfunction and reduces bile salt toxicity

Objective:To design a multifactorial biological modulation approach targeting ischemia reperfusion injury to augment viability of porcine liver grafts from non–heart-beating donors (NHBD). Background Data:Liver Transplantation (LTx) from NHBD is associated with an increased risk of primary nonfunction (PNF) and biliary complications. In porcine NHBD-LTx, we previously reported a 50% risk of PNF and toxic bile formation in grafts exposed to ≥30′ warm ischemia (WI). Methods:Porcine livers exposed to 45′ WI were cold stored, transplanted and either modulated (n = 6) or not (controls, n = 9). In the modulation group, donor livers were flushed with warm Ringers (avoiding cold-induced vasoconstriction), streptokinase (eliminating stagnating thrombi), and epoprostenol (vasodilator, platelet aggregation inhibitor) prior to cold storage. In recipients, glycine (Kupffer cell stabilizer), α1-acid-glycoprotein (anti-inflammatory protein), MAPKinase-inhibitor (pro-inflammatory cytokine generation inhibitor), α-tocopherol and glutathione (anti-oxidants), and apotransferrin (iron chelator) were administrated intravenously. PNF, survival, lactate, transaminase, TNF-α, redox-active iron, and biliary bile salt-to-phospholipid ratio were monitored. Results:No PNF was observed in modulated versus 55% in control pigs (P = 0.025). Survival was 83% in modulated versus 22% in control pigs (P = 0.02). At 180′ postreperfusion, lactate was lower in modulated (5.4 ± 1.9 mmol/L) versus control pigs (9.4 ± 2.2 mmol/L; P = 0.011). At 60′ postreperfusion, there was a trend for lower AST in modulated versus control pigs at 60′ (939 ± 578 vs. 1683 ± 873 IU/L; P = 0.089). Postreperfusion, TNF-α remained stable in modulated pigs (49 ± 27 pg/mL at 15′ and 85 ± 26 pg/mL at 180′; P = 0.399) but increased in control pigs (107 ± 36pg/mL at 15′ and 499 ± 216 pg/mL at 180′; P = 0.023). At 180′ postreperfusion, redox-active iron was higher in control pigs versus modulated pigs (0.21±0.18 vs. 0.042±0.062 μm; P = 0.038). Biliary bile salt-to-phospholipid ratio post-LTx was lower in modulated versus control pigs (1128 ± 447 vs. 4836 ± 4619; P = 0.05). Conclusions:A multifactorial biological modulation eliminates PNF, improves liver function and increases survival. Biochemically, TNF-α and redox-active iron are suppressed and biliary bile salt toxicity is reduced. Translating this strategy clinically may lead to wider and safer use of NHBD.

The extent of vacuolation in non-heart-beating porcine donor liver grafts prior to transplantation predicts their viability

Livers exposed to prolonged warm ischemia (WI), such as those from non–heart‐beating donors (NHBDs), are at higher risk of primary graft nonfunction (PNF). In a pig model of liver transplantation (LTx) from NHBDs, hepatocellular vacuolation, focal hepatocyte dropout, congestion, and sinusoidal dilatation appeared on biopsies taken after exposure to WI. In functioning grafts, vacuolation and sinusoidal dilatation were reversible after LTx, in contrast to PNF grafts. We studied whether the extent of these morphological signs and particularly vacuolation, present on pre‐LTx biopsies, was associated with WI length and able to predict PNF, hepatocellular damage, and survival. Pre‐LTx biopsies from pig livers exposed to incremental periods of WI were reviewed retrospectively. The extent of vacuolation was quantified blindly by a pathologists semiquantitative score, validated by stereological point counting and digital image analysis, and then used to predict PNF and hepatocellular damage. On biopsies taken after WI, stereological point counting and digital analysis scoring contributed significantly in predicting PNF (P = 0.027 and P = 0.043, respectively) versus the pathologists semiquantitative score (P = 0.058). Stereological point counting and digital image analysis predicted the extent of hepatocellular damage (P < 0.0001 and P = 0.001) versus the pathologists semiquantitative score (P = 0.085). In conclusion, the extent of parenchymal vacuolation present on WI liver grafts reflects the severity of hepatocellular damage and predicts pig liver graft viability before LTx. Further studies are now warranted to evaluate whether these anoxic changes that are associated with liver graft viability in pigs also apply to human NHBD liver biopsies. Liver Transpl 14:1256–1265, 2008.

Hypothermic Machine Perfusion of the Liver: Is It More Complex than for the Kidney?

BACKGROUND Hypothermic machine perfusion (HMP) is superior to simple cold storage (SCS) for the preservation of kidney grafts. Whether HMP is superior to SCS for liver preservation is not known. Before a HMP system can be used clinically for the liver, its superiority to SCS needs to be demonstrated in an in vivo large animal transplant model. OBJECTIVE The aim was to compare outcomes after liver transplantation (LT) following preservation by SCS or HMP using technology/perfusion conditions similar to those for kidney HMP. METHODS Pig livers were perfused via the hepatic artery and portal vein for 4 hours with nonoxygenated 4°C University of Wisconsin machine perfusion solution. In the SCS group, flushed livers were stored in histidine-tryptophan-ketoglutarate solution. After preservation by SCS (n = 6) or HMP (n = 8) and LT, we assessed graft and recipient survivals, pH and lactate, hepatocellular damage [aspartate aminotransferase (AST)], Kupffer cell activation (β-galactosidase), tumor necrosis factor (TNF) α production, endothelial cell function (hyaluronic acid), and expression of Krüppel-like factor (KLF) 2 and 4, which are mediators of the flow-dependent vasoprotective endothelial phenotype. RESULTS No primary graft nonfunction was observed; livers recovered equally well from the postanhepatic metabolic acidosis in both groups. Pig survival was 5/6 (83%) in the SCS versus 2/8 (12.5%) in the HMP group (P = .04). Livers from both groups recovered equally well from the postanhepatic metabolic acidosis. AST in liver rinse-out samples obtained before LT were lower in the HMP than in the SCS group (P < .05). After reperfusion, AST and β-galactosidase were equally increased in both groups (P = .13 and 0.962, respectively); TNF-α and hyaluronic acid levels were higher after HMP versus SCS (P = .001 and 0.043, respectively). KLF-2 and -4 expressions were equally up-regulated after reperfusion in the SCS and HMP groups. CONCLUSIONS In this in vivo model, liver HMP with subsequent transplantation was feasible. However, we did not demonstrate an advantage of HMP, using perfusion conditions shown to be effective for the kidney, over SCS. Despite similar immediate graft function, TNF-α generation, and endothelial cell dysfunction were more pronounced after HMP.

Improving the function of liver grafts exposed to warm ischemia by the leuven drug protocol: Exploring the molecular basis by microarray

Livers exposed to warm ischemia (WI) before transplantation are at risk for primary nonfunction (PNF), graft dysfunction, and ischemic biliary strictures, all associated with ischemia/reperfusion injury (IRI). Our multifactorial approach, Leuven drug protocol (LDP), has been shown to reduce these effects and increase recipient survival in WI/IRI‐damaged porcine liver transplantation. The aim was the identification of the molecular mechanisms responsible for the hepatoprotective effects of the LDP. Porcine livers were exposed to 45 minutes of WI, cold‐stored for 4 hours, transplanted, and either modulated (LDP group; n = 3) or not modulated (control group; n = 4). In the LDP group, the donor livers were flushed with streptokinase and epoprostenol before cold perfusion; the recipients received intravenous glycine, a‐1‐acid‐glycoprotein, FR167653 (a mitogen‐activated protein kinase inhibitor), a‐tocopherol, glutathione, and apotransferrin. Liver samples were taken before WI and 1 hour after reperfusion. Gene expression was determined with microarrays and molecular pathways and key regulatory genes were identified. The number of genes changed between baseline and 1 hour after reperfusion was 686 in the LDP group and 325 in the control group. The extra genes in the LDP group belonged predominantly to pathways related to cytokine activity, apoptosis, and cell proliferation. We identified 7 genes that were suppressed in the LDP group. These genes could be linked in part to the administered drugs. New potential drug targets were identified on the basis of genes induced in the control group but unaffected in the LDP group and interactions predicted by the literature. In conclusion, the LDP primarily resulted in the suppression of inflammation‐regulating genes in IRI. Furthermore, the microarray technique helped us to identify additional gene targets. Liver Transpl 18:206–218, 2012.

The release of microRNA-122 during liver preservation is associated with early allograft dysfunction and graft survival after transplantation

Early allograft dysfunction (EAD) after liver transplantation (LT) is associated with inferior graft survival. EAD is more prevalent in grafts from donation after circulatory death (DCD). However, accurate prediction of liver function remains difficult because of the lack of specific biomarkers. Recent experimental and clinical studies highlight the potential of hepatocyte‐derived microRNAs (miRNAs) as sensitive, stable, and specific biomarkers of liver injury. The aim of this study was to determine whether miRNAs in graft preservation fluid are predictive for EAD after clinical LT and in an experimental DCD model. Graft preservation solutions of 83 liver grafts at the end of cold ischemia were analyzed for miRNAs by reverse transcription polymerase chain reaction. Of these grafts, 42% developed EAD after transplantation. Results were verified in pig livers (n = 36) exposed to different lengths of warm ischemia time (WIT). The absolute miR‐122 levels and miR‐122/miR‐222 ratios in preservation fluids were significantly higher in DCD grafts (P = 0.001) and grafts developing EAD (P = 0.004). In concordance, the miR‐122/miR‐222 ratios in perfusion fluid correlate with serum transaminase levels within the first 24 hours after transplantation. Longterm graft survival was significantly diminished in grafts with high miR‐122/miR‐222 ratios (P = 0.02). In the porcine DCD model, increased WIT lead to higher absolute miR‐122 levels and relative miR‐122/miR‐222 ratios in graft perfusion fluid (P = 0.01 and P = 0.02, respectively). High miR‐122/miR‐222 ratios in pig livers were also associated with high aspartate aminotransferase levels after warm oxygenated reperfusion. In conclusion, both absolute and relative miR‐122 levels in graft preservation solution are associated with DCD, EAD, and early graft loss after LT. As shown in a porcine DCD model, miRNA release correlated with the length of WITs. Liver Transplantation 23 946–956 2017 AASLD.

Liver transplantation from non-heart-beating donors: current status and future prospects in an experimental model.

Abstract The widening gap between supply and demand for liver transplantation has prompted many transplant centers to use donors after cardiac death or non-heart-beating donors. These livers - contrary to “classical” brain-dead donors - are exposed to an unavoidable period of warm ischemia, jeopardizing graft function post-transplantation. In a newly developed preclinical model of liver transplantation, we studied - in a biologically unmodified environment - the exact tolerance of the liver to warm ischemia. Following the evidence that liver transplantation from non-heart-beating donors is feasible and safe, provided that warm and cold ischemia are kept short, a clinical programme of liver transplantation from non-heart-beating donors was successfully initiated in our and other Belgian centers. Recently, we demonstrated that the tolerance of such livers to warm ischemia could be substantially improved when some of the previously identified mechanisms leading to graft non-function were tackled by a multi-factorial pharmacological strategy. Meanwhile, cold storage has proven to be insufficient to optimally preserve organs from non-heart-beating donors. As an alternative, machine perfusion preservation was found to consistently improve outcome in kidney transplantation from non-heart-beating donors. Similarly, machine perfusion preservation could improve the preservation of livers, allowing to predict viability prior to transplantation and to ameliorate tolerance to warm ischemia. At present, the definition and development of optimal machine perfusion settings are under investigation at our institution.

Differential gene expression profile of porcine livers subjected to warm ischemia alone.

BACKGROUND Livers exposed to warm ischemia (WI) are increasingly used for transplantation. The molecular mechanisms activated by WI alone (prior to procurement and transplantation) are not understood. To elucidate the pathways involved, we used microarrays to investigate the gene expression in porcine livers exposed to WI. METHODS Porcine livers (n = 6 group) were randomly subjected to WI periods of 15, 30 or 45 minutes. mRNA was extracted and gene expression determined by microarray analysis. Using bioinformatics software, we identified differentially expressed genes and related molecular pathways. We used the corresponding human annotation of the porcine microarray for the functional analysis. RESULTS Between 0 and 15 minutes of WI, 3530 genes were altered with a 2-log-fold change of <-0.58 or >+0.58 and P < .05. Between 0 and 30 minutes of WI, 4141 genes were differentially expressed; and between 0 and 45 minutes of WI, 2814 genes. At each time point, ∼50% of genes were up-regulated, whereas 50% were down-regulated. After pathway mapping, we found that the same pathways were induced for observed clustering of in the three WI periods: cell death, proliferation, inflammation, and metabolism pathways. Among the top genes that were up-regulated after 15 minutes of WI, the majority started to return to but did not reach baseline expression with increasing WI. A similar pattern was observed for the top suppressed genes. CONCLUSIONS WI causes rapid changes in gene expression that affect several molecular pathways. This phenomenon seems to plateau at 15 to 30 minutes of WI. These new insights in the timing and the nature of molecular pathways induced by WI alone may help to design specific interventions to alter these changes and improve the outcome of livers from cardiac death donors.

The Synthesis Of Coagulation Factors During Normothermic Machine Perfusion Of Livers Is Impaired By Ischemia In Pigs And Might Predict Graft Viability

Normothermic Machine Perfusion (NMP) of livers is a promising tool to test graft viability during preservation. Coagulation factors are synthetized by the liver, hence are candidate markers of function. We evaluated the production of coagulation factors during NMP, tested if ischemia alters their synthesis, and correlated their levels with markers of hepatocyte damage (AST) and sinusoidal endothelial injury {Hyaluronic acid (HAc)}. Method Porcine livers exposed to 60 min Warm Ischemia (WI60, n=5) or not (WI0, n=5) were NMP perfused for 6 h. The concentration of Factors V (FV), VIII (FVIII), and X (FX) in the perfusate was measured at 15, 30min, 1, 2, 3, and 6h of NMP and compared within and between groups (between-within ANOVA). Levels of AST and HAc were correlated to FV and FVIII. Mean ±SD is given. Results FV increased significantly over time in both groups, although with a different kinetics, as a plateau was reached at 2h of NMP in WI0 only. FV concentration was inferior in WI60 vs. WI0 (p<0.0001) (Figure 1A). Similarly, FVIII raised over time significantly in both WI0 and WI60, reaching a plateau at 3h of NMP only in WI0. The production of FVIII was inferior in WI60 vs. WI0 (p=0.006) (Figure 1B). FX raised steadily without a plateau in WI0, while no significant increase was observed in WI60, in which the concentration of FX was inferior (p=0.001) (Figure 1C). The area under the curve of AST was greater in WI60 (3077) vs WI0 (405.3, p=0.0006). FV was inversely correlated to AST release at all time points except 6h. HAc levels increased over time [11.3 ±5.3 ng/mL at 15min to 101.8 ±75.5 ng/mL at 6h in WI0 (p=0.045), and from 51.5 ±6.9 ng/mL at 15min to 303.2 ±107.7 ng/mL at 6h in WI60 (p=0.01)] and were higher in WI60 vs WI0 (p=0.004). FVIII was inversely correlated to HAc levels at 1h (r:-0.58, p=0.02) and 2h (r:-0.59, p=0.03) of NMP. Discussion Coagulation factors are actively produced during NMP and their synthesis was impaired during NMP of severely WI damaged porcine livers. In particular, FV and FVIII, produced by hepatocytes and sinusoidal cells, respectively, can be used as markers of function of the hepatocellular and endothelial compartment, since they were inversely related to markers of hepatocellular injury (AST) and sinusoidal cell damage (HAc). FX synthesis during NMP should be further investigated, since its production is dependent on vitamin K, which was not supplemented in the perfusion system. The correlation of our findings to post-transplant outcomes warrants further investigations. Conclusion The production of coagulation factors during NMP of human livers and their role in the assessment of viability deserve to be explored. Figure. No caption available.

Hepatocellular uptake of cyclodextrin-complexed curcumin during liver preservation: A feasibility study

The increasing demand for donor organs and the decreasing organ quality is prompting research toward new methods to reduce ischemia reperfusion injury (IRI). Several strategies have been proposed to protect preserved organs from this injury. Before curcumin/dextrin complex (CDC), a potent antioxidant and anti‐inflammatory agent, can be used clinically we need to better understand the intracellular uptake under hypothermic conditions on a rat model of liver donation after circulatory death (DCD) and brain death (DBD). To be able to use the fluorescence of CDC for quantification the stability of CDC in different preservation solutions at 4°C or 37°C was investigated. Livers from Wistar rats were procured after being flushed‐out through the portal vein using CDC‐enriched preservation solutions and stored at 4°C for variable periods. The CDC signal was stable in different preservation solutions over a period of 4 h and allowed the rapid and lasting uptake of curcumin into cells. After 4 h of preservation, CDC was no longer visible microscopically, and HPLC analysis showed very low to non‐detectable tissue levels of CDC, proving metabolization during preservation. However, the distribution of CDC was not affected by warm ischemia damage (p = 0.278) nor by flushing the livers before or after 4 h of cold storage and without a warm preflush. Finally, curcumin reduced oxidative stress, lowered histological injury and did not change gene expression after WI/cold storage. Therefore, the use of CDC flush solution for the initial organ flush can offer a promising approach to the enhancement of liver preservation and the maintenance of its quality.