Assessing warm ischemic injury of pig livers at hypothermic machine perfusion.

BACKGROUND: Livers originating from donation after circulatory death (DCD) donors are exposed to warm ischemia (WI) before liver transplantation (LTx). Currently, there are no objective tests to evaluate the damage sustained before LTx. This study aims to identify surrogate markers for liver injury that can be assessed during hypothermic machine perfusion (HMP) preservation. In addition, we want to use mathematical equation modeling combining these markers to improve our assessment of DCD livers for transplantation. MATERIALS AND METHODS: Porcine livers were exposed to incremental periods of WI (0-120 min) and subsequently HMP preserved for 4 h. Biochemical and hemodynamic parameters were repeatedly measured in the perfusate during HMP. Subsequently, to mimic LTx, normothermic isolated-liver perfusion was applied for 2 h and the injury assessed using a morphological score. RESULTS: With increasing WI periods, the perfusate became more acidotic, and levels of aspartate aminotransferase (AST), liver fatty acid binding protein, redox-active iron, and arterial vascular resistance increased. A damage index, combining AST and pH (damage index = 2 - 37 × ß(AST) - 257 × ß(pH)) based on multifactorial analysis of the changing pattern of these markers, had increased sensitivity and specificity to reflect WI and reperfusion injury. CONCLUSIONS: This proof of concept study demonstrated the potential role for objective evaluation of DCD porcine livers during HMP and the advantage to use multifactorial analysis on the markers' changing pattern.

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.

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.

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 > or =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), alpha1-acid-glycoprotein (anti-inflammatory protein), MAPKinase-inhibitor (pro-inflammatory cytokine generation inhibitor), alpha-tocopherol and glutathione (anti-oxidants), and apotransferrin (iron chelator) were administrated intravenously. PNF, survival, lactate, transaminase, TNF-alpha, 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-alpha 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 +/- 36 pg/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 mum; 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-alpha 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 pathologist's 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 pathologist's 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 pathologist's 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.

Structural and functional aspects of the liver and liver sinusoidal cells in relation to colon carcinoma metastasis.

Nowadays, liver metastasis remains difficult to cure. When tumor cells escape and arrive in the liver sinusoids, they encounter the local defense mechanism specific to the liver. The sinusoidal cells have been widely described in physiologic conditions and in relation to metastasis during the past 30 years. This paper provides an "overview" of how these cells function in health and in diseases such as liver metastasis.

Immuno-localization of Fas and FasL in rat hepatic endothelial cells: influence of different fixation protocols.

Immunocytochemistry has been widely used to localize molecules involved in apoptosis. In this short report, we describe with the aid of confocal laser scanning microscopy the immunolocalization of Fas and FasL on liver sinusoidal endothelial cells, and show how the localization of these two molecules differ when the cells are fixed with different fixation protocols. Methanol fixation shows diffuse staining of Fas and FasL in the cytoplasm, as well as in the nucleus. In contrast, paraformaldehyde fixation reveals the presence of Fas and FasL polarized at one side of the cell and only in the cytoplasm. After fixation with a combination of paraformaldehyde and glutaraldehyde the polarization is still present although the fluorescence is concentrated and located as bright dots in the cytoplasm. In conclusion, paraformaldehyde preserves the (nuclear) membrane-associated structures better then methanol and results in a more accurate localization of Fas and FasL. Understanding the different outcome of these common used fixation protocols will assist investigators to select the most suitable method for visualizing membrane-bound Fas and FasL.

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.

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.

TGF-beta1-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.

Primary graft nonfunction and Kupffer cell activation after liver transplantation from non-heart-beating donors in pigs.

More extensive use of non-heart-beating donors (NHBD) could reduce mortality on liver transplantation waiting lists, but this is associated with more primary nonfunction (PNF). We assessed which parameters are involved in the development of PNF in livers from NHBD in a previously validated pig liver transplantation model, in which livers were transplanted after exposure to incremental periods of warm ischemia. The risk of PNF was unacceptably high (>50%) when livers were exposed to >30 minutes' warm ischemia before a short cold ischemic period. This study examined how PNF is affected by Kupffer cell activation (beta-galactosidase), the generation of cytokines tumor necrosis factor alpha and interleukin 6, antioxidant mechanisms (ascorbic acid, alpha-tocopherol, reduced glutathione), circulating redox-active iron, and sinusoidal endothelial cell function (hyaluronic acid clearance). Kupffer cells were more activated in PNF recipients, as suggested by higher beta-galactosidase levels (15 minutes after reperfusion), and secondarily, by higher production of tumor necrosis factor alpha and interleukin 6 (180 minutes after reperfusion). In addition, alpha-tocopherol and reduced glutathione were lower, and ascorbic acid and redox-active iron higher in PNF recipients. Finally, PNF grafts displayed progressively decreasing hyaluronic acid clearance (suggesting sinusoidal endothelial cell dysfunction) and parenchymal edema. Consequently, a reduced-flow phenomenon was documented. In grafts from NHBD that are destined to fail, beta-galactosidase activity (a surrogate of Kupffer cell activation) is higher, proinflammatory cytokines are overproduced, some antioxidant mechanisms fail, and circulating redox-active iron is more rapidly released. A no-flow phenomenon is eventually observed in these failing grafts.

The visualization of hepatic vasculature by X-ray micro-computed tomography.

In this study, X-ray micro-computed tomography (CT) was used to reconstruct the fine structure macro- and microvasculature in three dimensions in contrast-enhanced rat liver samples. The subsequent application in the experimental CC531s colorectal cancer model was concurrent with results obtained from confocal microscopy in earlier studies. The en bloc stains osmium tetroxide in combination with uranyl acetate provided an excellent contrasting result for hepatic tissue after a trial of several contrasting agents. X-ray micro-CT allowed us to image the large blood vessels together with the branching sinusoids of hepatic tissue in three dimensions. Furthermore, interruption of the microvasculature was noted when rats were injected with CC531s colorectal cancer cells indicating the presence of hepatic metastases.

Nitric oxide from rat liver sinusoidal endothelial cells induces apoptosis in IFN gamma-sensitized CC531s colon carcinoma cells.

BACKGROUND/AIMS: Investigation of apoptosis is pivotal in searching for mechanisms that eliminate colon cancer cells getting trapped in liver sinusoids at the time of surgical removal of the primary tumor. This study focuses on nitric oxide (NO), Fas/FasL and the involvement of interferon-gamma (IFNgamma) in liver sinusoidal endothelial cells (LSECs) and in the colon carcinoma cell line CC531s. METHODS: Apoptosis was quantified and visualized in vitro by specific DNA fragmentation, specific staining and electron microscopy. In vivo experiments were also conducted. RESULTS: In co-cultures of LSECs with CC531s, apoptosis of CC531s was observed only when they were pre-treated with IFNgamma, and was unaffected by blocking the Fas/FasL pathway. However, LSECs continuously produced NO, and apoptosis was inhibited by NO-inhibitors (NMMA and dexamethasone). When IFNgamma-sensitized CC531s were injected into rats, liver weight was lower, in contrast to control conditions where liver weight was higher. CONCLUSIONS: (i) LSECs induce apoptosis in IFNgamma-sensitized CC531s in vitro; (ii) LSECs express FasL; (iii) Fas on CC531s becomes active after IFNgamma-treatment; however, (iv) blocking the Fas/FasL pathway had no effect; (v) apoptosis was inhibited by NO-inhibitors; (vi) the immune system uses this IFNgamma-activated pathway to support LSECs in killing tumor cells.

CC531s colon carcinoma cells induce apoptosis in rat hepatic endothelial cells by the Fas/FasL-mediated pathway.

The mechanisms involved in colorectal carcinoma with liver metastasis are not well known. Metastasizing colon carcinoma cells express more FasL than primary colon carcinoma cells and cancer cells induce apoptosis in hepatocytes by the Fas/FasL pathway. Therefore, this study focused on Fas/FasL expression and functionality in rat liver sinusoidal endothelial cells (LSECs) and CC531s colon carcinoma cells in vitro and in vivo. RT-PCR and immunochemistry revealed Fas and FasL in LSECs and CC531s, respectively. Functionality of Fas was assessed in vitro by incubation with human recombinant FasL (1-100 ng/ml) with or without enhancer. At concentrations of 10 and 100 ng/ml with enhancer, respectively 21% and 44% of endothelial cells showed signs of apoptosis using Hoechst 33342/propidium iodide staining and electron microscopy. In co-cultures, apoptosis could be detected in endothelial cells neighboring the CC531s and could be inhibited by an antagonistic FasL antibody. Moreover, 18 h after mesenteric injection of CC531s, the sinusoidal endothelium revealed disruption. In conclusion, (i). CC531s cells induce apoptosis in LSECs in vitro by using Fas/FasL; (ii). CC531s cells damage the sinusoidal endothelial lining in vivo; and (iii). this might provide FasL-positive tumor cells a gateway towards the hepatocytes.

Antimycin A-induced defenestration in rat hepatic sinusoidal endothelial cells.

Liver sinusoidal endothelial cells (LSECs) possess fenestrae arranged in sieve plates. Hepatic endothelial fenestrae are open pores approximately 100 to 200 nm in diameter. Alterations in their number or diameter by hormones, xenobiotics, and diseases have important implications for hepatic microcirculation and function. Numerous reports of hepatotoxin-induced defenestration suggest that the cytoskeleton and the energy status of hepatic endothelial cells play a key role in the regulation of fenestrae. Therefore, we investigated the effect of antimycin A, an inhibitor of mitochondrial energy production, on the number of fenestrae in cultured LSECs using high-resolution microscopy and immunocytochemistry. Prolonged incubation (greater than 30 min) with antimycin A resulted in defenestrated cells and coincided with the appearance of F-actin dots, whereas the distribution of G-actin remained unchanged. Adenosine triphosphate (ATP) was depleted dramatically to less than 5% within 30 minutes within the LSECs. After treatment with antimycin A, unusual elevated fenestrated complexes were apparent, organized as a meshwork of anastomosing fenestrae at the center of and above the sieve plates. The position and appearance of these novel structures and their association with defenestration suggest that they are implicated in the process of defenestration. In conclusion, the results of experiments with antimycin A suggest that ATP is needed to maintain fenestrae and the underlying fenestrae-associated cytoskeleton rings that maintain fenestrae patency. Antimycin A-induced defenestration of LSECs is associated with the development of a structure in the sieve plate that appears to be intrinsically involved in defenestration.

Interactions between rat colon carcinoma cells and Kupffer cells during the onset of hepatic metastasis.

Liver sinusoids harbor populations of 2 important types of immunocompetent cells, Kupffer cells (KCs) and natural killer (NK) cells, which are thought to play an important role in controlling hepatic metastasis in the first 24 hr upon arrival of the tumor cells in the liver. We studied the early interaction of KCs, NK and CC531s colon carcinoma cells in a syngeneic rat model by confocal laser scanning microscopy. Results showed a minority of KCs (19% periportal and 7% pericentral) involved in the interaction with 94% of tumor cells and effecting the phagocytosis of 92% of them. NK cell depletion decreased the phagocytosis of tumor cells by KCs by 33% over a period of 24 hr, leaving 35% of the cancer cells free, as compared to 6% in NK-positive rats. Surviving cancer cells were primarily located close to the Glisson capsule, suggesting that metastasis would initiate from this region.