Addition of simvastatin to cold storage solution prevents endothelial dysfunction in explanted rat livers

Authors

  • Lucia Russo,

    1. Hepatic Hemodynamic Lab, Liver Unit, IMDIM, Hospital Clínic de Barcelona, IDIBAPS, Centro de Investigación Biomédica en Red en Enfermedades Hepáticas y Digestivas (CIBERehd), University of Barcelona, Barcelona, Spain
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    • These authors contributed equally to this work.

  • Jorge Gracia-Sancho,

    Corresponding author
    1. Hepatic Hemodynamic Lab, Liver Unit, IMDIM, Hospital Clínic de Barcelona, IDIBAPS, Centro de Investigación Biomédica en Red en Enfermedades Hepáticas y Digestivas (CIBERehd), University of Barcelona, Barcelona, Spain
    • Hepatic Hemodynamic Laboratory, Hospital Clínic de Barcelona, IDIBAPS, Esther Koplowitz Centre, Rosselló 149, Room 4.5, 08036 Barcelona, Spain
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    • These authors contributed equally to this work.

  • Héctor García-Calderó,

    1. Hepatic Hemodynamic Lab, Liver Unit, IMDIM, Hospital Clínic de Barcelona, IDIBAPS, Centro de Investigación Biomédica en Red en Enfermedades Hepáticas y Digestivas (CIBERehd), University of Barcelona, Barcelona, Spain
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  • Giusi Marrone,

    1. Hepatic Hemodynamic Lab, Liver Unit, IMDIM, Hospital Clínic de Barcelona, IDIBAPS, Centro de Investigación Biomédica en Red en Enfermedades Hepáticas y Digestivas (CIBERehd), University of Barcelona, Barcelona, Spain
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  • Juan Carlos García-Pagán,

    1. Hepatic Hemodynamic Lab, Liver Unit, IMDIM, Hospital Clínic de Barcelona, IDIBAPS, Centro de Investigación Biomédica en Red en Enfermedades Hepáticas y Digestivas (CIBERehd), University of Barcelona, Barcelona, Spain
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  • Guillermo García-Cardeña,

    1. Center for Excellence in Vascular Biology, Department of Pathology, Harvard Medical School and Brigham and Women's Hospital, Boston, Massachusetts, USA
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  • Jaime Bosch

    Corresponding author
    1. Hepatic Hemodynamic Lab, Liver Unit, IMDIM, Hospital Clínic de Barcelona, IDIBAPS, Centro de Investigación Biomédica en Red en Enfermedades Hepáticas y Digestivas (CIBERehd), University of Barcelona, Barcelona, Spain
    • Hepatic Hemodynamic Laboratory, Liver Unit, Hospital Clínic i Provincial, Villarroel 170, 08036 Barcelona, Spain
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    • Fax: +34932279856


  • Potential conflict of interest: Nothing to report.

  • Supported by grants from the Instituto de Salud Carlos III (PS 09/01261 to J.B. and FIS 11/00235 to J.G.-S.) and Ministerio de Ciencia e Innovación (SAF 2010/17043 to J.C.G.-P.). G.M. is a recipient of a fellowship from Instituto de Salud Carlos III (PFIS 09/00540), and J.G.-S. of a contract from the Programa Ramón y Cajal, Ministerio de Ciencia e Innovacion, Spain. Ciberehd is funded by Instituto de Salud Carlos III.

Abstract

Pathophysiological alterations in the endothelial phenotype result in endothelial dysfunction. Flow cessation, occurring during organ procurement for transplantation, triggers the endothelial dysfunction characteristic of ischemia/reperfusion injury, partly due to a reduction in the expression of the vasoprotective transcription factor Kruppel-like Factor 2 (KLF2). We aimed at (1) characterizing the effects of flow cessation and cold storage on hepatic endothelial phenotype, and (2) ascertaining if the consequences of cold stasis on the hepatic endothelium can be pharmacologically modulated, improving liver graft function. Expression of KLF2 and its vasoprotective programs was determined in (i) hepatic endothelial cells (HEC) incubated under cold storage conditions with or without the KLF2-inducer simvastatin, and (ii) rat livers not cold stored or preserved in cold University of Wisconsin solution (UWS) supplemented with simvastatin or its vehicle. In addition, upon warm reperfusion hepatic vascular resistance, endothelial function, nitric oxide vasodilator pathway, apoptosis, inflammation, and liver injury were evaluated in not cold stored livers or livers preserved in cold UWS supplemented with simvastatin or vehicle. Expression of KLF2 and its vasoprotective programs decrease in HEC incubated under cold storage conditions. Cold-stored rat livers exhibit a time-dependent decrease in KLF2 and its target genes, liver injury, increased hepatic vascular resistance, and endothelial dysfunction. The addition of simvastatin to the storage solution, maintained KLF2-dependent vasoprotective programs, prevented liver damage, inflammation, and oxidative stress and improved endothelial dysfunction. Conclusion: Our results provide a rationale to evaluate the beneficial effects of a vasoprotective preservation solution on human liver procurement for transplantation. (Hepatology 2012)

Liver transplantation is the only life-saving therapy for most types of advanced liver failure. Despite the advancement in surgical techniques, postoperative care, and immunosuppressive therapies, which have improved short-term and long-term graft survival, approximately 20% of liver transplants are associated with serious clinical problems.1 Moreover, liver transplantation is limited by the shortage of adequate organs for clinical use, which have led to the use of “marginal” livers from nonhealthy steatotic donors or nonheart-beating donors. However, marginal livers are much more prone to primary graft failure after transplantation.2 Hepatic ischemia/reperfusion (I/R) injury is considered one of the main determinants of the outcome after liver transplantation.3, 4

The process of hepatic I/R injury is a sequence of events involving many interconnected factors occurring in a variety of cell types. Liver endothelial cells are particularly vulnerable to I/R injury and develop serious alterations during cold storage, such as retraction, cell body detachment, and apoptosis, which are magnified upon warm reperfusion.5, 6 It is currently accepted that hepatic endothelium damage occurring during cold preservation represents the initial factor leading to hepatic I/R injury, determining poor graft microcirculation, platelet activation, persistent vasoconstriction, up-regulation of adhesion molecules, oxidative stress, Kupffer cell activation, neutrophil infiltration, and hepatocyte death.7, 8

Different mechanisms for endothelial damage during cold storage and/or warm reperfusion have been described.9, 10 We have recently unraveled that lack of hemodynamic stimulation occurring during cold storage conditions is the main detrimental effect of organ preservation for transplantation on the endothelial phenotype.11 In fact, flow cessation per se results in a significant reduction in endothelial vasoprotective pathways leading to cell activation and apoptosis. These negative effects of cold storage conditions, observed in cultured endothelial cells, are partly due to the loss of expression of the vasoprotective transcription factor Kruppel-like Factor 2 (KLF2) and can be prevented by adding a KLF2-inducer, such as simvastatin,12 to the cold preservation solution.11

Considering that endothelial protection during cold storage represents a key factor for a successful transplantation, and that induction of KLF2-derived transcriptional programs confers endothelial protection, the main purpose of the present study was to evaluate the effects of cold storage on the hepatic endothelial vasoprotective phenotype and if supplementing a cold preservation solution with the KLF2-inducer simvastatin ameliorates the hepatic I/R injury observed upon reperfusion.

Abbreviations

DHE: dihydroethidium; eNOS: endothelial nitric oxide synthase; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; HEC: hepatic endothelial cells; HO-1: hemeoxygenase-1; ICAM-1: intercellular adhesion molecule 1; I/R: ischemia/reperfusion; KLF2: Kruppel-like Factor 2; LDH: lactate dehydrogenase; NO: nitric oxide; TM: thrombomodulin; UWS: University of Wisconsin solution.

Materials and Methods

Animals.

Male Wistar rats from Charles River Laboratories SA (Barcelona, Spain) weighing 275-300 g were used. The animals were kept in environmentally controlled animal facilities at the Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS). All experiments were approved by the Laboratory Animal Care and Use Committee of the University of Barcelona and were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, NIH Publication 86-23, revised 1996).

Hepatic Endothelial Cells Isolation and Cold Preservation.

Rat hepatic endothelial cells (HEC) were isolated as described.13 Briefly, after perfusion of the livers with 0,015% collagenase A and isopycnic sedimentation of the resulting dispersed cells through a two-step density gradient of Percoll (25%-50%), monolayer cultures of HEC were established by selective attachment on a collagen I substrate. Cells were cultured (37°C, 5% CO2) in Roswell Park Memorial Institute (RPMI) 1640 as described.13 Highly pure and viable cells were used.

After 2 hours of isolation, HEC were washed twice with phosphate-buffered saline (PBS) and lysed (no cold storage group) or incubated 16 hours at 4°C in University of Wisconsin solution (UWS) supplemented with simvastatin 1 μM (Calbiochem, Darmstadt, Germany) or its vehicle (dimethyl sulfoxide 0.1% vol/vol) (n = 4 per group). The dose of simvastatin used has been validated.11, 12

Small Interfering RNA (siRNA) Experiments.

siRNA transfection was performed as described with minor modifications.11, 14 Briefly, HECs were transfected with an siRNA targeting rat KLF2 (5 nM, s157429, Life Technologies, Carlsbad, CA), or with a control siRNA (5 nM, 4390843, Life Technologies) using siPORT transfection agent (Life Technologies) according to the manufacturer's instructions. At 24 hours posttransfection, cells were treated for an additional 16 hours with either 1 μM simvastatin or its vehicle.

Cold Preservation of Liver.

Rats were anesthetized with ketamine hydrochloride (100 mg/kg intraperitoneally; Merial Laboratories, Barcelona, Spain) plus midazolam (5 mg/kg intraperitoneally; Laboratorios Reig Jofré, Barcelona, Spain). Afterwards the abdomen was opened, liver was exsanguinated with Krebs' buffer, and flushed by way of the portal vein with cold UWS supplemented with simvastatin (10 μM) or its vehicle.

Rat livers (n = 7 per group) were harvested and one lobe from each liver was immediately snap-frozen and the other three were incubated at 4°C for 1, 6, or 16 hours in UWS supplemented with simvastatin or its vehicle. Then liver lobes were snap-frozen for molecular studies.

Liver Vascular Studies.

Liver vascular responses were assessed in the isolated, in situ liver perfusion system, as described.15 Briefly, after cannulation of the bile duct, livers were perfused through the portal vein with Krebs' buffer in a recirculation fashion at a constant flow rate of 30 mL/min with a total volume of 100 mL. An ultrasonic transit-time flow probe (model T201; Transonic Systems, Ithaca, NY) and a pressure transducer (Edwards Lifesciences, Irvine, CA) were placed online, immediately ahead of the portal inlet cannula, to continuously monitor portal flow and perfusion pressure. Another pressure transducer was placed immediately after the thoracic vena cava outlet for measurement of outflow pressure. The flow probe and the two pressure transducers were connected to a PowerLab (4SP) linked to a computer using the Chart v. 5.0.1 for Windows software (ADInstruments, Mountain View, CA). The average portal flow, inflow and outflow pressures were continuously sampled, recorded, and afterwards blindly analyzed under code. After 20 minutes of stabilization the livers were flushed with cold UWS or with cold UWS supplemented with simvastatin (10 μM), then cold stored for 16 hours in UWS or in UWS supplemented with simvastatin (n = 7 per group).

After cold storage livers were exposed at room temperature (22°C) for 20 minutes to mimic the warm ischemia period and reperfused by way of the portal vein with Krebs' buffer (37°C). During the first 10 minutes of reperfusion (initial stabilization period), portal flow was progressively increased up to 30 mL/min. The perfusion preparations were continuously monitored for 60 minutes. Afterwards, liver endothelial function was evaluated by performing flow pressure curves (increases of 5mL/min every 2 minutes).16 Intrahepatic vascular resistance (IVR) was calculated as: (inflow portal pressure − outflow portal pressure) / portal flow.

In an independent group of rats (n = 5 per group), following warm reperfusion hepatic endothelial function was evaluated analyzing endothelium-dependent vasorelaxation to incremental doses of acetylcholine (ACH; 10−7 to 10−5M) after preconstriction with methoxamine (10−4M).17, 18

Control livers (no cold storage) were perfused, flushed with UWS, harvested, and immediately reperfused ex vivo.

Aliquots of the perfusate were sampled for the measurement of transaminases and lactate dehydrogenase (LDH). Bile output (reported as μL of bile/g of liver) was evaluated at the end of the study.

Analysis of Hepatic Transaminases and LDH.

Hepatic injury was assessed in terms of transaminases and LDH levels analyzed with standard methods at the Hospital Clinic of Barcelona's CORE lab.

Measurement of Cyclic Guanosine Monophosphate (cGMP).

Levels of cGMP, a marker of NO bioavailability, were analyzed in liver homogenates using an enzyme immunoassay (Cayman Chemical, Ann Arbor, MI) as described.19 The results are expressed as pmol/mg tissue.

Dihydroethidium Staining.

In situ superoxide (Omath image) levels were evaluated with the oxidative fluorescent dye dihydroethidium (DHE; Molecular Probes).20 Briefly, liver cryosections (10 μm) were incubated with DHE (10 μmol/L) in PBS. Fluorescence images were obtained with a laser scanning confocal microscope (TCS-SL DMIRE2, Leica), and quantitative analysis was performed with ImageJ 1.43m software (National Institutes of Health, Bethesda, MD).

Histological Analysis.

Liver samples were fixed in 10% formalin, embedded in paraffin, sectioned (thickness of 2 μm), and slides were stained with hematoxylin and eosin (H&E) to analyze the hepatic parenchyma.

The samples were photographed and analyzed using a microscope equipped with a digital camera and the assistance of Axiovision software (Zeiss, Jena, Germany).

RNA Processing and Real-Time TaqMan Polymerase Chain Reaction (PCR) Analysis.

Total RNA from HEC was isolated and purified using the Trizol method (Invitrogen, El Prat de Llobregat, Barcelona, Spain). Total RNA from rat tissue was isolated and purified using RNeasy Mini Kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. RNA quality was verified using Agilent's 2100 Bioanalyzer. RNA was reverse-transcribed to complementary DNA (cDNA) using the QuantiTect Reverse Transcription kit (Qiagen). cDNA templates were amplified by real-time TaqMan PCR on an ABI Prism 7900 sequence Detection System (Applied Biosystems, Foster City, CA). Expression of KLF2 and its target genes eNOS, thrombomodulin (TM), and hemeoxygenase (HO-1) and Collagen-I was analyzed using predesigned gene expression assays obtained from Applied Biosystems according to the manufacturer's protocol and reported relative to endogenous control 18S. All PCR reactions were performed in duplicate and using nuclease-free water as no template control.

Western Blotting.

Liver samples were processed as described.21 Aliquots from each sample containing equal amounts of protein (40-100 μg) were run on 8%-15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. Equal loading was ensured by Ponceau staining. The blots were subsequently blocked for 1 hour and probed overnight at 4°C with antibodies recognizing eNOS (BD Transduction Laboratories, Lexington, KY), phosphorylated eNOS at Ser1176 (BD Transduction Laboratories), cleaved Caspase-3 (Cell Signaling Technology, Beverly, MA), or ICAM-1 (R&D Systems, Minneapolis, MN), all 1:1,000, followed by an incubation with their corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10,000, Stressgen, Victoria, BC, Canada) for 1 hour at room temperature. Blots were revealed by chemiluminescence. Protein expression was determined by densitometric analysis using the Science Lab 2001, Image Gauge (Fuji Photo Film, Düsseldorf, Germany). Blots were assayed for glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Santa Cruz Biotech, Santa Cruz, CA) content as standardization of sample loading.

Drugs and Reagents.

Collagenase was from Roche Diagnostics (Mannheim, Germany). Percoll was from Amersham Biosciences (Uppsala, Sweden). Collagen type I was from Invitrogen. Reagents for cell culture were provided by Biological Industries (Kibbutz Beit Haemek, Israel).

Statistical Analysis.

Statistical analyses were performed with the SPSS 16.0 for Windows statistical package (Chicago, IL). All results are expressed as mean ± standard error of the mean. Comparisons between groups were performed with analysis of variance followed by Tukey's test or with Student's t test or the Mann-Whitney t test when adequate. Differences were considered significant at P < 0.05.

Results

Simvastatin Maintains the Expression of KLF2 and Its Vasoprotective Target Genes in the Liver During Cold Storage.

Rat liver lobes cold stored for 1, 6, and 16 hours in UWS exhibited a significant reduction in KLF2 expression compared with their corresponding control liver lobes (Fig. 1A). KLF2 reduction was accompanied by a significant decrease in the expression of its vasoprotective target genes eNOS, TM, and HO-1 (Fig. 1B). Simvastatin addition to UWS totally prevented the decay of hepatic KLF2, eNOS, TM, and HO-1 during cold storage (Fig. 1A,B). Considering that after 16 hours of cold storage there was a maximal decrease of the vasoprotective genes that was completely abrogated by adding simvastatin to cold storage solution, this timepoint was chosen for all following experiments.

Figure 1.

Simvastatin maintains KLF2 and its vasoprotective target genes expression in livers during cold storage. (A) KLF2 and (B) eNOS, TM, and HO-1 mRNA expression was determined in livers not cold stored or stored for 1, 6, or 16 hours in UWS supplemented with simvastatin (10 μM) or its vehicle (*P < 0.05 versus no cold storage and simvastatin; n = 7 per condition).

Simvastatin Preserves the Expression of KLF2 and Its Vasoprotective Target Genes in HECs During Cold Storage.

As shown in Fig. 2, freshly isolated HECs cold stored for 16 hours in UWS exhibited a significant reduction in the expression of KLF2, eNOS, and TM compared with cells not cold stored. Addition of simvastatin to UWS maintained the endothelial expression of KLF2 and its vasoprotective target genes during cold storage. Importantly, the ability of simvastatin to maintain the expression of the studied vasoprotective genes was annulled when KLF2 expression was muted by siRNA silencing (Fig. 2).

Figure 2.

Simvastatin induces hepatic endothelial KLF2 and its vasoprotective target genes expression during cold storage. Top: KLF2, eNOS, and TM mRNA expression was determined in freshly isolated HECs preserved at 4°C for 0 hours (no cold storage) or 16 hours in UWS supplemented with simvastatin (1 μM) or its vehicle (*P < 0.05 versus no cold storage and simvastatin; n = 4 per condition). Bottom: KLF2 and its vasoprotective target genes expression in HEC transfected with specific siRNA against KLF2 or control and preserved for 16 hours in the presence of simvastatin (1 μM) (*P < 0.05 versus siRNA control; n = 5 per condition).

Simvastatin Addition to UWS Significantly Protects Liver Function and Viability During Cold Storage and Warm Reperfusion.

To evaluate the effects of simvastatin addition to UWS on hepatic injury derived from cold preservation and warm reperfusion, hepatic architecture distortion, hepatic function, bile production, and presence of oxidative stress, inflammation, and apoptosis were analyzed.

Histological examination revealed that livers cold stored for 16 hours in UWS exhibited evident hepatocellular lesions, mainly in centrilobular areas, defined by loss of cohesion of cell plates, necrotic hepatocytes, presence of Councilman bodies, and anoxia-derived small fat vacuoles (Fig. 3A). These histological changes were much attenuated or absent in liver grafts cold stored for 16 hours in UWS supplemented with simvastatin.

Figure 3.

Simvastatin ameliorates I/R liver injury. Liver grafts not cold stored (control group) or cold stored for 16 hours in UWS supplemented with simvastatin (10 μM) or its vehicle were analyzed following warm reperfusion. Hepatic architecture status was assessed by H&E staining, 20× (top) and 40× (bottom) magnifications (A). Hepatic injury was measured by analyzing transaminases (ALT and AST), LDH levels (B), and bile production (C) (*P < 0.05 versus no cold storage and simvastatin; n = 7 per group).

As shown in Fig. 3B,C, cold-stored and warm-reperfused liver grafts released higher amounts of transaminases and LDH and a lower quantity of bile in comparison to grafts reperfused without previous cold preservation. These detrimental effects were not observed in liver grafts cold stored in UWS supplemented with simvastatin.

Figure 4 depicts significantly higher levels of Omath image, ICAM-1, and cleaved caspase-3 in cold-stored and warm-reperfused livers grafts compared with control livers, indicating increased oxidative stress, inflammation, and apoptosis, respectively. These negative events from cold storage were markedly attenuated, or entirely prevented, in liver grafts cold stored in simvastatin-containing UWS.

Figure 4.

Simvastatin prevents hepatic I/R injury-derived oxidative stress, inflammation, and cell death. (A) Top: representative confocal microscopy images of superoxide (Omath image) in situ detection in fresh sections from livers not cold stored (control group) or cold preserved for 16 hours in UWS supplemented with simvastatin (10 μM) or its vehicle, and afterwards warm reperfused. Bottom: liver sections described above fluorescence intensity analysis. (*P < 0.05 versus no cold storage and simvastatin; #P < 0.05 versus vehicle; n = 3 per condition). (B) Top: representative images of cleaved caspase-3 (apoptosis) and ICAM-1 (inflammation) immunoblots of liver grafts preserved as described above. Bottom: densitometric analysis of depicted proteins normalized to GAPDH (*P < 0.05 versus no cold storage and simvastatin; n = 7 per condition).

Simvastatin Addition to UWS Improves Liver Microcirculation and Prevents Endothelial Dysfunction on Reperfusion.

Livers cold stored for 16 hours exhibited a deteriorated microcirculation upon reperfusion, as demonstrated by significantly increased liver vascular resistance as compared to control livers (Fig. 5A). Cold storage-derived increments in liver vascular resistance were not observed in liver grafts cold preserved in the presence of simvastatin.

Figure 5.

Simvastatin improves hepatic microcirculation and endothelial dysfunction in livers cold stored and warm reperfused. (A) Intrahepatic vascular resistance (IVR) variation observed during warm reperfusion of livers not cold stored (control group) or livers cold preserved for 16 hours in UWS supplemented with simvastatin (10 μM) or its vehicle (n = 7 per group). (B) Endothelial function evaluation of liver grafts described in (A). Top: IVR increment due to portal flow increments (n = 7 per group). (C) Endothelium-dependent relaxation to ACH of liver grafts preserved as described in (A) (n = 5 per group) (*P < 0.05 versus no cold storage and simvastatin).

In addition, liver grafts stored for 16 hours in cold UWS exhibit endothelial dysfunction (Fig. 5B,C). As depicted in Fig. 5B, in response to portal flow increments between 35 and 60 mL/min control livers were able to maintain a constant hepatic vascular resistance, thus demonstrating normal flow-dependent vasodilatation of the liver vascular bed. However, cold-stored livers preserved in UWS did not accommodate portal flow increases, exhibiting a marked and significant increment in their vascular resistance. Remarkably, cold storage-derived endothelial dysfunction was entirely prevented in livers cold preserved in UWS supplemented with simvastatin.

Similarly, dose-response curves to ACH showed that cold-stored livers exhibit significantly reduced endothelial-derived vasodilatation in comparison to not cold-stored livers (Fig. 5C), further demonstrating the development of acute endothelial dysfunction during cold storage. This pathological phenomenon was prevented when livers were cold stored in the presence of simvastatin.

Liver microcirculation deterioration and development of endothelial dysfunction after cold preservation were accompanied by significant reductions in eNOS expression and activity and cGMP levels comparing to controls (Fig. 6), with no modification in collagen-I expression (1.00 ± 0.16 controls versus 0.92 ± 0.32 cold stored; NS), a known marker of hepatic stellate cell activation. Simvastatin addition to the cold-storage solution maintained hepatic eNOS expression and improved eNOS phosphorylation, which was associated with up-regulation of cGMP levels.

Figure 6.

Simvastatin preserves NO bioavailability during liver procurement. (A) Top: representative eNOS and p-eNOS western blots of liver grafts not cold stored or cold preserved for 16 hours in UWS supplemented with simvastatin (10 μM) or its vehicle. Bottom: protein densitometric analysis normalized to GAPDH. (B) cGMP levels determined in livers described in (A) (*P < 0.05 versus no cold storage and simvastatin; #P < 0.05 versus vehicle; n = 7 per group).

Discussion

The endothelium is the primary target of cold preservation and reperfusion injuries in liver transplantation. Liver sinusoidal endothelial injury involves cell activation, apoptosis, and detachment, leading to hepatic microcirculatory dysfunction.7, 22 Up to now, the reduction in endothelial function and viability during liver procurement for transplantation has been mostly attributed to the deleterious effects derived from ischemia.23 However, a recent study from our group demonstrated that absence of blood flow-derived shear stress stimuli per se, which occurs during organ procurement for transplantation, negatively affects the endothelial vasoprotective phenotype inducing acute endothelial dysfunction.11

This pioneering study created the rationale to investigate strategies for organ preservation based on machine perfusion of kidney or liver grafts.24, 25 Furthermore, it allows study of the molecular mechanisms leading to increased endothelial sensitivity to injury in the absence of shear stress, with the aim of discovery druggable targets to prevent endothelial and tissue damage during organ procurement. For this purpose, we analyzed the effects of shear stress interruption and cold storage on the hepatic endothelial phenotype and function, and developed a pharmacological strategy to maintain endothelial health in the setting of organ transplantation.

We first characterized the hepatic endothelial vasoprotective phenotype during cold storage, both at the tissue and cellular levels, by analyzing the KLF2-derived protective pathways. Our study demonstrates that during cold storage conditions the hepatic endothelial vasoprotective phenotype is rapidly lost. Indeed, the hepatic expression of KLF2 and its target genes eNOS, TM, and HO-1 is significantly reduced after just 1 hour or 6 hours of cold storage. Reduced expression of KLF2 and its transcriptional target progressively increased throughout cold storage. Although it is well established that within the liver, as well as in the vasculature, the expression of KLF2 is mainly endothelial,11, 26, 27 we further characterized the effects of shear stress termination and cold storage conditions on the vasoprotective phenotype in freshly isolated HECs. These in vitro experiments confirmed that once flow stimulus is terminated, and cells are preserved under cold-storage conditions, hepatic endothelial KLF2-derived vasoprotective pathways are significantly down-regulated.

To understand the pathophysiological consequences of an abnormal endothelial phenotype occurring during cold storage, we characterized the hepatic microcirculation status and the hepatic endothelial function during warm reperfusion. These experiments showed that upon reperfusion cold-stored liver grafts exhibit much higher hepatic vascular resistance, as compared to liver grafts not cold stored. Moreover, these liver grafts exhibit acute endothelial dysfunction. These microcirculatory abnormalities were accompanied by significant hepatic injury, as demonstrated by marked increments in: hepatic enzymes release, inflammation, apoptosis, oxidative stress, histological injury, and significant reduction in bile production. Our results are in agreement with previous reports describing increased vascular tone, apoptosis, and inflammation following cold storage and warm reperfusion,8,28-32 further supporting the concept that both the endothelium and the liver parenchyma are negatively affected by cold storage and warm reperfusion.

It is well known that successful graft function and patient recovery after transplantation depends on the degree of organ protection achieved during cold storage, being the composition of the organ preservation solution crucial to reach maximum protection.33, 34 A variety of studies have evaluated the possible beneficial effects of new or modified organ preservation solutions on liver function and viability upon reperfusion28, 35; however, none of them focused at improving endothelial protection during cold storage. In our study, we addressed this question by analyzing the possible beneficial effects of adding simvastatin, a drug known for it vasoprotective properties, to a standard solution for organ preservation.

Statins, or HMG-CoA reductase inhibitors, up-regulate KLF2-derived transcriptional programs improving endothelial function.12,19,27,36 These kinds of drugs have been described as prophylactic agents to treat I/R injuries.37 Moreover, we recently suggested that simvastatin could be used as a supplement for organ preservation solutions due to its capability to sustain the expression of KLF2-derived vasoprotective transcriptional pathways in cold-stored endothelial cells.11 Here we demonstrate that the addition of simvastatin to UWS, a commonly used cold-storage solution, maintains KLF2-derived vasoprotective pathways during short and long periods of cold liver ischemia. Furthermore, simvastatin addition to UWS dramatically improves the capacity of this solution to protect liver viability and function during cold storage and to inhibit the development of hepatic microcirculatory dysfunction and liver injury upon warm reperfusion. Specifically, liver grafts cold stored in the presence of simvastatin and afterwards warm reperfused exhibited significantly reduced hepatic injury, normal hepatic resistance, and improved endothelial function as compared to grafts cold stored without simvastatin in the preservation solution. Remarkably, the protective effects of simvastatin were observed in liver grafts cold stored for 16 hours, a period of time where UWS no longer provides protection,38, 39 thus opening up the possibility to lengthen liver procurement periods.

Liver function and viability protection conferred by simvastatin, defined as normalization of liver enzymes release and bile production, can be partly explained by the prevention of inflammation, apoptosis, and oxidative stress, as demonstrated by their surrogate markers ICAM-1, cleaved caspase-3, and Omath image. These results, which are in accordance with previous reports describing KLF2-derived antiinflammatory, antiapoptotic, and antioxidant effects,14, 40 suggest that the beneficial effects of simvastatin on liver function derive from its ability to maintain KLF2-derived vasoprotective pathways during cold storage.

To understand the molecular mechanisms responsible for the simvastatin-derived liver microcirculation protection, and considering that statins enhance endothelial NO production by upregulating eNOS expression and activity,41 and that NO donors protect livers against I/R injury,42 we characterized the NO pathway in the liver grafts included in the present study. These experiments demonstrated that simvastatin addition to cold-storage solution leads to an up-regulation of hepatic NO bioavailability, measured as its secondary messenger cGMP. The up-regulation in NO levels could derive from increased eNOS expression and activity, as suggested by increased expression of the biologically active phosphorylated eNOS together with reduced levels of its scavenger superoxide (Omath image).20 Altogether, these observations suggest that maintenance of an adequate NO generation may be responsible, at least in part, for preventing the increase in liver vascular resistance as well as for the normal endothelial function observed in liver grafts cold stored with simvastatin.

Two important clinical implications derive from the present study. First, it has been recently suggested that improvement in organ function posttransplantation achieved by machine continuous perfusion preservation may be partly derived from endothelial protection due to up-regulation of shear stress-sensitive protective genes.43 The data included in our study demonstrate that addition of a vasoprotective agent, such as simvastatin, to a liver cold-storage preservation solution represents a much easier and cost-effective alternative to machine perfusion preservation. Second, it is well known that cold-storage and warm-reperfusion injuries are especially severe, and are associated with serious morbidity and mortality when using expanded criteria donors or marginal ones.2 The new approach for better preservation of organs for transplantation described in the present study opens the possibility to improve the function of liver grafts from marginal donors by using vasoprotective preservation solutions, which would represent a main step forward to improve donor pools and overcome current problems of organ shortage.

Acknowledgements

The work was partly carried out at the Esther Koplowitz Centre, Barcelona. The authors thank Montserrat Monclús for technical assistance, Eugenio Rosado and Marcos Pasarín for helpful discussions, and Dr. Miquel Bruguera for expertise in liver histology. Contributions: L.R. designed the research, performed experiments, analyzed data, and wrote the article. J.G.-S. designed the research, conceived ideas, wrote the article, obtained funding, and codirected the study. H.G.-C. and G.M. performed experiments and analyzed data. J.C.G.-P. conceived ideas, critically revised the article, and obtained funding. G.G.-C. conceived ideas and critically revised the article. J.B. designed the research, conceived ideas, wrote the article, obtained funding, and codirected the study. All authors edited and reviewed the final article.

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