Katrien Vekemans

Rapid clearance of the circulating metastatic factor autotaxin by the scavenger receptors of liver sinusoidal endothelial cells

Autotaxin, also known as NPP2 (nucleotide pyrophosphatase/phosphodiesterase 2), is a secreted lysophospholipase-D that generates lysophosphatidic acid and thereby promotes the metastatic and invasive properties of tumor cell as well as angiogenesis. We show here that, in mice, NPP2 is cleared from the circulation within minutes and is retained by the liver sinusoidal endothelial cells (LSECs). The binding of NPP2 to isolated LSECs resulted in its degradation and could be competed for with ligands of the scavenger receptor family. Our finding that circulating NPP2 has a rapid turnover has important implications for its development as an anti-cancer target.

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.

Preserving the morphology and evaluating the quality of liver grafts by hypothermic machine perfusion: a proof-of-concept study using discarded human livers.

The wider use of livers from expanded criteria donors and donation after circulatory death donors may help to improve access to liver transplantation. A prerequisite for safely using these higher risk livers is the development of objective criteria for assessing their condition before transplantation. Compared to simple cold storage, hypothermic machine perfusion (HMP) provides a unique window for evaluating liver grafts between procurement and transplantation. In this proof‐of‐concept study, we tested basic parameters during HMP that may reflect the condition of human liver grafts, and we assessed their morphology after prolonged HMP. Seventeen discarded human livers were machine‐perfused. Eleven livers were nontransplantable (major absolute contraindications and severe macrovesicular steatosis in the majority of the cases). Six livers were found in retrospect to be transplantable but could not be allocated and served as controls. Metabolic parameters (pH, lactate, partial pressure of oxygen, and partial pressure of carbon dioxide), enzyme release in the perfusate [aspartate aminotransferase (AST) and lactate dehydrogenase (LDH)], and arterial/portal resistances were monitored during HMP. Nontransplantable livers released more AST and LDH than transplantable livers. In contrast, arterial/portal vascular resistances and metabolic profiles did not differ between the 2 groups. Morphologically, transplantable livers remained well preserved after 24 hours of HMP. In conclusion, HMP preserves the morphology of human livers for prolonged periods. A biochemical analysis of the perfusate provides information reflecting the extent of the injury endured. Liver Transpl, 2012.

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.

Artificial circulation of the liver: machine perfusion as a preservation method in liver transplantation.

Due to the sharp increase in liver transplant candidates and the subsequent shortage of suitable donor livers, an extension of the current donor criteria is necessary. Simple cold storage, the current standard in organ preservation has proven to be insufficient to preserve extended criteria donor livers. Therefore a renewed interest grew toward alternative methods for liver preservation, such as hypothermic machine perfusion and normothermic machine perfusion. These “new” preservation methods were primarily assessed in rat models, and only a few clinically relevant large animal models have been described so far. This review will elaborate on these alternative preservation methods. Anat Rec, 291:735–740, 2008.

TGF-β1-induced cardiac myofibroblasts are nonproliferating functional cells carrying DNA damages

TGF-beta1 induces differentiation and total inhibition of cardiac MyoFb cell division and DNA synthesis. These effects of TGF-beta1 are irreversible. Inhibition of MyoFb proliferation is accompanied with the expression of Smad1, Mad1, p15Ink4B and total inhibition of telomerase activity. Surprisingly, TGF-beta1-activated MyoFbs are growth-arrested not only at G1-phase but also at S-phase of the cell cycle. Staining with TUNEL indicates that these cells carry DNA damages. However, the absolute majority of MyoFbs are non-apoptotic cells as established with two apoptosis-specific methods, flow cytometry and caspase-dependent cleavage of cytokeratin 18. Expression in MyoFbs of proliferative cell nuclear antigen even in the absence of serum confirms that these MyoFbs perform repair of DNA damages. These results suggest that TGF-beta1-activated MyoFbs can be growth-arrested by two checkpoints, the G1/S checkpoint, which prevents cells from entering S-phase and the intra-S checkpoint, which is activated by encountering DNA damage during the S phase or by unrepaired damage that escapes the G1/S checkpoint. Despite carrying of the DNA damages TGF-beta1-activated MyoFbs are highly functional cells producing lysyl oxidase and contracting the collagen matrix.

Attempt to rescue discarded human liver grafts by end ischemic hypothermic oxygenated machine perfusion.

In a porcine liver transplant model, a brief period of oxygenated hypothermic machine perfusion (HMP) at the end of simple cold storage (SCS) has been shown to improve the viability of damaged liver grafts. To test the clinical validity of this strategy, we randomized SCS-discarded human liver grafts to either 4 hours of HMP (n = 13) or an additional 4 hours of SCS (n = 14). All livers were then warm reperfused to mimic ischemia-reperfusion injury ex vivo. The settings for HMP were: portal vein: 3 mm Hg, 300 mL/min and hepatic artery: 20 mm Hg, pO(2): 300 mm Hg. Perfusion used Kidney Machine Perfusion Solution at 4°C to 8°C. During warm reperfusion, aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) values were higher (P < .015) among the SCS versus HMP methods at all times. The AST slope was lower in HMP versus SCS (P = .01). The LDH slope tended to be lower for HMP versus SCS (P = .07). Morphological scores were not different between HMP and SCS. At the start of warm reperfusion, MAPK was lower in HMP versus SCS (P = .02). Endothelin-1 (EDN1) and ICAM-1 tended to be lower in HMP versus SCS (P = .1 and .07, respectively). No difference was noted in MAPK, EDN1, and ICAM-1 after 60 or 120 minutes of warm reperfusion. In conclusion, HMP down-regulated MAPK and tended to reduce EDN1 and ICAM-1 mRNA in human liver grafts. During warm reperfusion, HMP versus SCS livers showed reduced AST and LDH release but no morphological difference. Further optimization of liver HMP may require different timing/duration of perfusion and/or an higher perfusion temperature.

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.

Hypothermic Liver Machine Perfusion With EKPS-1 Solution vs Aqix RS-I Solution: In Vivo Feasibility Study in a Pig Transplantation Model

OBJECTIVE Hypothermic machine perfusion (HMP) is superior to simple cold storage for kidney preservation. We previous observed in a porcine liver transplantation model increased tumor necrosis factor-alpha (TNF-alpha) production eventually leading to poor recipient survival after HMP using standard kidney perfusion solution (KPS-1) compared with simple cold storage. We compared two solutions for HMP preservation of the liver: enriched KPS-1 (EKPS-1) and Aqix RS-I. METHODS Pig livers were obtained after cold flushing with histidine-tryptophan-ketoglutarate solution. Subsequently, the livers were subjected to dual-vessel perfusion with two preservation solutions: EKPS-1 (n = 6) and Aqix RS-I (n = 3). After HMP preservation and transplantation, graft and recipient survival, hepatocellular damage (aspartate aminotransferase concentration), TNF-alpha production, and endothelial cell damage (hyaluronic acid clearance) were recorded. RESULTS No primary graft nonfunction was observed. Recipient survival at postoperative day 3 was similar in both groups (33%). Aspartate aminotransferase concentration measured in serum samples after reperfusion was similar in both groups. After reperfusion, TNF-alpha concentration was higher and hyaluronic acid clearance was lower in the EKPS-1 group vs the Aqix RS-I group at 60, 120, and 180 minutes (all P < .05). CONCLUSION Hypothermic machine perfusion provided adequate longer term graft survival. After reperfusion, TNF-alpha production seems to be reduced, and endothelial cell dysfunction remains pronounced with Aqix RS-1 solution compared with EKPS-1 solution.

Potentiation of Adverse Effects of Cold by Warm Ischemia in Circulatory Death Donors for Porcine Liver Transplantation

BACKGROUND Wider use of donors after circulatory death (DCD) could reduce mortality on the liver transplantation waiting list. We previously reported that pig livers exposed to ≥ 30 minutes of warm ischemia followed by 4 hours of cold ischemia are at high risk of primary graft nonfunction. We sought to determine how prolonged cold ischemia, after a short, normally well-tolerated period of warm ischemia affects graft function and recipient survival using a porcine model of liver transplantation. MATERIALS AND METHODS Livers were transplanted after exposure to no warm plus 4 hours cold ischemia (group 1); 15 minutes of warm and 4 hours of cold ischemia (group 2); no warm and 8 hours of cold ischemia (group 3); or 15 minutes of warm and 8 hours of cold ischemia (group 4). Recipient survival, graft dysfunction incidence, liver function (prothrombin time), hepatocellular damage (aspartate aminotransferase), sinusoidal cell function (hyaluronic acid), and inflammation (tumor necrosis factor-α) were recorded after transplantation. Biopsies were scored for ischemia-reperfusion injury. RESULTS Day 4 survival in group 4 was 0% versus 100%, 83%, and 100% in groups 1, 2, and 3, respectively. Recipients in group 4 exposed to short warm but prolonged cold ischemia displayed severe graft dysfunction, the highest peak transaminase, the greatest inflammatory response, more sinusoidal endothelial cell dysfunction and, the worst histologic score for ischemia-reperfusion injury. CONCLUSIONS Liver grafts from DCD donors, even when exposed to short periods of warm ischemia, did not tolerate prolonged cold ischemia well and should be transplanted without delay.