Effects of Exogenous Endothelin-1 Application on Liver Perfusion in Native and Transplanted Porcine Livers

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Effects of Exogenous Endothelin-1 Application on Liver Perfusionin Native and Transplanted Porcine Livers1

Thomas Kraus,*,2 Arianeb Mehrabi,* Markus Golling,* Fabian Schaffer,† Octavian Bud,*Martha-Maria Gebhard,† Christian Herfarth,* and Ernst Klar*

*Department of Surgery and †Department of Experimental Surgery, University of Heidelberg, Heidelberg, Germany

Journal of Surgical Research 93, 272–281 (2000)doi:10.1006/jsre.2000.5972, available online at http://www.idealibrary.com on

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Purpose. This study was designed to assess and dif-ferentiate the impact of progressivly increasing portalvenous endothelin-1 (ET) plasma concentrations onhepatic micro- and macroperfusion of native porcinelivers (Group A) and liver grafts after experimentaltransplantation (Group B).

Methods. A standardized gradual increment in sys-temic ET plasma concentration (0–58 pg/ml) was in-duced by continuous ET-1 infusion into the portal veinin both groups (A: n 5 10, B: n 5 10). Control animalseceived only saline (n 5 5, each group). Hepatic mi-rocirculation (HMC) was quantified by thermodiffu-ion electrodes, hepatic artery flow (HAF), and portalenous flow (PVF) by Doppler flowmetry.Results. No changes in ET or perfusion parametersere observed in controls. The mean ET level afterrthotopic liver transplantation (OLT) in Group B waslevated (baseline: 3.8 6 2.4 pg/ml) compared withroup A (2.8 6 1.9 pg/ml). With rising ET levels HAFecreased progressively in Group A from 205 6 97baseline) to 160 6 72 ml/min, and in Group B from61 6 87 to 146 6 68 ml/min. PVF decreased in Group Arom 722 6 253 to 370 6 198 ml/min, and in Group Brom 846 6 263 to 417 6 203 ml/min. Baseline HMC inroup A was 86 6 15 and decreased significantly to9 6 9 ml/100 g/min, and baseline MC in Group B was0 6 22 and decreased to 44 6 32 ml/100 g/min. Noignificant alteration in systemic circulation wasoted at the ET concentrations investigated.Conclusions. Significant impairment of hepaticicro- and macrocirculation was detected after in-

uction of systemic ET levels above 9.4 pg/ml both inative and in transplanted livers. Disturbance of

1 This study was supported by the Forschungsschwerpunkt Trans-plantation, University of Heidelberg.

2 To whom correspondence should be addressed at Chir. Univ.Klinik Heidelberg, INF 110, 69120 Heidelberg, Germany. Fax:(0)6221-411666. E-mail: Thomas_Kraus©ukl.uni-heidelberg.de.

2720022-4804/00 $35.00Copyright © 2000 by Academic PressAll rights of reproduction in any form reserved.

ion April 11, 2000

MC was caused predominantly by reduction ofortal venous flow, while the effect of ET on HAFas less pronounced. Characteristics of flow impair-ent in transplanted and native livers were analo-

ous after short cold ischemic graft storage (6 h).© 2000 Academic Press

Key Words: endothelin; liver perfusion; hepatic bloodflow; microcirculation; porcine liver transplantation;thermodiffusion.

INTRODUCTION

Patients with different forms of chronic and acuteliver disease often show increased systemicendothelin-1 (ET) concentrations compared withhealthy subjects [1–5]. The liver appears to be both asource of ET generation and a major target for ETimpact, particularly during hepatic injury states [6, 7].A variable degree of hepatic injury is regularly encoun-tered in the early postoperative course after orthotopicliver transplantation (OLT) due to postischemic reper-fusion [8]. Correspondingly, elevated systemic ET lev-els have been detected after OLT [9–13]. ET elicits awide range of vascular and metabolic effects in theliver [15–17]. The known spectrum of ET effects and itssecretory stimuli render very attractive the assump-tion that increased ET generation is involved in themediation of postischemic graft damage [7]. Differentmechanisms could contribute to ET-mediated hepaticinjury [7].

Most research has so far concentrated on the enor-mous vasoconstrictive potential of ET peptides in var-ious tissues. Studies that focus specifically on the im-pact of ET peptides on liver blood flow are very scarce
and based mostly on intravital microscopy in smallanimal models. Existing data allow us to conclude thatET induces contraction of portal venous vasculature

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and possibly even of activated sinusoidal pericytes[18–26]. Postischemic hepatic reperfusion after OLTmay activate hepatic endothelial cells and sinusoidalpericytes. This could render elevated ET secretion afterOLT particularly pathogenic [25, 26]. While the rele-vance of minor portal venous ET concentration changeson hepatic perfusion regulation is not fully understood,we know that high ET levels cause hepatic necrosis dueto severe vasospasms [27].

This is the first investigation to study the effect of ETon liver micro- and macroperfusion in a large animalmodel. We tested the hypothesis that elevation of por-tal venous ET concentration leads to impairment ofhepatic micro- and macrocirculation even at very lowsystemic levels, not yet associated with a depression ofsystemic circulation. As it has been claimed that ETeffects on hepatic perfusion can be markedly influencedby injury-derived cell activation, immunological fac-tors, and neural responses (all are characteristic fea-tures of liver transplantion), flow data were obtainedduring standardized exogenous ET stimulation both innative livers and in liver grafts early after experimen-tal OLT. For evaluation of hepatic parenchymal micro-circulation during exogenous ET application enhancedthermal diffusion electrodes were used alongsideDoppler flow probes for detection of macrocirculation inhepatic artery and portal vein. Data thus allowed sub-differentiation of flow volumes in different vascularcompartments, responsible for hepatic parenchymalperfusion changes in both groups.

MATERIAL AND METHODS

Group A: Investigation of the physiological situation of native liv-ers. A median laparotomy was performed in 15 pigs under generalanesthesia (German Landrace, aged 12–19 weeks, weight 21–26 kg).After premedication (azaperone 1–2 mg im), anesthesia was inducedwith 10 mg/kg iv ketamine, 0.25 mg/kg iv midazolamine hydrochlo-ride, and 0.08 mg/kg iv pancuronium bromide and continued withfentanyl 0.05 mg/kg/h iv infusion. Animals were placed on mechan-ical respiration (oxygen 0.5–1 liters/min, nitrous oxide/isoflurane1.5–2 liters/min). Body temperature was stabilized above 35°C usingheated blankets (Warm-Touch, Mallinckrodt Medical).

Group B: Investigation of liver grafts. An uneventful experimen-tal OLT was performed in 15 pairs of young pigs (German Landrace,aged 10–18 weeks, weight 20–25 kg). Anesthesia was equivalent tothat given for Group A. UW solution (4°C) was used for graft pres-ervation. Cold ischemia time was standardized (6 6 0.5 h). A porto-jugular shunt was inserted during the anhepatic period to reducesplanchnic congestion. Cuff techniques were applied for portal andinfrahepatic caval anastomoses. Biliary reconstruction involved anend-to-end choledochocholedochostomy. No perioperative immuno-suppression was administered. Only animals with an uneventfulpostoperative course were investigated on Day 5 according to the ETperfusion protocoll. Transplanted livers were reexposed by medianrelaparotomy.

Portal venous infusion of endothelin or saline (control groups).The splenic vein was cannulated in both groups using an infusion

KRAUS ET AL.: EXOGENOUS END

catheter (Braun Melsungen, Germany). The tip of the catheter waspropagated to the confluence of the portal and superior mesentericveins. By cannulation via the distal splenic vein mechanical spasms

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of the portal vein were reduced. After a period of hemodynamicstabilisation (20 min) a low-volume portal venous infusion wasstarted and continued over 45 min using an electronically controlledintensive care perfuser. Infusion included either pure saline (controlgroups) or previously dissolved ET peptides in saline (Fa. Novabio-chem, Strassburg, France) in identical fluid aliquots (50 ml). Type ofinfusion was randomized and blinded in both transplanted and nontransplanted animals. Saline and saline–ET solution were stored at220°C in labeled perfuser syringes. Infusion volume was pread-justed to guarantee a standardized ET dosage of 400 pmol/min (ap-proximately 20 pmol/kg/min, average animal weight 22 kg). Thenumbers of animals investigated were (Group A) n 5 10, (A controls)n 5 5, (Group B) n 5 10, and (B controls) n 5 5.

Analysis of plasma ET concentrations. Systemic ET plasma con-centration was retrospectively determined by radioimmunoassay(RIA) (ET1/2-RIA, BI-14210, Biomedica GmbH, Vienna, Austria)before onset of infusion (baseline) and at 15, 30, and 45 min. Prior toRIA ET was extracted by a solid-phase extraction on reverse-phasesilica (Sep-Pak C-18-cartidges, Waters/Millipore), preconditioned byconsecutive washing steps. The eluate was dried and concentratedwith a stream of nitrogen (37°C). Calibration standards (ET-lyophilized), HPLC-purified 125I-labeled ET tracer, highly specificnd sensitive rabbit anti-(porcine) ET antibodies, and extracts wereissolved in sodium phosphate buffer. Separation of the bound ETractions was achieved by a second antibody precipitation step afterreincubation at room temperature for 30 min (anti-rabbit Ig immu-oprecipitating reagent, Biomedica Salugia, Italy). The detection

imit of the ET1/2-RIA was 0.1 fmol/ml. Cross-reactivity of big ET toT(1–3) was , 1%. Cross-reactivity of the ET1/2-RIA to ET-2 andT-3 was 142 and 98%, respectively. Intraassay and interassayoefficients of variation for ET1/2-RIA were 3 and 10.9%. Recovery ofT was 90.8%. All measurements were performed in duplicate. Fur-

her details of the assay have been previously published [28].

Monitoring of systemic circulation. During infusion experimentsean systemic arterial pressure (MAP) and central venous pressure

CVP) were recorded via indwelling catheters fed into the commonarotid artery and internal jugular vein. Heart rate (HR) was mon-tored by surface ECG (Hellige Monitoring Station, Germany).

Measurement of hepatic macrocirculation. Transit-time Dopplerow probes (Transonic Inc., Ithaca, NY) were employed for continu-us quantification of blood flow in the common hepatic artery (HAF)nd portal vein (PVF). Transhepatic blood flow (THF) was calculatedy adding portal and arterial flow volumes (HAF 1 PVF). The liver

weight was measured at autopsy for calculation of parenchymousflow values per 100 g tissue weight. Transhepatic arterial and/orportocaval shunt fraction [%-THF] was calculated with the formula[(THF-MC)/THF] at different points during ET infusion.

Measurement of hepatic microcirculation. Hepatic parenchymalmicrocirculation (HMC) was continuously determined with en-hanced thermal diffusion electrodes (TD, Thermal Technologies Inc.,Cambridge, MA) implantated into the left median porcine liver lobe(depth 15 mm). TD was recently validated and proved to be sensitiveand reliable for measurement of HMC. Details of TD technology andvalidation have recently been published [29, 30]. HMC and ultra-sonic flow data were stored on-line on MS-DOS-based microcomput-ers.

Statistical analysis. Data are expressed as absolute and meanvalues 6 SD. Distribution of values for intra- and interindividualanalysis were compared using the t test according to Student forpaired and unpaired data analysis. In the case of gross differences ofvariance the Welsh test was used. P values , 0.01 were accepted asndicating statistically significant differences. For all analysis SASoftware (SAS-Vs. 6.12; SAS Institute, Cary, NC) was used.

Animal care. During the experiments all animals received hu-

273HELIN-1 AND LIVER PERFUSION

ane care in compliance with the National Research Council9s cri-eria for humane care, as outlined in the Guide for the Care and Usef Laboratory Animals prepared by the National Institutes of Health

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(NIH Publication 86-23, revised 1985). Animals were sacrificed im-mediately after infusion experiments.

RESULTS

Plasma Endothelin Concentrations during ControlledET Infusion

Portal venous and systemic plasma ET concentra-tions could be modulated in a standardized way byexogeous ET application, reaching equivalent levels atanalogous points during infusion in both groups. Base-line ET concentration before ET application was low inboth groups. The baseline ET level in Group A was2.8 6 1.9; in Group B levels were slightly elevatedcompared with the physiological situation (3.8 6 2.4pg/ml). Maximum levels after 45 min of ET infusionwere 46 6 32 in Group A and 41 6 23 pg/ml in GroupB. Differences of mean ET levels at all analogouspoints during ET infusion were insignificant, thus al-lowing a valid comparison of hemodynamic flow valuesbetween groups. ET levels in controls (Control-A/-B)were comparable to baseline levels before ET infusionin Group A (Fig. 1).

Systemic Hemodynamics during ET Infusion

FIG. 1. Mean 6 SD systemic ET plasma concentrations (pg/mlinfusion (controls, n 5 5, each group) in healthy pigs (Group A, n 5(Group B, n 5 10). *P , 0.01 versus baseline levels at this and abetween Groups A and B are not significant.

274 JOURNAL OF SURGICAL RESEAR

No significant alteration in systemic circulation wasdetected during ET infusion in neither groups nor con-

trols. There was cardiocirculatory stability within therange of ET concentrations induced. CVP ranged be-tween 2 and 5 cm H2O. Differences in MAP and HRvalues were insignificant (Fig. 2).

Hepatic Macrocirculation during ET Infusion

A continuous decline in HAF and PVF during ETinfusion was observed in both groups. Flow reductionwas more pronounced in the portal vein compared withHAF reduction in both groups: Mean baseline HAF inGroup A was 205 6 97 ml/min and decreased to 160 62 ml/min during ET infusion (21% HAF reduction). Inransplanted livers (Group B) mean baseline HAF was61 6 87 ml/min and decreased to 146 6 68 ml/minuring ET infusion (9% HAF reduction). A slight re-uction in HAF was preexistent in liver transplantsompared with Group A, but differences in HAF at allonsecutive analogous points during ET applicationere insignificant (Fig. 3).In native livers baseline PVF was 722 6 253 ml/min

nd decreased to 370 6 198 ml/min during ET infusion(48% mean PVF flow reduction). In Group B baselinePVF was 846 6 263 ml/min and declined to 417 6 203ml/min during ET application (50% mean PVF flow

t different time points during portal venous ET infusion or saline) and early postoperative course after porcine liver transplantationter points during infusion. #P , 0.01 versus controls. Differences

: VOL. 93, NO. 2, OCTOBER 2000

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reduction). No significant differences in PVF could bedetected at all consecutive points between groups dur-ing ET infusion (Fig. 4). Intraindividual HAF and PVF

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changes reached statistical significance at all pointslater than 10 min after onset of ET infusion comparedwith baseline.

Hepatic Microcirculation during ET Infusion

Baseline values of HMC in native livers (Group A)were 86 6 15 and 90 6 22 ml/100 g/min in liver trans-plants (Group B). During ET infusion a rapid decline inHMC was demonstrated in both groups. HMC in GroupA was reduced to 29 6 9, and in Group B to 44 6 32

l/100 g/min, after ET infusion. The relative reductionn HMC in native livers was 66% during ET infusionnd 51% in transplants. Reduction of HMC reachedtatistical significance at systemic ET levels above.4 6 5 pg/ml, compared with intraindividual baseline.MC impairment was less pronounced in Group B

han in Group A at high ET concentrations; differencesere insignificant (Fig. 5). Transhepatic arterialnd/or portocaval shunt fraction [%-THF] before anduring ET application ranged between 1.7 and 15.5%.ean shunt fraction in Group A before ET applicationas 8.8 6 2%, and in Group B, 9.5 6 3%. Mean shunt

raction in Group A after maximum ET application was.6 6 2%, and in Group B, 8.0 6 4%. Changes in shunt

FIG. 2. Mean 6 SD arterial pressure (MAP mm Hg) and heart rhealthy pigs (Group A) and after liver transplantation (Group B). D


ractions at different points during ET applicationere insignificant after intra- and interindividual com-arison.

DISCUSSION

Hemodynamic effects of exogenous ET applicationhave been analyzed in multiple prior investigations.Persisting vasoconstriction was documented in the pe-ripheral vasculature [33–36]. A positive inotropic effectwas found in the heart but high levels can lead tocardiodepression, secondary to coronary constriction[35, 37]. Renal vasoconstriction was detected above athreshold of 28 pmol/liter [38]. In contrast, only veryfew studies have focused on hepatic effects of ET. Nodata at all were available on hepatic ET effects in largeanimals. Only one group analyzed the cardiovascularimpact of exogenous ET application in a porcine modelbut without quantification of liver perfusion [36].

In our study portal venous infusion of exogenous ETled to a marked reduction in HMC at systemic ETplasma concentrations above a threshhold of 10 pg/ml.Systemic circulation was not affected within the ETconcentration range achieved. These rather low sys-temic ET concentrations were deliberately establishedto simulate physiological ET levels, as previously des-ribed [31, 32]. Reference data for data comparison con-cerning ET impact on liver perfusion are available onlyfor small animal models: In rats, exogenous portalvenous ET infusion (30–1000 pmol/kg) induced an in-

HR (1/min) at different points during portal venous ET infusion inrences between groups and time points are not significant.

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crement in portal vein pressure above 100 pmol/kg.Systemic ET concentration was not analyzed [39]. Assystemic ET levels in rats lie around 1 pmol/liter [40],

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one can argue that such concentrations are rather un-physiological. However, HMC impairment in rats afterportal venous ET application has also been shown atlower ET dosages between 2 and 10 pmol/liter/min,

FIG. 3. Mean 6 SD hepatic arterial flow (HAF, ml/min) at differafter liver transplantation (Group B), and in control animals of both#P , 0.01 baseline Group A versus Group B.


which were comparable to those in our current inves-tigation [26]. More indirect proof for an effect of ET onHMC is derived from studes of ET antagonists.

Ethanol-induced and postischemic disturbances ofHMC were found to be reduced after prior applicationof an ET antiserum or a pharmacological receptor an-tagonists [41, 42].

points during portal venous ET infusion in healthy pigs (Group A),oups. *P , 0.01 versus baseline values of same group and control.

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Comparison and interpretation of existing hepatic flowdata during exogenous ET application is problematic, asvariable species, ET dosages, and modes of infusion have

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been used. Furthermore, despite the application of iden-tical systemic ET dosages, locally effective ET concentra-tions can be modified by regional variation of clearancecapacity in tissues [11, 43]. ET effects are further depen-

FIG. 4. Mean 6 SD portal venous flow (PVF, ml/min) at differentliver transplantation (Group B), and in control animals of both gr

ifferences between groups A and B at analogous time points are n


dent on the quantity of regional ET receptor expression,which can be up- or downregulated [38]. Finally, ETeffects are often biphasic and long-lasting [33, 43]. Effects

of low levels can mask physiological effects of higher ETdosages in later stages, when a gradual increment of ETconcentrations is investigated, as in our study. Further-more, ET can be cleared from systemic plasma by the

nts during portal venous ET infusion in healthy pigs (Group A), afters. *P , 0.01 versus baseline values. #P , 0.01 versus controls.ignificant.

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liver due to receptor binding, metabolism, and biliaryexcretion [43]. This suggests that effective intrahepaticET concentrations during portal venous ET application

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probably are underestimated by ET measurements insystemic plasma. No samples from the portal vein norliver biopsies have yet been collected during our investi-

FIG. 5. Mean 6 SD hepatic parenchymal microcirculation (HMCime points during portal venous ET infusion in healthy pigs (Grouproups. *P , 0.01 versus baseline values, #P , 0.01 versus controls

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gation, as this would have altered splanchnic and hepaticperfusion due to vascular irritation. The portal venousroute was chosen for ET application to mimic physiolog-

ical portal venous ET influx to the liver, as the splanchnicvasculature is a major source of endogenous ET genera-tion [31, 32, 38]. Despite all these limits of data interpre-

measured by thermodiffusion electrodes (ml/100 g/min) at differentafter liver transplantation (Group B), and in control animals of bothifferences between groups A and B at analogous time points are not


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tation, there is now sufficient scientific proof that ETpeptides can exert a direct impact on HMC.

Which factors triggered by ET lead to HMC impair-

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ment have not been fully clarified. Different patho-physiological mechanisms appear to be involved. First,there is a contractile impact of ET on portal venous andsplanchnic vasculature. ET might further influencecontraction of hepatic sinusoidal pericytes (Ito cells).The dominant role of hepatic pericytes for mediation ofhepatic ET effects has been claimed by various inves-tigators, while its impact on HMC currently remainscontroversial [45–47]. ET-mediated Ito cell contractioncould potentially aggravate HMC via sinusoidal flowobstruction, explaining histological features of con-gested sinusoids after postischemic reperfusion [48]. Itmay be of particular interest that unspecific activationof Ito cells by various modes of liver injury appears tobe essential for development of contractile potential[18, 19, 26].

Liver transplantation can induce ET secretion, Itocell activation, and ET receptor upregulation. Theoret-ically, these factors should render transplanted liversparticularly sensitive to ET-mediated HMC impair-ment, after either endogenous or exogenous ET appli-cation. No data exist so far to clarify whether and towhat extent liver transplantation does have a sensitiz-ing impact for ET effects on the liver or prehepaticvessels. Impairment of HMC in our study appeared tobe a function of portal venous flow reduction and in-creased intrahepatic vascular resistance in parallel. Itwas characterized by flow decline in prehepatic portalvein and artery. PVF reduction during ET applicationwas the dominant factor for transhepatic flow impair-ment, declining by up to 48% during ET infusion inGroup A and up to 50% in Group B compared withbaseline values. HAF declined only by 21% in Group A,and by 9% in Group B. Relative HAF reduction duringET application was less pronounced in transplantedcompared with native livers. A minor preexisting dis-turbance of HAF and HMC before onset of ET applica-tion was detected in liver grafts, compared with nativeorgans. This could potentially be caused by a slightelevation of mean baseline ET concentrations in theearly course after OLT, as has been previously de-cribed. Despite this minor preexisting flow derange-ment, no further aggravating or sensitizing effects ofOLT on ET-induced mediation of hepatic blood flowcould be shown in our investigation. Characteristics ofhepatic parenchymal flow were analogous in bothgroups within the range of ET concentrations investi-gated.

These observations render the assumption of cellactivation prerequisites for Ito cell modulation of he-patic blood flow rather less relevant or could point to acomparably minor impact of sinusoidal cell contractionon hepatic flow, compared with the dimension of flow


changes induced by ET in prehepatic macrocirculation.In the presence of the strong vasoactive mediator ET,other potential flow-modifying factors, such as hepatic

denervation and immunological stimuli, appear tohave a comparably minor impact. However, it must beremembered that OLT was performed after a veryshort cold ischemia of only 6 h in our study. Graftfunction of animals investigated was good. ET applica-tion in animals with more severely damaged livergrafts could potentially have led to altered results inthis respect. This could have also been the case if ETinfusions had been further prolonged.

It also has to be pointed out that a valid differenti-ation between various modes of intrahepatic flowobstruction, i.e., by external vascular compression(edema, necrosis), sinusoidal constraction, or intrahe-patic vascular (arterial or venous) constriction is be-yond the analytical scope of the current study. TDmeasures integrated hepatic tissue perfusion, distantfrom major vessels, as proven by analysis of thermaltissue properties. HAF and PVF macroperfusion wereonly quantified in extrahepatic segments. HMC distur-bance after OLT was further characterized by an aug-mented intrahepatic shunt perfusion. This can also bea consequence of postischemic liver damage, as previ-ously described [44]. On the other hand we could notdetect a significant increase in intrahepatic shunt frac-tions during ET application. Differences in shunt frac-tions between groups were insignificant.

HMC impairment is regularly encountered afterOLT and appears to be of relevance for long-term graftfunction. Most HMC flow changes after OLT are dy-namic and potentially reversible. The dynamic reactiv-ity of HMC in liver grafts modulated by administrationof ET can also be interpreted as a sign of graft vitalityin our study. This could be lost in severe graft failure ordystrophy. ET peptides play a relevant part in themultifactorial pathology of postischemic liver graftreperfusion. Even if no sensitizing impact of OLT onhepatic ET effects could be documented, this does notreduce the estimated relevance of ET peptides for me-diation of postischemic HMC disturbances. Hepatopro-tective effects of ET antagonists were also recentlydescribed after porcine OLT [50, 51]. Ongoing evalua-tion of ET-related hepatic pathophysiology appears tobe beneficial for improvement of graft function andsurvival after OLT.

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Effects of Exogenous Endothelin-1 Application on Liver Perfusion in Native and Transplanted Porcine Livers