Cell Transplantation, Vol. 21, pp. 1349–1360, 2012 0963-6897/12 $90.00 + .00Printed in the USA. All rights reserved. DOI: http://dx.doi.org/10.3727/096368911X623853Copyright 2012 Cognizant Comm. Corp. E-ISSN 1555-3892

www.cognizantcommunication.com

Beneficial Effects of Ischemic Preconditioning on Pancreas Cold Preservation

Anthony R. Hogan,*† Marco Doni,*‡ R. Damaris Molano,* Melina M. Ribeiro,* Angela Szeto,*Lorenzo Cobianchi,*‡ Elsie Zahr-Akrawi,* Judith Molina,* Alessia Fornoni,*§¶ Armando J. Mendez,*§#

Camillo Ricordi,*†§#**††‡‡ Ricardo L. Pastori,*§# and Antonello Pileggi*†**‡‡

*Diabetes Research Institute, University of Miami, Miami, FL, USA†DeWitt-Daughtry Family Department of Surgery, University of Miami Leonard M. Miller School of Medicine, Miami, FL, USA

‡Division of Hepatobiliary and Pancreatic Surgery, Department of Surgery, University of Pavia,IRCCS Fondazione “San Matteo” Hospital, Pavia, Italy

§Department of Medicine, University of Miami Leonard M. Miller School of Medicine, Miami, FL, USA¶Division of Nephrology, University of Miami Leonard M. Miller School of Medicine, Miami, FL, USA

#Division of Endocrinology, University of Miami Leonard M. Miller School of Medicine, Miami, FL, USA**Department of Microbiology and Immunology, University of Miami Leonard M. Miller School of Medicine, Miami, FL, USA

††Jackson Memorial Hospital Transplant Institute, University of Miami, Miami, FL, USA‡‡Department of Biomedical Engineering, University of Miami, Miami, FL, USA

Ischemic preconditioning (IPC) confers tissue resistance to subsequent ischemia in several organs. The pro-tective effects are obtained by applying short periods of warm ischemia followed by reperfusion prior toextended ischemic insults to the organs. In the present study, we evaluated whether IPC can reduce pancre-atic tissue injury following cold ischemic preservation. Rat pancreata were exposed to IPC (10 min of warmischemia followed by 10 min of reperfusion) prior to �18 h of cold preservation before assessment of organinjury or islet isolation. Pancreas IPC improved islet yields (964 ± 336 vs. 711 ± 204 IEQ/pancreas; p =0.004) and lowered islet loss after culture (33 ± 10% vs. 51 ± 14%; p = 0.0005). Islet potency in vivo waswell preserved with diabetes reversal and improved glucose clearance. Pancreas IPC reduced levels ofNADPH-dependent oxidase, a source of reactive oxygen species, in pancreas homogenates versus controls(78.4 ± 45.9 vs. 216.2 ± 53.8 RLU/µg; p = 0.002). Microarray genomic analysis of pancreata revealed upreg-ulation of 81 genes and downregulation of 454 genes (greater than twofold change) when comparing IPC-treated glands to controls, respectively, and showing a decrease in markers of apoptosis and oxidative stress.Collectively, our study demonstrates beneficial effects of IPC of the pancreas prior to cold organ preservationand provides evidence of the key role of IPC-mediated modulation of oxidative stress pathways. The use ofIPC of the pancreas may contribute to increasing the quality of donor pancreas for transplantation and toimproving organ utilization.

Key words: Ischemic preconditioning; Pancreas; Cold ischemia; Cold preservation; Islets of Langerhans;Oxidative stress; NADPH oxidase; NADPH-dependent superoxide (NOX); Microarrays; Transcriptome; Rat

INTRODUCTION shortage of donor pancreata particularly impacts clinicalislet transplantation, where the number of islets obtainedfrom a donor pancreas remains quite variable and notRestoration of β-cell function is a desirable goal for

patients with insulin-requiring diabetes. Currently, β-cell completely reproducible (55); approximately 50% ofhuman glands processed with the intent to transplantreplacement is achieved in selected cases of diabetes by

transplantation of vascularized pancreata or as isolated yield islets deemed adequate for transplantation (51),and generally islets obtained from more than one donorislet grafts (islet transplantation) (47). Despite the steady

increase in organ donation following brain death over pancreas are needed to attain good metabolic controlafter implant (20).the last two decades, pancreas recovery rates remain

unsatisfactory and thus far lower than those of other Multiple variables can influence both the yield andthe quality of islets obtained from a single gland, includ-solid organs, with an underutilization of potentially

transplantable glands (www.optn.org) (25,52). The ing donor and organ characteristics, as well as the methods

Received April 8, 2011; final acceptance July 20, 2011. Online prepub date: February 2, 2012.Address correspondence to Antonello Pileggi, M.D., Ph.D., Associate Professor of Surgery, Microbiology & Immunology and Biomedical Engineer-ing, Director, Preclinical Cell Processing & Translational Models Program, Cell Transplant Center-Diabetes Research Institute, University of Miami,1450 NW 10th Avenue (R-134), Miami, FL 33136, USA. Tel: (305) 243-2924; Fax: (305) 243-4404; E-mail: apileggi@med.miami.edu

1349

1350 HOGAN ET AL.

used for pancreas recovery and preservation (3,27, Pancreatectomy41,49,51). It has been recognized that the duration of As described previously (49), surgery in donor ratspancreas cold ischemia inversely correlates with the was performed under general anesthesia, using an intra-number of islets recovered from a donor pancreas muscular mixture of ketamine hydrochloride 80–100(27,41,49). The ischemic damage endured by the exo- mg/kg (Ketaset; Wyeth-Fort Dodge) and xylazinecrine pancreas may be exacerbated at the time of pancre- 5–10 mg/kg (Anased; Akron) in saline solution. Theatic digestion. This digestion, during the islet isolation common bile duct (CBD) was cannulated with a poly-process, results in the induction of stress-activated inflam- ethylene (PE-50) catheter secured by a suture and thematory pathways and reduces the mass of functionally pancreas was dissected en-block with adjacent stomach,transplantable islets (49). Implementation of strategies duodenum, and spleen. The abdominal viscera were per-aimed at reducing organ ischemic injury may allow for a fused in a retrograde fashion with ice-cold University ofbetter utilization of donor pancreata for transplantation. Wisconsin (UW; ViaSpan; DuPont) preservation solu-Several approaches have been explored in recent years tion, via 24-gauge catheter cannulation of the abdominalthat have generally relied on modifications of organ pro- aorta. Prior to UW infusion, the infrahepatic portal veincurement techniques or of the procurement solution used and inferior vena cava were incised to provide outflowfor organ storage leading to improved islet recovery tracts, and the suprahepatic inferior vena cava and aortarates from deceased donor pancreata (3,30). were occluded. Frozen saline slurry was loaded into the

Ischemic preconditioning (IPC) is emerging in recent abdomen to reduce the temperature of the pancreas dur-years as a means to induce organ resistance to subse- ing organ perfusion and retrieval. The explanted pan-quent noxious stimuli, including ischemic injury. Ini- creas was separated from its vascular pedicle and placedtially described by Murry et al. in the myocardium (39) into a sterile jar containing UW. The jar was kept onand then extended to other organs (liver, kidney, brain, ice for a cold ischemic preservation of 18 h before isletetc.) (8,9,17,19,62), IPC is a cytoprotective surgical pro- isolation was performed.cedure consisting of exposing organs to brief periods ofwarm ischemia (inflow occlusion) followed by briefs

Ischemic Preconditioningperiod of warm reperfusion.

The ischemic preconditioning procedure was per-The effects of IPC have not yet been studied exten-formed prior to CBD cannulation and en-block dissec-sively on the pancreas or on the islets of Langerhans. Intion. This entailed placement of micro-bulldog clampsrodent models of acute pancreatitis, IPC of the pancreason the celiac trunk (CT) and the superior mesentericresulted in a decrease in the severity of pancreatic dam-artery (SMA) (Fig. 1). The arterial supply to the pan-age via a reduction of inflammation and coagulationcreas (and adjacent organs) was occluded for 10 min(11,45,64).(warm ischemia). The clamps were subsequentlyIn the present study, we have evaluated the effect ofremoved and 10 min of reperfusion allowed before ini-IPC of the pancreas in a rodent model of cold ischemictiating the pancreatectomy.preservation (49) through the assessment of multiple

molecular and functional parameters. We observed thatIPC of the pancreas prior to cold preservation can Islet Isolationimprove islet yields and recovery possibly via the modu- As reported previously (49), the dissociation enzymelation of the levels of oxidative stress mediators in the solution (Liberase; Roche) was injected through thegland. Additionally, IPC of the pancreas resulted in the CBD catheter after the ampulla of Vater was clamped.differential expression of multiple molecular markers The distended gland was dissected free from nonpancre-assessed by gene expression that help shedding new atic tissue. Pooled glands were subjected to digestion atlight on the mechanisms underlying IPC-mediated cyto- 37°C for 21 min. This was followed by purification onprotection in the pancreas. Euroficoll discontinuous density gradients (Mediatech).

Islets were cultured in supplemented CMRL mediaMATERIALS AND METHODS(Gibco-Invitrogen) at 37°C and 5% CO2 (10).Animals

Lewis rats (males, 260–280 g) and athymic nu/nuIslet Countsmice (males, 20–22 g)(both from Harlan, Indianapolis,

IN) were housed in virus antibody-free rooms in isolated The zinc-binding dye dithizone (Sigma-Aldrich) wasused to stain islet samples (29), which were subse-cages with free access to autoclaved water and food at

the division of Veterinary Resources of the University quently scored for size range (54). An algorithm wasthen utilized to convert islet counts to the “ideal” 150-of Miami (UM). All animal procedures were performed

under protocols approved and monitored by the IACUC. µm islet (islet equivalent; IEQ) and then multiplied for

PANCREAS ISCHEMIC PRECONDITIONING 1351

Figure 1. Technique for the induction of ischemic preconditioning (IPC) of the rat pancreas. (A) Retroperitoneal vessels areidentified. Along the infrarenal abdominal aorta (AA) the emergence of the celiac trunk (CT) and superior mesenteric artery (SMA)are isolated and prepared for inflow occlusion. In the image are indicated the inferior vena cava (IVC), the liver, the right kidney(RK), duodenum (D), and pancreas. (B) Microvascular clamps are applied on CT and SMA during the inflow occlusion phase ofischemic preconditioning (IPC).

the dilution factor to estimate the total IEQ obtained for tolerance test was performed to assess the metabolic per-formance of transplanted islets. Briefly, after overnighteach condition (54).fasting, animals received an intraperitoneal glucose

Islet Recovery After Culture bolus (2 mg/kg of body weight) and glycemia was mea-After isolation, islet aliquots were cultured in supple- sured with portable glucometers on whole blood (tail

mented CMRL media at 37°C, 5% CO2. After overnight vein) for 120 min in conscious animals. The area underculture, counts allowed for the estimation of the percent the curve (AUC) of glycemic values after bolus was cal-islet recovery over the amount plated on the day of iso- culated for individual animals to evaluate glucose clear-lation, which is a surrogate measure of islet quality ance between experimental groups (22).(23,49).

NADPH-Dependent Oxidase AssayIslet Transplantation Pancreata were either subjected or not subjected to IPC.

Pancreas biopsies were snap-frozen immediately afterA single intravenous streptozotocin (200 mg/kg;Sigma) injection was used to render nude mice diabetic. pancreatectomy and stored at −80°C until assayed. The

nicotinamide adenine dinucleotide phosphate (NADPH)-Glycemia was monitored using portable glucometers(OneTouchUltra2; Lifescan) on whole blood samples dependent superoxide activity was measured by luci-

genin-enhanced chemiluminescence in tissue homoge-obtained from the tail vein. Nonfasting glycemic values≥ 350 mg/dl in three consecutive readings confirmed the nates (60). Briefly, pancreatic tissue was suspended in

phosphate-buffered saline (PBS) containing 2 mM ofoccurrence of diabetes after induction and achievementof normoglycemia (defined as the first of at least three diethylenetriamine pentaacetic acid with the addition

of protease inhibitors (pepstatin, aprotonin, leupeptin,nonfasting glycemic values ≤200 mg/dl on 3 consecutivedays) after islet transplantation. Islets were implanted and phenylmethanesulphonylfluoride, all from Sigma-

Aldrich). Tissue was homogenized and sonicated (twounder the left kidney capsule of diabetic mice under gen-eral anesthesia obtained with isofluorane (Aerrane, 5-s pulses using a cup-horn sonicator probe at �50%

power). Protein concentrations were determined by theBaxter) 2–3% mix with oxygen, as described previously(49). Nephrectomy of the graft-bearing kidney was per- BCA Protein Assay (Pierce). The reaction was initiated

by addition of NADPH (100 µM final concentration)formed in all animals achieving normoglycemia toexclude residual function of the native pancreas, which and dark-adapted lucigenin (5 µM final concentration;

Sigma). Light emission was recorded over several min-was confirmed by prompt resumption of hyperglycemia(48). Postoperative analgesia was obtained by subcuta- utes using a microtiter plate luminometer (Centro-

LB960; Berthold, Germany). Data were expressed asneous injections of buprenorphine (Buprenex; Reckitt-Benckiser) for the first 48 h. An intraperitoneal glucose relative light units (RLU) normalized to protein content

1352 HOGAN ET AL.

and corrected for by a sample blank. Each experiment RESULTSwas performed in duplicate. The specificity of lucigenin- Ischemic Preconditioning Increases the Islet Yieldsenhanced chemiluminscent superoxide detection was Following Cold Pancreas Preservationconfirmed by adding the flavoprotein NADPH oxidase

Islets were obtained in parallel, side-by-side isola-inhibitor diphenyleneiodonium (10 µM) and the respira-

tions (n = 9 individual isolations per group) from poolstory mitochondrial chain inhibitor rotenone (50 µM) to

of rat pancreata that were subjected or not subjected toblock specific superoxide pathways (Sigma) (32,60).

IPC prior to 18-h cold preservation (from 4.7 ± 0.1 and6.3 ± 0.8 donors, respectively). Significantly higher islet

Gene Arrays yields on a per donor basis were obtained from pan-Pancreatic biopsies were stored in RNAlater creata exposed to IPC (964 ± 112 IEQ/donor; n = 42)

(Ambion/Applied-Biosystems) immediately after pan- when compared to controls (711n = 68 IEQ/donor; n =createctomy following or not following IPC treatment 57; p = 0.004) (Fig. 2A).(n = 5 animals per group). Specimens were processed

Pancreatic Ischemic Preconditioning Improves Isletfor RNA isolation and gene arrays by Genome Explora-Recovery After Overnight Culturetions USA (Memphis, TN). Briefly, total RNA was iso-

lated using RNA STAT-60 (Tel-Test; Friendswood, TX) Assessment of the islet counts following the firstfrom �100 mg of frozen pancreatic tissue. Immediately postisolation overnight culture is a surrogate marker ofprior to cDNA synthesis, RNA sample purity and con- islet quality (23,49). Islet recovery after overnight cul-centration were determined by dual-beam UV spectro- ture (calculated as the percent of islets counted on dayphotometry (OD260/280); RNA integrity was determined 1 compared to that plated on day 0) was significantlyby capillary electrophoresis (RNA-6000 Nano Lab-on- lower in the control group (33.7 ± 3.3%; n = 9), whena-Chip kit and a Bioanalyzer 2100; Agilent). The RNA compared to the IPC group (51.3 ± 4.9%; n = 9; p =was processed and labeled according to standard reverse 0.0005) (Fig. 2B).transcription in vitro transcription methods. First- andsecond-strand cDNA were synthesized from total RNA Preserved In Vivo Potency of Islets Isolated(2.0 µg) using T7-oligo(dT)primer (5′-GGCCAGTG From Ischemic PancreataAATTGTAATACGACTCACTATAGGGAGGCGG-3′) Rat pancreata exposed to 18-h cold ischemia fromand cRNA was synthesized, labeled with digoxigenenin- IPC-treated and control donors were processed to isolate11-UTP (Roche) and purified using the NanoAmp RT- islets. After isolation, the islets were cultured overnightIVT Labeling Kit (Applied-Biosystems). Labeled cRNA and then separated into 100 IEQ aliquots. The aliquots(10 µg) was fragmented and hybridized to the Rat were transplanted into chemically induced diabetic nudeGenome Survey Microarray for 16 h at 55°C (Chemilu- mice in order to assess the effects of pancreatic IPC onminescence Detection Kit; Applied-Biosystems), which islet function in vivo (Fig. 3A). Reversal of diabetes incontains 60-mer oligonucleotide probes representing mice after transplantation of islets obtained from controlapproximately 27,000 rat genes in public and Celera data- (n = 10 mice) and IPC-treated (n = 9 mice) pancreatabases. Arrays were washed and stained with anti-digoxi- occurred in 90% and 77.7% of recipients with a mediangenin-AP Fab fragments (Roche). Chemiluminescent of 2 and 3 days, respectively (p = NS). Additionally,signals were measured using the AB1700 Microarray glucose clearance during intraperitoneal glucose toler-Analyzer (Applied-Biosystems). Analysis of expression ance test showed better islet potency in the IPC groupdata, quality control metrics, and statistical analysis when compared to control (Fig. 3B), with the AUC ofwere performed in Bioconductor R using the ABarray glycemic values 9,495 ± 1,292 versus 12,090 ± 2,006package (Applied-Biosystems). Additional gene annota- mg min dl−1, respectively.tion, gene ontology and pathway analysis was performedusing the Celera Panther database. Reduced NADPH-Dependent Oxidase Activity

in Pancreas After Ischemic PreconditioningStatistical Analysis NADPH-dependent oxidase (NOX) is an enzyme that

catalyzes the formation of reactive oxygen species (ROS)Data were analyzed using Microsoft Excel,SigmaPlot-v9.0 (Systat) and Prismv4.00 (GraphPad). (32,59,60). NOX is present in endothelial cells and

smooth muscle cells. To evaluate whether IPC couldComparisons between two experimental groups weredone using paired or unpaired (whenever appropriate) modulate oxidative pathways in pancreatic tissue, biop-

sies were collected right after the completion of pancrea-Student’s t-test. Survival curves were compared usinglog rank test. Data are presented as means ± SE. Statisti- tectomies from glands exposed or not exposed to IPC

and processed to determine NOX activity. IPC-treatedcal significance was considered for p < 0.05.

PANCREAS ISCHEMIC PRECONDITIONING 1353

Figure 2. Effects of ischemic preconditioning (IPC) on islet yield and recovery. (A) Islet yields after isolation (n = 9 per group)from glands exposed to 18 h of cold preservation in University of Wisconsin (UW) solution without (Control, n = 57 pancreata) orafter IPC treatment (IPC; n = 42 pancreata). (B) Islets obtained from pancreata exposed to 18-h cold ischemia without (Control) orwith IPC treatment (IPC) were plated soon after isolation with loss estimated following overnight culture. Data are representativeof 9 independent experiments. Paired Student’s t-test: *p = 0.004; **p = 0.0005. IEQ, islet equivalents.

pancreata expressed significantly lower levels of NOX This analysis elicited 81 genes with >twofold increaseand 454 genes with >twofold decrease when comparingactivity compared to non-IPC pancreata (78.4 ± 19.6 vs.

216.2 ± 19.6 RLU/µg of proteins, respectively; n = 5 IPC-treated glands to controls, respectively (n = 5 pergroup). Table 1 summarizes the most representativepancreata per group; p = 0.003) (Fig. 4).genes identified by our analysis (expressed as fold

Ischemic Preconditioning Is Associated With changes when comparing IPC to control) and groupeda Differential Gene Expression in the Pancreas based on known gene function.

To identify potential molecular signatures associatedDISCUSSIONwith IPC-mediated cytoprotection in the pancreas, we

performed gene expression arrays to assess RNA tran- The ultimate goal of insulin-requiring diabetes thera-pies is attaining optimal metabolic control to stabilizescription on control and IPC-treated pancreata that were

collected immediately after pancreatectomy. The thresh- and prevent the progression of diabetic complications.Transplantation of pancreatic islets, as either vascular-old for statistical significance (p < 0.05) of differential

gene expression was set to twofold in either direction. ized whole organ or isolated cell clusters, offers the

Figure 3. In vivo potency of islets obtained following 18-h of cold pancreas preservation with or without previous IPC treatment.(A) Time to diabetes reversal of athymic nu/nu (nude) mice induced diabetic by streptozotocin and transplanted with 100 IEQ ofislets isolated from untreated glands (Control; n = 10) or IPC-treated glands (IPC; n = 9) exposed to cold ischemia. (B) Meanglycemic values ± SE during an intraperitoneal glucose tolerance test in recipients of rat islets isolated from pancreata treated ornot treated with IPC (n = 5 mice per group). The mean area under the curve of glycemic values for control and IPC group was12,090 ± 2,006 versus 9,495 ± 1,292 mg min dl−1), respectively.

1354 HOGAN ET AL.

preservation and to increase the number of isletsobtained from a single donor pancreas. These includethe implementation of improved donor selection andorgan allocation criteria, pancreas recovery techniquesthat reduce injury during pancreatectomy (30,51), theuse of different preservation solutions (3,43) and oxy-gen-enhancing moieties during organ preservation (2,16,26,53), intraductal injection of preservation solutions(38,57) with antioxidants (1) or protease inhibitors (44),among other strategies.

Ischemic preconditioning represents a natural meansof inducing organ resistance to subsequent ischemicinjury that has been described as efficacious for severalFigure 4. NADPH-dependent superoxide activity in pancreatic

tissue after ischemic preconditioning. Pancreatic biopsies were organs (8,9,17,19,39,62). The effects of and mechanismsobtained from rat glands exposed (IPC) or not exposed to IPC underlying IPC on the pancreas have not been studied(control) prior to pancreatectomy (n = 5 glands per group). extensively. A decrease in the severity of inflammationNicotinamide adenine dinucleotide phosphate (NADPH)-

and coagulation has been described after IPC treatmentdependent superoxide (NOX) activity was measured on tissuein a model of induced pancreatitis (11,45,64).homogenates by lucigenin-enhanced chemiluminescence. Data

are expressed as relative light units (RLU) normalized to pro- In the present study, we evaluated the effects of IPCtein content and corrected for by a sample blank. Paired Stu- prior to cold pancreas preservation injury in a rat modeldent t-test: *p = 0.003. (49). This model of extended cold preservation is associ-

ated with measurable reductions in islet yields after iso-lation and recovery after overnight culture, increasedoption of achieving restoration of β-cell function with a

more physiologic metabolic control than exogenous activation of stress signal transduction pathways, as wellas impaired in vivo potency, when compared to organsinsulin therapy. One of the limitations of β-cell replace-

ment therapies is the current shortage of pancreata for exposed to short ischemia (49). Our current study indi-cates that IPC can significantly improve pancreas qualitytransplantation obtained from heart-beating donors after

cerebral death. after extended cold preservation, as shown by theincreased islet isolation yields in our model. Islet recov-Over the last three decades, the steady increase in

success rates of organ transplantation has been paral- ery after overnight islet culture is a surrogate marker ofislet quality based on the fact that if islets are damagedleled by improved rates of deceased multiorgan donation

both in the US (based on United Network for Organ during the isolation process (i.e., due to pancreatic ische-mic damage) fewer islets will be recovered (49). Indeed,Sharing—UNOS statistics) and abroad. Unfortunately,

the number of donor pancreata recovered for transplan- under control conditions (namely, 18 h of cold preserva-tion without preconditioning), a significantly lowertation (either as whole organ or as isolated islets)

remains unsatisfactory to date (25,52). For instance, number of islets was recovered when compared to IPC-treated pancreata exposed to the same period of coldfrom a pool of >8,000 multiorgan donors available in

2006 through UNOS, approximately 2,000 pancreata preservation. This finding extends our preliminary obser-vations on a smaller sample size (21) and suggests thatwere recovered with <1,500 used for transplant

(www.optn.org). In the US alone, underutilization of IPC treatment prior to cold ischemia may lead to lessdamaged islets after isolation and, therefore, a reductionpancreata potentially “optimal” for transplantation has

been recognized: from the pool of pancreata available in islet loss in culture. Islets isolated from control orIPC-treated pancreata that were subjected to 18-h coldover a 4-year period, 22.3% of “optimal” glands were

used for whole organ transplant, while from the remain- preservation were assessed in vivo using a model of sub-optimal islet transplantation into chemically induced dia-ing pool, 48.5% were considered “suitable islet donors”

(i.e., 11% “optimal” and 89% “standard”), but only betic immunodeficient mice (49). We have previouslyshown that islets obtained from pancreata exposed to2.1% of them were eventually used for islet transplanta-

tion (52). prolonged cold ischemic preservation are severelyimpaired, when compared to those from short ischemicSeveral factors that may influence the quality of pan-

creata and islets for transplantation have been recog- glands in this model (50). In the present study, glucoseclearance in response to intraperitoneal challengenized (3,27,41,49,51,56). The duration of cold ischemia

inversely correlates with organ quality, islet yields, and showed a trend toward better function in recipients ofislets from IPC-treated pancreata with decreased AUCpotency obtained from a donor pancreas (3,27,49). Vari-

ous approaches have been proposed to improve organ than controls, suggesting improved glucose clearance.

PANCREAS ISCHEMIC PRECONDITIONING 1355

Table 1. Ischemic Preconditioning (IPC)-Mediated Differential Gene Expression in the Pancreas

FoldFunction/Symbol Gene Name Gene ID Change*

AngiogenesisAngptl2 Angiopoietin-like 2 21719386 4.17

Cell adhesionClstn3 Calsyntenin 3 22188001 −7.32Cdh22 Cadherin 22 21633870 −7.03Lu Lutheran blood group (Auberger b antigen included) 21398855 −6.52Mag Myelin-associated glycoprotein 20815669 −3.69

Cell differentiationNrg2 Neuregulin 2 21844607 −9.12Chrd Chordin 20776266 −8.94Sema6b Sema domain, transmembrane domain, cytoplasmic domain, (semaphorin) 6B 21972816 −5.65Cdk5rap2 Cyclin-dependent kinase 5 (CDK5) activator-binding protein 21723808 −5.17Klk8 Kallikrein 8 (neuropsin/ovasin) 21882549 −4.95Fgfr1 Fibroblast growth factor receptor 1 21014605 −4.81Dzip1 Deleted in azoospermia (DAZ) interacting protein 1 22420040 −4.28Ddx21a DEAD (Asp-Glu-Ala-Asp) box polypeptide 21a 21916357 −2.47Frag1 Fibroblast growth factor (FGF) receptor activating protein 1 21385343 −2.35Grn Granulin 21842753 −2.08

Cell signalingRgs2 Regulator of G-protein signaling 2 20830772 6.09Tek Endothelial-specific receptor tyrosine kinase 22330185 4.26Camk1 Calcium/calmodulin-dependent protein kinase I 21347280 −9.37Araf1 V-raf oncogene homolog 1 (murine sarcoma 3611 virus) 21751145 −8.22Adcyap1 Adenylate cyclase activating polypeptide 1 20800830 −6.15Taar1 Trace-amine-associated receptor 1 20899571 −5.64Ppp1r1b Protein phosphatase 1, regulatory (inhibitor) subunit 1B 22132190 −5.56Rap2a RAS related protein 2a 21018723 −4.97Epha8 Ephrin (Eph) receptor A8 21868692 −4.71Irs2 Insulin receptor substrate 2 22371221 −4.2Pkig Protein kinase inhibitor, gamma 21620597 −4.08Rasgrf1 RAS protein-specific guanine nucleotide-releasing factor 1 21212924 −3.61Gpr20 G protein-coupled receptor 20 21426773 −3.39Prkca Protein kinase C, alpha 20711520 −2.88Pkn3 Protein kinase N3 22394399 −2.71Gtpbp4 Guanosine triphosphate (GTP) binding protein 4 21541278 −2.15Syngap1 Synaptic Ras GTPase activating protein 1 homolog (rat) 21159510 −2.13

Cell metabolismPrkag2 Protein kinase, AMP-activated, gamma 2 noncatalytic subunit 21011193 3.06Insig1 Insulin induced gene 1 21545770 2.89Pgap1 Glucose-6-phosphate isomerase (GPI) deacylase 22307014 −10.91Ces5 Carboxylesterase 5 22182833 −6.47Igf1r Insulin-like growth factor 1 receptor 21679085 −5.66Colq Collagen-like tail subunit (single strand of homotrimer) of asymmetric acetylcholinesterase 21616866 −3.78Pld2 Phospholipase D2 21594820 −3.61Gphn Gephyrin 21396855 −3.53Pfkl Phosphofructokinase, liver, B-type 22195504 −2.03

Apoptosis relatedCflar Caspase 8 and fas-associated death domain (FADD)-like apoptosis regulator 20893535 3.03Card9 Caspase recruitment domain family, member 9 21811219 −7.08Gzmb Granzyme B 21539290 −5.88Gzmg Granzyme G 22073786 −3.27Gzmc Granzyme C 21369614 −2.77Casp9 Caspase 9 20849613 −2.34

(continued)

1356 HOGAN ET AL.

Table 1. Continued

FoldFunction/Symbol Gene Name Gene ID Change*

Stress responseHspa1a Heat shock 70kD protein 1A 22357696 13.38Hspa1b Heat shock 70kD protein 1B (mapped) 22210564 8.93Ccl2 Chemokine (C-C motif) ligand 2 20907835 8.76Ccl7 Chemokine (C-C motif) ligand 7 20948272 6.83Myd116 Myeloid differentiation primary response gene 116 22152051 5.43Gadd45g Growth arrest and DNA-damage-inducible 45 gamma 22095143 4.12Vof16 Ischemia related factor vof-16 20911041 2.46Ngb Neuroglobin 21369173 −18.43Tsarg1 Testis spermatogenesis apoptosis related protein 1 21881027 −12.39Mgl1 Macrophage galactose N-acetyl-galactosamine specific lectin 1 21979497 −8.26Tnfrsf8 Tumor necrosis factor receptor superfamily, member 8 21916973 −5.88Proc Protein C 21803961 −5.82Ncf1 Neutrophil cytosolic factor 1 (p47phox) 21546613 −5.52Znf14 Zinc finger protein 14 (KOX 6) 21472789 −5.04Klrb1a Killer cell lectin-like receptor subfamily B, member 1A (mapped) 22204047 −4.75Tnfrsf12a Tumor necrosis factor receptor superfamily, member 12a 21622297 −4.63Cd69 CD69 antigen 21548910 −4.37Saa4 Serum amyloid A 4 21641522 −3.96Socs1 Suppressor of cytokine signaling 1 21078402 −3.05Ccl4 Small inducible cytokine A4 21677876 −2.89Gpx6 Glutathione peroxidase 6 22172436 −2.88Ccl5 Chemokine (C-C motif) ligand 5 20896278 −2.87Map3k11 Mitogen-activated protein kinase kinase kinase 11 21208015 −2.12

Transcript regulationFos FBJ murine osteosarcoma viral oncogene homolog 22022961 23.12Nr4a1 Nuclear receptor subfamily 4, group A, member 1 20970853 16.09Junb Jun-B oncogene 22336310 7.26Bhlhb2 Basic helix–loop–helix domain containing, class B2 22013413 7.65Jun Jun oncogene 22313734 5.41Copeb Core promoter element binding protein 21391739 4.57Bhlhb3 Basic helix–loop–helix domain containing, class B3 22140391 4.19Jund Jun D proto-oncogene 20933026 2.13Phox2a Paired-like homeobox 2a 22118192 −5Crabp2 Cellular retinoic acid binding protein 2 20759589 −4.77Ubtf Upstream binding transcription factor, RNA polymerase I 20930584 −3.98Nfil3 Nuclear factor, interleukin 3 regulated 21240715 −3.65

ProteolysisMcpt1 Mast cell protease 1 20711693 −7.08Nln Neurolysin (metallopeptidase M3 family) 22189636 −4.35

*Fold change. Positive numbers indicate upregulation and negative numbers downregulation when comparing IPC to control pancreata.

Modulation of oxidative stress pathways by subject- islets. Involvement of the NOX pathway in postischemicoxidative stress has been recognized for the myocar-ing organs to IPC prior to cold ischemic damage has

been proposed as one of the putative mechanisms under- dium, where an association between decreased NOXactivity and improved myocardial function has beenlying its cytoprotective effects (14,61). We observed that

NOX activity in glands obtained soon after the pan- demonstrated after IPC (14). Evidence of increasedNOX activity in aging mice has been implicated ascreatectomy (following in situ perfusion with cold UW

and before initiation of cold preservation) was signifi- source of the oxidative damage that causes cerebrovas-cular dysfunction (46). Conversely, increased levels ofcantly reduced in IPC compared with control. This sug-

gests a role for the IPC-induced modulation of oxidative NOX activity have been associated with the favorableeffects of IPC in decreasing myocardial infarct (MI) sizestress pathways in the pancreas that may have contrib-

uted to the observed improved outcomes in isolated (5). Additionally, inhibition of NOX activity has been

PANCREAS ISCHEMIC PRECONDITIONING 1357

shown to abolish the hepatoprotective effects of IPC previously reported that CCL4 (macrophage inflammatoryprotein-1α; MIP-1α) production by islets in vitro after(61). The improved outcome associated with the reduc-

tion of NOX activity observed in our study may be isolation is significantly increased after 18 h of coldischemic preservation, when compared to short coldrelated to the intrinsic difference between the pancreas

and other tissues, as well as the experimental design preservation (49). Also, increased CCL5 (regulated uponactivation, normal T cell expressed and secreted;tested. Indeed, the previous study in the MI model was

performed in conditions of warm ischemic damage (5), RANTES) gene expression has observed in rodent isletsexposed to cytokines in vitro and was associated withwhile ours were done by cold perfusion with preserva-

tions solution at the time of organ recovery. Also, the poorer outcome after transplantation (58). The downreg-ulation of Ccl4 and Ccl5 gene expression by IPC in theprevious study on hepatic cytoprotection were per-

formed using an ex vivo perfusion system to mimic present study supports a possible anti-inflammatoryeffect in the pancreas.ischemia-reperfusion (61). Notably, in the pathogene-

sis of atherosclerosis, within the atheroma, low levels The activation of the nuclear factor-κB (NF-κB)pathway in tissues exposed to ischemia and reperfusionof reactive oxygen species promote growth, moderate

levels cause senescence, and high levels induce apo- injury may lead to both initial cytoprotective effects andto intense and deleterious inflammation upon reperfu-ptosis of smooth muscle cells (31,32). This observation

points to the different role of NOX in different stages sion (28). In our study, pancreatic IPC was associatedwith the upregulation of Nfkbia, which encodes an inhib-of pathophysiological conditions. Although not for-

mally addressed in our study, it is reasonable to assume itor of NF-κB that has been shown to decrease islet celldeath (15,18,33). In IPC-treated glands, the expressionthat the low-to-moderate levels seen in the IPC-treated

pancreata, at the very least, do not harm the tissue, of Vof16 was increased. The expression of Vof16,encoding for the ischemia related factor Vof-16, haswhereas the high levels within the control pancreata

might be harmful. been described in the rat brain following chronic ische-mia (63).Molecular arrays have become accessible research

tools to assist in the study of the mechanisms underlying Pancreatic IPC was associated with a sharp reductionof Ncf1 expression. The cytosolic factor p47-phox,complex biological phenomena. These arrays may also

lead to the discovery of potential targets for therapeutic encoded by the neutrophil cytosolic factor (NCF1) gene,is an essential component of the NADPH-oxidase sys-interventions. Because of the lack of in-depth molecular

studies in the context of pancreas ischemia and IPC in tem: Ncf1 transcription is necessary for the activation ofNOX (35). This observation is consistent with thethe scientific literature, we performed a gene array anal-

ysis on glands obtained after pancreatectomy. This observed reduction of NOX activity after IPC in the pan-creas. Ncf1 gene deficiency is associated with decreasesrevealed upregulation of 81 genes and downregulation

of 454 genes (using a cut-off of >twofold change) when in atherosclerotic lesion formation via a reduction inNOX-induced production of reactive oxygen speciescomparing IPC-treated glands to controls, respectively.

Our data indicate that the changes in mRNA synthesis (ROS) (4). Furthermore, the downregulation of tran-scripts for serum amyloid A4 (Saa4) and macrophageinduced by IPC of the pancreas favor cell survival,

based on an observed reduction of genes involved in the galactose N-acetyl-galactosamine specific lectin 1 (Mgl1),which are expressed in inflammatory and fibrotic dis-induction of apoptosis (Table 1). In addition, transcripts

involved in cellular differentiation, signal transduction, eases (34,65), was observed in IPC-treated glands.Members of the heat shock protein gene transcriptand metabolic processes were downregulated (Table 1).

These observations are in agreement with differential family (Hspa1a, Hspa1b, Hspa4) were upregulated afterIPC, suggesting a beneficial stabilization of existing pro-gene expression arrays reported in other models of IPC

of the retina and liver, which have demonstrated a con- teins as a cytoprotective mechanism during the subse-quent period of ischemic injury (6,36,42). In particular,trolled arrest of metabolic functions as a possible under-

lying mechanism of the ischemic tolerance induced by the overexpression of Hspa1a (heat shock 70kD protein)has been associated with the cytoprotective effects ofIPC (24,42).

The expression of stress response genes was signifi- IPC in other organs (58,66), including a rat model ofpancreatitis (64).cantly downregulated by IPC treatment of the pancreas:

several chemokine C-C motif ligands (Ccl2, Ccl4, Ccl5, Pancreatic IPC was associated with the downregula-tion of tumor necrosis factor (TNF) receptors (Tnfrsf8,Ccl7) and cellular adhesion transcripts [cadherin 22

(Cdh22), calsyntenin 3 (Clstn3), Lutheran blood group Tnfrsf12a), which are involved in the induction of cellu-lar apoptosis and TNF production (13,40). The expres-(Lu), myelin-associated glycoprotein (Mag)] that are

involved in immunoregulatory processes were differen- sion of five proapoptotic genes [caspase recruitmentdomain family, member 9 (Card9), caspase 9 (Casp9),tially expressed (7,37). For example, using the same

model of cold pancreas preservation in rats, we have granzyme B, G, and C (Gzmb, Gzmg, and Gzmc)] was

1358 HOGAN ET AL.

downregulated, while the expression of Cflar [CASP8 REFERENCESand fas-associated death domain (FADD)-like apoptosis 1. Avila, J.; Barbaro, B.; Gangemi, A.; Romagnoli, T.;regulator; c-Flip-1], a potent apoptosis inhibitor was Kuechle, J.; Hansen, M.; Shapiro, J.; Testa, G.; Sankary,

H.; Benedetti, E.; Lakey, J.; Oberholzer, J. Intra-ductalupregulated, thus indicating a strict regulation of apopto-glutamine administration reduces oxidative injury duringsis by the IPC. A previous study in rats has shown thathuman pancreatic islet isolation. Am. J. Transplant. 5(12):pancreatic grafts exposed to IPC have increased cellular 2830–2837; 2005.

apoptosis after transplantation (12). This discrepancy 2. Avila, J. G.; Wang, Y.; Barbaro, B.; Gangemi, A.; Qi, M.;from our results may be explained by the multiple differ- Kuechle, J.; Doubleday, N.; Doubleday, M.; Churchill, T.;

Salehi, P.; Shapiro, J.; Philipson, L. H.; Benedetti, E.;ences in the experimental design, including the fact thatLakey, J. R.; Oberholzer, J. Improved outcomes in isletwe assessed gene expression profiles resulting from theisolation and transplantation by the use of a novel hemo-IPC protocol on biopsies obtained immediately after the globin-based O2 carrier. Am. J. Transplant. 6(12):2861–

treatment, whereas the previous study evaluated apopto- 2870; 2006.sis after transplantation, therefore including the effect of 3. Baertschiger, R. M.; Berney, T.; Morel, P. Organ preser-

vation in pancreas and islet transplantation. Curr. Opin.surgical and reperfusion injury. Additionally, we haveOrgan Transplant. 13(1):59–66; 2008.previously demonstrated that �18 h of cold preservation

4. Barry-Lane, P. A.; Patterson, C.; van der Merwe, M.; Hu,of the pancreas results in apoptosis induction primarily Z.; Holland, S. M.; Yeh, E. T.; Runge, M. S. p47phox isin the acinar tissue and not in islet cells (49); in the required for atherosclerotic lesion progression in ApoE(−/−)transplant study (12), cold ischemia was 6 h followed mice. J. Clin. Invest. 108(10):1513–1522; 2001.

5. Bell, R. M.; Cave, A. C.; Johar, S.; Hearse, D. J.; Shah,by reperfusion injury.A. M.; Shattock, M. J. Pivotal role of NOX-2-containingCollectively, our study demonstrates the beneficialNADPH oxidase in early ischemic preconditioning.effects of ischemic preconditioning of the pancreas prior FASEB J. 19(14):2037–2039; 2005.

to cold organ preservation and provides initial evidence 6. Benjamin, I. J.; Kroger, B.; Williams, R. S. Activation ofof the key role of IPC-mediated modulation of oxidative the heat shock transcription factor by hypoxia in mamma-

lian cells. Proc. Natl. Acad. Sci. USA 87(16):6263–6267;stress pathways. Additional pathways and differential1990.gene expression patterns identified in our microarray

7. Bless, N. M.; Huber-Lang, M.; Guo, R. F.; Warner, R. L.;analysis will be of assistance to future studies that aim Schmal, H.; Czermak, B. J.; Shanley, T. P.; Crouch, L. D.;at a better understanding of IPC-mediated cytoprotection Lentsch, A. B.; Sarma, V.; Mulligan, M. S.; Friedl, H. P.;of the pancreas. Ward, P. A. Role of CC chemokines (macrophage inflam-

matory protein-1 beta, monocyte chemoattractant protein-The use of IPC of the pancreas may improve the1, RANTES) in acute lung injury in rats. J. Immunol.quality of a donor pancreas for transplantation and there-164(5):2650–2659; 2000.fore improve organ utilization. Also, the enhancement 8. Clavien, P. A.; Selzner, M.; Rudiger, H. A.; Graf, R.;

of pancreatic preservation may increase the efficiency of Kadry, Z.; Rousson, V.; Jochum, W. A prospective ran-islet isolations and allow for the recovery of a higher domized study in 100 consecutive patients undergoing

major liver resection with versus without ischemic precon-number of functional islets to attain insulin indepen-ditioning. Ann. Surg. 238(6):843–852; 2003.dence from a single donor organ.

9. Clavien, P. A.; Yadav, S.; Sindram, D.; Bentley, R. C.Protective effects of ischemic preconditioning for liverresection performed under inflow occlusion in humans.ACKNOWLEDGMENTS: This work was possible thanks to theAnn. Surg. 232(2):155–162; 2000.generous support of the Diabetes Research Institute Founda-

10. Cobianchi, L.; Fornoni, A.; Pileggi, A.; Molano, R. D.;tion (www.DiabetesResearch.org). Cell processing and animalSanabria, N. Y.; Gonzalez-Quintana, J.; Bocca, N.; Marzorati,procedures were performed at the Pre-clinical Cell ProcessingS.; Zahr, E.; Hogan, A.; Ricordi, C.; Inverardi, L. Ribofla-and Translational Models Core, partially supported by thevin inhibits IL-6 expression and p38 activation in isletJuvenile Diabetes Research Foundation International (to C.R.cells. Cell Transplant. 17(5):559–566; 2008.and A.P.) (4-2004-361; 4-2008-811; 17-2010-5) and by the

11. Dembinski, A.; Warzecha, Z.; Ceranowicz, P.; Tomaszewska,Diabetes Research Institute Foundation (to A.P.). The Univer-R.; Dembinski, M.; Pabianczyk, M.; Stachura, J.; Konturek,sity of Miami Institutional Animal Care and Use CommitteeS. J. Ischemic preconditioning reduces the severity ofhas an Animal Welfare Assurance on file with the Office ofischemia/reperfusion-induced pancreatitis. Eur. J. Pharmacol.Laboratory Animal Welfare (OLAW), National Institutes of473(2–3):207–216; 2003.Health (assurance number: A-3224-01, effective 12/4/02) and

12. Drognitz, O.; Liu, X.; Obermaier, R.; Neeff, H.; vonis accredited by the Association for Assessment and Accredita-Dobschuetz, E.; Hopt, U. T.; Benz, S. Ischemic precondi-tion of Laboratory Animal Care. The authors are grateful totioning fails to improve microcirculation but increasesDrs. Miguel A. Perez-Pinzon, Luca A. Inverardi, David Dellaapoptotic cell death in experimental pancreas transplanta-Morte, Kunjan R. Dave, Ami P. Raval, Hirohito Ichii, andtion. Transpl. Int. 17(6):317–324; 2004.Christopher A. Fraker for constructive scientific discussions

13. Duckett, C. S.; Thompson, C. B. CD30-dependent degra-on this study. Special thanks to Sergio San Jose, Weijun An,dation of TRAF2: Implications for negative regulation ofYelena Gadea, Irayme Labrada, and Kevin Johnson for theirTRAF signaling and the control of cell survival. Genesexcellent technical assistance. The authors declare no conflictsDev. 11(21):2810–2821; 1997.of interest.

PANCREAS ISCHEMIC PRECONDITIONING 1359

14. Duda, M.; Konior, A.; Klemenska, E.; Beresewicz, A. 28. Latanich, C. A.; Toledo-Pereyra, L. H. Searching for NF-kappaB-based treatments of ischemia reperfusion injury.Preconditioning protects endothelium by preventing ET-1-

induced activation of NADPH oxidase and xanthine oxi- J. Invest. Surg. 22(4):301–315; 2009.29. Latif, Z. A.; Noel, J.; Alejandro, R. A simple method ofdase in post-ischemic heart. J. Mol. Cell. Cardiol. 42(2):

400–410; 2007. staining fresh and cultured islets. Transplantation 45(4):827–830; 1988.15. Eldor, R.; Yeffet, A.; Baum, K.; Doviner, V.; Amar, D.;

Ben-Neriah, Y.; Christofori, G.; Peled, A.; Carel, J. C.; 30. Lee, T. C.; Barshes, N. R.; Brunicardi, F. C.; Alejandro,R.; Ricordi, C.; Nguyen, L.; Goss, J. A. Procurement ofBoitard, C.; Klein, T.; Serup, P.; Eizirik, D. L.; Melloul,

D. Conditional and specific NF-kappaB blockade protects the human pancreas for pancreatic islet transplantation.Transplantation 78(3):481–483; 2004.pancreatic beta cells from diabetogenic agents. Proc. Natl.

Acad. Sci. USA 103(13):5072–5077; 2006. 31. Li, J.; Kon, L. M.; Guillory, R. J. Papain proteolysisreleases a soluble NADPH dependent diaphorase activity16. Fraker, C. A.; Alejandro, R.; Ricordi, C. Use of oxygen-

ated perfluorocarbon toward making every pancreas from bovine neutrophil membranes. FEBS Lett. 424(3):188–192; 1998.count. Transplantation 74(12):1811–1812; 2002.

17. Franchello, A.; Gilbo, N.; David, E.; Ricchiuti, A.; 32. Li, J. M.; Shah, A. M. Differential NADPH- versusNADH-dependent superoxide production by phagocyte-Romagnoli, R.; Cerutti, E.; Salizzoni, M. Ischemic pre-

conditioning (IP) of the liver as a safe and protective tech- type endothelial cell NADPH oxidase. Cardiovasc. Res.52(3):477–486; 2001.nique against ischemia/reperfusion injury (IRI). Am. J.

Transplant. 9(7):1629–1639; 2009. 33. Liu, D.; Cardozo, A. K.; Darville, M. I.; Eizirik, D. L.Double-stranded RNA cooperates with interferon-gamma18. Giannoukakis, N.; Rudert, W. A.; Trucco, M.; Robbins,

P. D. Protection of human islets from the effects of inter- and IL-1 beta to induce both chemokine expression andnuclear factor-kappa B-dependent apoptosis in pancre-leukin-1beta by adenoviral gene transfer of an Ikappa B

repressor. J. Biol. Chem. 275(47):36509–36513; 2000. atic beta-cells: Potential mechanisms for viral-inducedinsulitis and beta-cell death in type 1 diabetes mellitus.19. Heizmann, O.; Loehe, F.; Volk, A.; Schauer, R. J. Ische-

mic preconditioning improves postoperative outcome after Endocrinology 143(4):1225–1234; 2002.34. Liu, T.; Dhanasekaran, S. M.; Jin, H.; Hu, B.; Tomlins,liver resections: A randomized controlled study. Eur. J.

Med. Res. 13(2):79–86; 2008. S. A.; Chinnaiyan, A. M.; Phan, S. H. FIZZ1 stimulationof myofibroblast differentiation. Am. J. Pathol. 164(4):20. Hogan, A.; Pileggi, A.; Ricordi, C. Transplantation: Cur-

rent developments and future directions; the future of clin- 1315–1326; 2004.35. Lopes, L. R.; Hoyal, C. R.; Knaus, U. G.; Babior, B. M.ical islet transplantation as a cure for diabetes. Front.

Biosci. 13:1192–1205; 2008. Activation of the leukocyte NADPH oxidase by proteinkinase C in a partially recombinant cell-free system. J.21. Hogan, A. R.; Doni, M.; Ribeiro, M. M.; Molano, R. D.;

Cobianchi, L.; Molina, J.; Zahr, E.; Ricordi, C.; Pastori, Biol. Chem. 274(22):15533–15537; 1999.36. Massip-Salcedo, M.; Casillas-Ramirez, A.; Franco-Gou,R. L.; Pileggi, A. Ischemic preconditioning improves islet

recovery after pancreas cold preservation. Transplant. R.; Bartrons, R.; Ben Mosbah, I.; Serafin, A.; Rosello-Catafau, J.; Peralta, C. Heat shock proteins and mitogen-Proc. 41(1):354–355; 2009.

22. Ichii, H.; Pileggi, A.; Molano, R. D.; Baidal, D. A.; Khan, activated protein kinases in steatotic livers undergoingischemia-reperfusion: Some answers. Am. J. Pathol.A.; Kuroda, Y.; Inverardi, L.; Goss, J. A.; Alejandro, R.;

Ricordi, C. Rescue purification maximizes the use of 168(5):1474–1485; 2006.37. Menten, P.; Proost, P.; Struyf, S.; Van Coillie, E.; Put,human islet preparations for transplantation. Am. J. Trans-

plant. 5(1):21–30; 2005. W.; Lenaerts, J. P.; Conings, R.; Jaspar, J. M.; De Groote,D.; Billiau, A.; Opdenakker, G.; Van Damme, J. Differen-23. Ichii, H.; Sakuma, Y.; Pileggi, A.; Fraker, C.; Alvarez,

A.; Montelongo, J.; Szust, J.; Khan, A.; Inverardi, L.; tial induction of monocyte chemotactic protein-3 in mono-nuclear leukocytes and fibroblasts by interferon-alpha/betaNaziruddin, B.; Levy, M. F.; Klintmalm, G. B.; Goss,

J. A.; Alejandro, R.; Ricordi, C. Shipment of human islets and interferon-gamma reveals MCP-3 heterogeneity. Eur.J. Immunol. 29(2):678–685; 1999.for transplantation. Am. J. Transplant. 7(4):1010–1020;

2007. 38. Munn, S. R.; Kaufman, D. B.; Field, M. J.; Viste, A. B.;Sutherland, D. E. Cold-storage preservation of the canine24. Kamphuis, W.; Dijk, F.; van Soest, S.; Bergen, A. A.

Global ge Globne expression profiling of ischemic pre- and rat pancreas prior to islet isolation. Transplantation47(1):28–31; 1989.conditioning in the rat retina. Mol. Vis. 13:1020–1030;

2007. 39. Murry, C. E.; Jennings, R. B.; Reimer, K. A. Precondi-tioning with ischemia: A delay of lethal cell injury in25. Krieger, N. R.; Odorico, J. S.; Heisey, D. M.; D’Alessandro,

A. M.; Knechtle, S. J.; Pirsch, J. D.; Sollinger, H. W. ischemic myocardium. Circulation 74(5):1124–1136;1986.Underutilization of pancreas donors. Transplantation

75(8):1271–1276; 2003. 40. Nakayama, M.; Ishidoh, K.; Kojima, Y.; Harada, N.;Kominami, E.; Okumura, K.; Yagita, H. Fibroblast growth26. Kuroda, Y.; Kawamura, T.; Suzuki, Y.; Fujiwara, H.;

Yamamoto, K.; Saitoh, Y. A new, simple method for factor-inducible 14 mediates multiple pathways of TWEAK-induced cell death. J. Immunol. 170(1):341–348; 2003.cold storage of the pancreas using perfluorochemical.

Transplantation 46(3):457–460; 1988. 41. Nano, R.; Clissi, B.; Melzi, R.; Calori, G.; Maffi, P.;Antonioli, B.; Marzorati, S.; Aldrighetti, L.; Freschi, M.;27. Lakey, J. R.; Warnock, G. L.; Rajotte, R. V.; Suarez-

Alamazor, M. E.; Ao, Z.; Shapiro, A. M.; Kneteman, Grochowiecki, T.; Socci, C.; Secchi, A.; Di Carlo, V.;Bonifacio, E.; Bertuzzi, F. Islet isolation for allotransplan-N. M. Variables in organ donors that affect the recovery

of human islets of Langerhans. Transplantation 61(7): tation: Variables associated with successful islet yield andgraft function. Diabetologia 48(5):906–912; 2005.1047–1053; 1996.

1360 HOGAN ET AL.

42. Navarro-Sabate, A.; Peralta, C.; Calvo, M. N.; Manzano, Mintz, D. H.; Lacy, P. E. Islet isolation assessment in manand large animals. Acta Diabetol. Lat. 27(3):185–195;A.; Massip-Salcedo, M.; Rosello-Catafau, J.; Bartrons, R.

Mediators of rat ischemic hepatic preconditioning after 1990.55. Ricordi, C.; Lakey, J. R.; Hering, B. J. Challenges towardcold preservation identified by microarray analysis. Liver

Transpl. 12(11):1615–1625; 2006. standardization of islet isolation technology. Transplant.Proc. 33(1–2):1709; 2001.43. Noguchi, H.; Naziruddin, B.; Onaca, N.; Jackson, A.;

Shimoda, M.; Ikemoto, T.; Fujita, Y.; Kobayashi, N.; 56. Saito, Y.; Goto, M.; Maya, K.; Ogawa, N.; Fujimori, K.;Kurokawa, Y.; Satomi, S. Brain death in combination withLevy, M. F.; Matsumoto, S. Comparison of modified Cel-

sior solution and M-Kyoto solution for pancreas preserva- warm ischemic stress during isolation procedures inducesthe expression of crucial inflammatory mediators in thetion in human islet isolation. Cell Transplant. 19(6/7):

751–758; 2010. isolated islets. Cell Transplant. 19(6):775–782; 2010.57. Sawada, T.; Matsumoto, I.; Nakano, M.; Kirchhof, N.;44. Noguchi, H.; Ueda, M.; Hayashi, S.; Kobayashi, N.;

Nagata, H.; Iwanaga, Y.; Okitsu, T.; Matsumoto, S. Com- Sutherland, D. E.; Hering, B. J. Improved islet yield andfunction with ductal injection of University of Wisconsinparison of M-Kyoto solution and histidine-tryptophan-

ketoglutarate solution with a trypsin inhibitor for pancreas solution before pancreas preservation. Transplantation75(12):1965–1969; 2003.preservation in islet transplantation. Transplantation 84(5):

655–658; 2007. 58. Schroppel, B.; Zhang, N.; Chen, P.; Chen, D.; Bromberg,J. S.; Murphy, B. Role of donor-derived monocyte chem-45. Obermaier, R.; von Dobschuetz, E.; Drognitz, O.; Hopt,

U. T.; Benz, S. Ischemic preconditioning attenuates capil- oattractant protein-1 in murine islet transplantation. J. Am.Soc. Nephrol. 16(2):444–451; 2005.lary no-reflow and leukocyte adherence in postischemic

pancreatitis. Langenbecks Arch. Surg. 389(6):511–516; 59. Sorescu, D.; Szocs, K.; Griendling, K. K. NAD(P)H oxi-dases and their relevance to atherosclerosis. Trends Cardi-2004.

46. Park, L.; Anrather, J.; Girouard, H.; Zhou, P.; Iadecola, C. ovasc. Med. 11(3–4):124–131; 2001.60. Szeto, A.; Nation, D. A.; Mendez, A. J.; Dominguez-Nox2-derived reactive oxygen species mediate neurovas-

cular dysregulation in the aging mouse brain. J. Cereb. Bendala, J.; Brooks, L. G.; Schneiderman, N.; McCabe,P. M. Oxytocin attenuates NADPH-dependent superoxideBlood Flow Metab. 27(12):1908–1918; 2007.

47. Pileggi, A.; Alejandro, R.; Ricordi, C. Islet transplanta- activity and IL-6 secretion in macrophages and vascularcells. Am. J. Physiol. Endocrinol. Metab. 295(6):E1495–tion: Steady progress and current challenges. Curr. Opin.

Organ Transplant. 11(1):7–13; 2006. 1501; 2008.61. Tejima, K.; Arai, M.; Ikeda, H.; Tomiya, T.; Yanase, M.;48. Pileggi, A.; Molano, R. D.; Berney, T.; Ichii, H.; San Jose,

S.; Zahr, E.; Poggioli, R.; Linetsky, E.; Ricordi, C.; Inverardi, Inoue, Y.; Nishikawa, T.; Watanabe, N.; Ohtomo, N.;Omata, M.; Fujiwara, K. Induction of ischemic toleranceL. Prolonged allogeneic islet graft survival by protopor-

phyrins. Cell Transplant. 14(2–3):85–96; 2005. in rat liver via reduced nicotinamide adenine dinucleotidephosphate oxidase in Kupffer cells. World J. Gastroent-49. Pileggi, A.; Ribeiro, M. M.; Hogan, A. R.; Molano, R. D.;

Cobianchi, L.; Ichii, H.; Embury, J.; Inverardi, L.; Fornoni, erol. 13(38):5071–5078; 2007.62. Thornton, M. A.; Zager, R. A. Brief intermittent reperfu-A.; Ricordi, C.; Pastori, R. L. Impact of pancreatic cold pres-

ervation on rat islet recovery and function. Transplantation sion during renal ischemia: Effects on adenine nucleo-tides, oxidant stress, and the severity of renal failure. J.87(10):1442–1450; 2009.

50. Poggioli, R.; Faradji, R. N.; Ponte, G.; Betancourt, Lab. Clin. Med. 115(5):564–571; 1990.63. Tohda, M.; Watanabe, H. Molecular cloning and charac-A.; Messinger, S.; Baidal, D. A.; Froud, T.; Ricordi, C.;

Alejandro, R. Quality of life after islet transplantation. terization of a novel sequence, vof-16, with enhancedexpression in permanent ischemic rat brain. Biol. Pharm.Am. J. Transplant. 6(2):371–378; 2006.

51. Ponte, G. M.; Pileggi, A.; Messinger, S.; Alejandro, A.; Bull. 27(8):1228–1235; 2004.64. Warzecha, Z.; Dembinski, A.; Ceranowicz, P.; Konturek,Ichii, H.; Baidal, D. A.; Khan, A.; Ricordi, C.; Goss, J. A.;

Alejandro, R. Toward maximizing the success rates of S. J.; Dembinski, M.; Pawlik, W. W.; Tomaszewska, R.;Stachura, J.; Kusnierz-Cabala, B.; Naskalski, J. W. andhuman islet isolation: Influence of donor and isolation fac-

tors. Cell Transplant. 16(6):595–607; 2007. others. Ischemic preconditioning inhibits development ofedematous cerulein-induced pancreatitis: Involvement of52. Porrett, P. M.; Yeh, H.; Frank, A.; Deng, S.; Kim, J. I.;

Barker, C. F.; Markmann, J. F. Availability of suitable cyclooxygenases and heat shock protein 70. World J.Gastroenterol. 11(38):5958–5965; 2005.islet donors in the United States. Transplantation 84(2):

280–282; 2007. 65. Wu, J.; Kobayashi, M.; Sousa, E. A.; Liu, W.; Cai, J.;Goldman, S. J.; Dorner, A. J.; Projan, S. J.; Kavuru,53. Qin, H.; Matsumoto, S.; Klintmalm, G. B.; De Vol, E. B.

A meta-analysis for comparison of the two-layer and Uni- M. S.; Qiu, Y.; Thomassen, M. J. Differential proteomicanalysis of bronchoalveolar lavage fluid in asthmatics fol-versity of Wisconsin pancreata preservation methods in

islet transplantation. Cell Transplant. 20(7):1127–1137; lowing segmental antigen challenge. Mol. Cell. Proteomics4(9):1251–1264; 2005.2011.

54. Ricordi, C.; Gray, D. W.; Hering, B. J.; Kaufman, D. B.; 66. Yeh, C. H.; Hsu, S. P.; Yang, C. C.; Chien, C. T.; Wang,N. P. Hypoxic preconditioning reinforces HIF-alpha-Warnock, G. L.; Kneteman, N. M.; Lake, S. P.; London,

N. J.; Socci, C.; Alejandro, R.; Zeng, Y.; Scharp, D. W.; dependent HSP70 signaling to reduce ischemic renal fail-ure-induced renal tubular apoptosis and autophagy. LifeViviani, G.; Falqui, L.; Tzakis, A.; Bretzel, R. G.; Federlin,

K.; Pozza, G.; James, R. F. L.; Rajotte, R. V.; Di Carlo, Sci. 86(3–4):115–123; 2010.V.; Morris, P. J.; Sutherland, D. E. R.; Starzl, T. E.;