Platelets and Platelet-derived SerotoninPromote Tissue Repair After

Normothermic Hepatic Ischemia in MiceAntonio Nocito,1 Panco Georgiev,1 Felix Dahm,1 Wolfram Jochum,2 Michael Bader,3

Rolf Graf,1 and Pierre-Alain Clavien1

Hepatic ischemia and reperfusion (I/R) leads to the formation of leukocyte–platelet aggregates.Upon activation, platelets generate reactive oxygen species and release proapoptotic and proin-flammatory mediators as well as growth factors. In cold hepatic ischemia, adhesion of platelets toendothelial cells mediates sinusoidal endothelial cell apoptosis. Furthermore, platelet-derivedserotonin mediates liver regeneration. We hypothesized that platelets may contribute to reper-fusion injury and repair after normothermic hepatic ischemia. The aim of this study was to assessthe impact of platelets in normothermic hepatic I/R injury using models of impaired plateletfunction and immune thrombocytopenia. Inhibition of platelet function in mice was achievedvia clopidogrel feeding. Immune thrombocytopenia was induced via intraperitoneal injection ofanti-CD41 antibody. Platelet-derived serotonin was investigated using mice lacking tryptophanhydroxylase 1. Mice were subjected to 60 minutes of partial hepatic ischemia and various timepoints of reperfusion. Hepatic injury was determined via AST and histological analysis of thenecrotic area as well as leukocyte infiltration. Liver regeneration was determined via proliferatingcell nuclear antigen and Ki67 immunohistochemistry. Neither inhibition of platelet function norplatelet depletion led to a reduction of I/R injury. Liver regeneration and repair were significantlyimpaired in platelet-depleted animals. Mice lacking peripheral serotonin were deficient in hepa-tocyte proliferation, but otherwise displayed normal tissue remodeling. Conclusion: Plateletshave no direct impact on the pathogenesis of normothermic I/R injury. However, they mediatetissue repair and liver regeneration. Furthermore, platelet-derived serotonin is a mediator ofhepatocyte proliferation in the postischemic liver, but has no impact on tissue remodeling.(HEPATOLOGY 2007;45:369-376.)

Platelets are specialized blood constituents that playpivotal roles in physiological and pathological pro-cesses, including hemostasis, inflammation, host

defense, and wound healing. Upon activation, platelets

release inflammatory mediators, growth factors, and pro-teases that can either enhance or limit tissue injury de-pending on the specific context. For example, inhibitionof thrombocyte activity reduces ischemic tissue injury inthe heart,1 lung,2 and pancreas.3

In the liver, platelets and leukocytes synergistically trig-ger sinusoidal endothelial cell apoptosis upon reperfusionof cold preserved organs.4-9 During transplantation, theliver is subjected to a combination of hypothermic (cold)ischemia, intentionally applied to protect the liver graft,and normothermic (warm) ischemia.10,11 Apart from thetransplantation setting, warm ischemia also occurs duringliver resection or in shock. In contrast to cold ischemia,warm ischemia is tolerated only for a short period andrapidly leads to hepatocellular injury and tissue necro-sis.12,13

Little is known about the impact of platelets on nor-mothermic ischemia/reperfusion (I/R) injury of the liver.Mice deficient for P-selectin, an adhesion molecule criti-cal to the postischemic platelet–endothelial cell interac-tion,14 display reduced platelet and neutrophilsequestration and a better survival following warm isch-

Abbreviations: I/R, ischemia/reperfusion; PCNA, proliferating cell nuclear anti-gen; PMN, polymorphonuclear leukocyte; TNF-�, tumor necrosis factor-�; Tph1,tryptophan hydroxylase 1.

From the Departments of 1Visceral and Transplantation Surgery and 2Pathology,Swiss Hepato-Pancreato-Biliary Center, University Hospital Zurich, Zurich, Swit-zerland; and the 3Max Delbruck Center for Molecular Medicine, Berlin, Germany.

Received August 17, 2006; accepted October 25, 2006.Supported by the Olga Mayenfisch Foundation, Zurich, Switzerland (A. N.); the

Swiss National Foundation (3200B0-109906), Bern, Switzerland (P. A. C.); andthe Gebert Ruef Foundation, Basel, Switzerland (W. J.).

This study was presented at the 93rd Annual Meeting of the Swiss SurgicalSociety, where it was awarded the 2006 Research Prize.

Address reprint requests to: Pierre Alain Clavien, M.D., Ph.D., Department of Visceraland Transplantation Surgery, University Hospital Zurich, Ramistrasse 100, CH-8091 Zu-rich, Switzerland. E-mail: clavien@chir.unizh.ch; fax: (41) 44-255-44-49.

Copyright © 2007 by the American Association for the Study of Liver Diseases.Published online in Wiley InterScience (www.interscience.wiley.com).DOI 10.1002/hep.21516Potential conflict of interest: Nothing to report.Supplementary material for this article can be found on the HEPATOLOGY website

(http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html).

369

emia.15 In addition, the inhibition of platelet adhesion bythe administration of anti-fibrinogen antibody decreasesshort-term liver injury after ischemia.16 However, theseexperimental approaches also inhibit leukocyte-mediatedeffects; thus, the specific contribution of platelets to warmI/R injury of the liver is still unknown. Ischemic liverinjury also induces tissue repair, a process that includesinflammation, tissue resorption, and remodeling, as wellas hepatocyte proliferation. Interestingly, the initiation ofhepatocellular proliferation and liver regeneration haverecently been shown to involve the release of platelet-derived serotonin.17

Consequently, platelets could exert opposing effects bymediating ischemic liver injury on the one hand and byfacilitating the ensuing tissue repair process on the otherhand. Therefore, using an established protocol of phar-macological platelet inhibition and a new model of plate-let depletion, we evaluated the role of platelets innormothermic ischemia and reperfusion injury of theliver. We further investigated how platelets influencepostischemic inflammation, and whether they play a crit-ical role in the repair process following warm hepatic isch-emia. Finally, we assessed the impact of platelet-derivedserotonin in mediating tissue repair following an ischemicinsult.

Materials and Methods

Animals. Experiments were performed on 8-week-oldto 12-week-old male wild-type (Harlan, the Netherlands)and tryptophan hydroxylase 1 (Tph1)�/� mice (kindlyprovided by M. Bader18), both with a C57BL/6 back-ground. Animals were kept in the animal facility of theUniversity Hospital of Zurich with access to standardchow and water ad libitum. All procedures were approvedby the Veterinary Office of the Canton of Zurich andwere performed in accordance with institutional animalcare guidelines.

Inhibition of Platelet Function. Inhibition of plate-let adhesion and aggregation with clopidogrel (Sanofi-Synthelabo-Recherche, Toulouse, France; 5 mg/kg/day)was performed as described.17 Clopidogrel or equal vol-umes of saline (controls) were administered via daily ga-vage starting 5 days before ischemia and continued untilharvesting.

Immune Thrombocytopenia Model. Thrombocyto-penia was induced in C57BL/6 mice via intraperitonealinjection of 1 mg/kg anti-CD41 (glycoprotein GPIIb,clone MWReg 30, BD Biosciences, Franklin Lakes, NJ)diluted in 200 �l phosphate-buffered saline. This strategyleads to a reduction of platelets below 10% of their initialvalue (see Supplementary Figure). Control mice were

treated via intraperitoneal injection of an equivalentamount of nonimmune IgG2 (BD Biosciences, FranklinLakes, NJ). Whole blood samples (20 �l) were collectedfrom the tail vein before injection and 24, 48, 72, and 96hours after injection and diluted in 20 �l EDTA (12mM). Blood cell counts were assessed using a Coulter AcTdiff counter (Beckman Coulter, Nyon, Switzerland). Sus-tained platelet depletion (7 d) was achieved via additionaldaily injection of 0.5 mg/kg anti-CD41 starting on thefirst postoperative day.

Partial Hepatic Ischemia. A model of 70% hepaticischemia was used in all experiments as described.19

Group size was n � 5 unless otherwise indicated. Micewere anesthetized with inhalation of isoflurane/O2

(Halocarbon Laboratories, River Edge, NJ), and a con-stant gas mixture was maintained with a vaporizer sys-tem (Provet, Basel, Switzerland). After midlinelaparotomy, the liver was freed from its ligaments. Sub-sequently all structures of the portal triad to the left andmedian hepatic lobes were occluded for 60 minuteswith a microvascular clamp (Aesculap, San Francisco,CA). In this model, mesenteric congestion is preventedby allowing intestinal blood flow through the right andcaudate lobes. Reperfusion was initiated via removal ofthe clamp.

Serum AST Levels. Blood samples were obtainedfrom the inferior vena cava after different periods of reper-fusion and were immediately centrifuged at 2,000g for 6minutes. AST levels were measured using a serum multi-ple biochemical analyzer (Ektachem DTSCII; Johnson &Johnson Inc., Rochester, NY).

Histological Examination. Liver tissue was immer-sion-fixed in 4% buffered formaldehyde embedded inparaffin, sectioned, and stained with hematoxylin-eosinusing standard histological techniques. In addition, slideswere immunostained for myeloperoxidase (polyclonalrabbit antibody; NeoMarkers, Fremont, CA), proliferat-ing cell nuclear antigen (PCNA) (polyclonal rabbit anti-body; clone PC-10, InnoGenex, San Ramon, CA), andKi-67 (monoclonal rabbit clone SP6; NeoMarkers) usingthe Ventana Discovery automated staining system withthe DAB Map kit (Ventana, Tucson, AZ). All immuno-stains were counterstained with hematoxylin. Polymor-phonuclear leukocyte (PMN) infiltration was scoredsemiquantitatively on a scale of 1 (none) to 4 (severe).PCNA-positive and Ki-67–positive cells were counted in10 randomly selected high-power fields (�400) per slide.All histologic analyses were performed by 2 investigatorsblinded with respect to the experimental group.

Quantitative Real-Time PCR. Total RNA was ex-tracted from 50 mg of liver tissue using TRIzol reagent

370 NOCITO ET AL. HEPATOLOGY, February 2007

(Invitrogen, Paisley, Scotland, UK) following the manu-facturer’s instructions. Five micrograms of RNA was re-verse-transcribed using the ThermoScript RT-PCRSystem (Invitrogen, Basel, Switzerland) kit yielding thecomplementary DNA template. Quantitative real-timePCR amplification and data analysis were performed us-ing an ABI Prism 7000 Sequence Detector System (PEApplied Biosystems, Rotkreuz, Switzerland). TaqMangene expression assays (PE Applied Biosystems, Rotkreuz,Switzerland) for IL-1� (Mm00434228_m1), IL-6 (Mm00446190_m1), Cxcl2 (Mm 00436450_m1), and tumornecrosis factor-� (TNF-�) (Mm 00443258_m1) wereused to quantify mRNA expression of the respectivegenes. Messenger RNA expression levels of each samplewere normalized to 18S RNA (TaqMan ribosomal RNAcontrol reagents, PE Applied Biosystems). The resultsshown represent fold induction after ischemia versus base-line levels in control mice before ischemia.

ELISA. TNF-� and IL-6 levels in serum were deter-mined by ELISA (DuoSet mouse TNF-� and IL-6; R&DSystems, Minneapolis, MN) according to the manufac-turer’s instructions.

Statistical Analysis. Data are expressed as themean � SD. Continuous variables were compared withthe Student t test, ordinal variables with the Mann-Whit-ney U test. A P value of 0.05 was considered significant.Data analysis was performed with SPSS 12.0.1 for Win-dows (SPSS Inc., Chicago, IL).

Results

Does Depletion or Functional Inhibition of Plate-lets Reduce Normothermic Hepatic I/R Injury? To testwhether platelets mediate normothermic hepatic I/R in-jury, platelets were depleted with anti-CD41 antibody orwere functionally inhibited with clopidogrel (starting 5 dbefore ischemia) before 1 hour of ischemia. Liver injurywas determined by AST release (Fig. 1A,C) after 1, 4, 24,and 48 hours of reperfusion as well as histological quan-tification of liver necrosis (Fig. 1B,D) after 4 and 24 hoursof reperfusion. No difference in AST release or histolog-ical liver injury was detected between control animals andeither strategy of platelet inhibition. These findings sug-gest that platelets do not contribute directly to ischemicliver injury.

Does Depletion or Functional Inhibition of Plate-lets Reduce Postischemic Leukocyte Infiltration?Platelets are known to interact with leukocytes and maytherefore play a role in the inflammatory reaction follow-ing hepatic ischemia. To assess this interaction, we quan-tified neutrophils in a semiquantitative fashion usingmyeloperoxidase immunostaining 24 hours after reperfu-sion. Whereas PMN infiltration was not affected by clo-pidogrel treatment (Fig. 2A), platelet-depleted animalsshowed significantly less leukocyte infiltration of the post-ischemic liver (Fig. 2B). A representative microphoto-graph is shown in Fig. 2C.

Fig. 1. Quantification of postischemic liver injury. (A) Serum AST levels after 60 minutes of ischemia and 1, 4, 24, and 48 hours of reperfusion(dashed line, control; solid line, clopidogrel-treated animals). (B) Extension of liver injury determined on liver sections via morphometric analysis ofthe necrotic area after 60 minutes of ischemia and 4 and 24 hours of reperfusion (black bars, clopidogrel-treated animals; white bars, control). (C)Serum AST levels after 60 minutes of ischemia and 1, 4, 24, and 48 hours of reperfusion (dashed line, control; solid line, platelet-depleted animals).(D) Histological liver injury after 60 minutes of ischemia and 4 and 24 hours of reperfusion (black bars, platelet-depleted animals; white bars, control).

HEPATOLOGY, Vol. 45, No. 2, 2007 NOCITO ET AL. 371

Does Depletion or Functional Inhibition of Plate-lets Reduce Postischemic Expression of InflammatoryMediators? Attraction of neutrophils to the site of isch-emic tissue injury is mediated by the release of cytokinesand chemokines. TNF-�, IL-6, and IL-1� are majorproinflammatory cytokines in the liver, while the chemo-kine Cxcl2 (also known as MIP-2) is a strong chemoat-tractant for neutrophils, which is involved in ischemicliver injury.20,21 The presence of transcripts coding forthese proinflammatory molecules was analyzed via real-time PCR. Whereas TNF-� and IL-1� transcripts (Fig.4A,C) were only slightly reduced, expression levels of IL-6and Cxcl2 (Fig. 4B,D) were significantly lower in platelet-depleted animals. Consistent with reduced neutrophil in-filtration, platelet depletion caused a marked reduction inpostischemic TNF-� and IL-6 release (Fig. 5), as deter-mined via ELISA. Importantly, inhibition of platelet ag-

gregation by clopidogrel did not affect the expression ofproinflammatory mediators (Fig. 3), in accordance withthe lack of effect on PMN infiltration (Fig. 2A). Takentogether, these data suggest that platelets are required forthe secretion of proinflammatory cytokines, which in turnare crucial for neutrophil infiltration.

Does Platelet Depletion Reduce Postischemic LiverRepair and Regeneration? Inflammation is crucial forthe containment and consecutive resorption of damagedtissue. In view of reduced inflammation and neutrophilinfiltration, we asked whether platelet depletion wouldalso delay tissue repair in the postischemic liver. To thisend, we quantified residual liver necrosis 7 days after isch-emic insult (Fig. 6). Whereas control animals displayedminimal residual necrosis (3% of liver tissue), platelet-depleted animals still showed more than 10% necrosis,indicating a delayed resolution.

Restitution of hepatic parenchyma after an ischemicinsult requires a combination of tissue resorption and re-modeling as well as hepatocellular proliferation. Becauseplatelets are critical mediators of liver regeneration afterhepatectomy,17 we hypothesized that they would also playa role in the restitution of hepatic parenchyma followingischemia. Quantification of hepatocyte regeneration by

Fig. 3. Effects of inhibition of platelet function on postischemicexpression of inflammatory mediators: expression of proinflammatorymediators in liver tissue after 60 minutes of ischemia followed by 4 hoursand 24 hours of reperfusion determined via real-time PCR. Transcriptlevels were normalized to livers receiving saline or clopidogrel treatmentwithout I/R and are given as fold induction (white bars, control; blackbars, clopidogrel-treated animals).

Fig. 2. Effect of depletion and functional inhibition of platelets onpostischemic inflammation: semiquantitative analysis of PMN infiltrationafter 60 minutes of ischemia and 24 hours of reperfusion determined viamyeloperoxidase immunostaining. (A) White bars indicate controls; blackbars indicate clopidogrel-treated animals. (B) White bars indicate con-trols; black bars indicate platelet-depleted animals (*P � .016). (C)Representative photomicrograph of myeloperoxidase staining (control[nonimmune IgG2]). The extensive necrotic areas show a dramaticinfiltration of PMNs; only a few vital hepatocytes can be detected aroundthe portal field. (Original magnification �100.)

372 NOCITO ET AL. HEPATOLOGY, February 2007

PCNA and Ki67 immunostaining revealed a significantreduction of hepatocellular proliferation in platelet-de-pleted animals only at 48 hours, pointing to an impairedrather than delayed liver regeneration (Fig. 7).

Does Platelet-Derived Serotonin Mediate Postisch-emic Liver Regeneration and Tissue Repair? Plateletsmediate liver regeneration through the release of seroto-nin.17 The finding that platelets are involved in postisch-emic liver repair and regeneration prompted us toinvestigate the role of platelet-derived serotonin usingTph1�/� mice. These animals have a disrupted gene for

Tph1, rendering them devoid of serotonin outside of thecentral nervous system.18 Initially, we tested whether alack of serotonin results in a reduction of I/R injury. As forplatelet depletion, serotonin deficiency did not decreasetissue injury (data not shown). We then analyzed neutro-phil infiltration and the expression of proinflammatorymarkers as well as the resolution of necrotic tissue. Incontrast to platelet-depleted animals, serotonin deficiencydid not affect the sequel of inflammation and tissue re-sorption; this is reflected by an equal amount of remnantnecrosis 7 days after the initial ischemic insult (Fig. 8A).Because serotonin has mitogenic properties, we furtheranalyzed the regenerative activity of hepatocytes andfound that the lack of serotonin significantly reduced he-patocyte proliferation (Fig. 8B,C). This further supports arole for serotonin in liver regeneration, while the presenceof other platelet-derived factors is instrumental for tissueresorption and remodeling.

Fig. 4. Effects of platelet depletion on postischemic expression ofinflammatory mediators: expression of proinflammatory mediators in livertissue after 60 minutes of ischemia followed by 4 hours and 24 hours ofreperfusion determined via real-time PCR. Transcript levels were normal-ized to livers receiving IgG2 or anti-CD41 treatment without I/R and aregiven as fold induction (white bars, control; black bars, platelet-depletedanimals). *P � .001 (panel B, 4 h), P � .02 (panel B, 24 h), P � .02(panel D, 4 h), P � .032 (panel D, 24 h).

Fig. 5. Effects of platelet depletion on postischemic serum levels ofproinflammatory mediators. Serum levels of (A) TNF-� (*below detectionlimit) and (B) IL-6 (*P � .033) after 60 minutes of ischemia followed by4 and 24 hours of reperfusion.

Fig. 6. Quantification of residual liver necrosis: extent of necrosis inliver tissue 7 days after the initial ischemic insult of 60 minutes. *P �.032.

Fig. 7. Regeneration of postischemic livers. Livers treated with IgG2(dashed line) and anti-CD41 (solid line) were analyzed for PCNA- andKi67-positive hepatocytes after 60 minutes of ischemia and 24 and 48hours of reperfusion as well as 7 days after the initial ischemic insult(n � 10 per group). The number of positive hepatocytes per high-powerfield (�400) is presented. *P � 0.013 (A), P � 0.035 (B).

HEPATOLOGY, Vol. 45, No. 2, 2007 NOCITO ET AL. 373

DiscussionThis study was designed to assess the role of platelets in

normothermic I/R injury of the liver. Neither abrogationof platelet aggregation nor platelet depletion reducedpostischemic tissue injury. Instead, postischemic inflam-mation, as well as liver regeneration and consequentlytissue repair, were strikingly impaired. In particular,platelet-derived serotonin mediates hepatocyte prolifera-tion, which is an integral component of postischemic tis-sue repair.

Research on the role of platelets in normothermic I/Rinjury has been limited by the absence of a suitable model.Although various protocols targeting platelets have beenavailable, thrombocytes either remained physicallypresent,22 or leukocytes were concomitantly affected.14,23

It was only recently that a pure model of platelet-deple-tion using a monoclonal antibody against a plateletepitope was described.17 Here, we established a model ofimmune thrombocytopenia using a commercially avail-able antibody directed against the platelet-associatedepitope GPIIb. Intraperitoneal administration of this an-tibody selectively reduced platelet counts by more than90%, whereas leukocyte and erythrocyte counts remainedunaffected (Supplementary Figure), indicating a specificthrombocytopenia. This approach may also be instru-mental in investigating platelet function in organs otherthan the liver. We also employed mice lacking Tph1,which is the rate-limiting enzyme for the synthesis of pe-ripheral serotonin. Using this model, we recently demon-strated platelet-derived serotonin to mediate hepatocyteproliferation after partial hepatectomy.17

We found that I/R injury as determined by AST levelsand liver tissue necrosis was not affected by the absence orimpaired function of platelets. This result stands in con-trast with previous studies.15,16 One of these studies usedanti-fibrinogen antibodies to inhibit platelet–endothelialcell interactions in a model of 90 minutes of ischemia andassessed reperfusion injury only up to 60 minutes.16 Tis-

sue necrosis and repair do not manifest themselves during1 hour of reperfusion and thus cannot be conclusivelyassessed at this time point. The short reperfusion timemight have been chosen because of the severe ischemicinsult, because 90 minutes of hepatic ischemia lead tosubtotal tissue necrosis. In pilot experiments, we assessedan ischemic period of 90 minutes, which was associatedwith high mortality in the platelet-depleted group (datanot shown). Furthermore, as the authors suggested, thebeneficial effects seen after functional platelet inhibitioncould be attributed to reduced leukocyte sequestration,because activated platelets have been reported to modu-late leukocyte adherence through secretion of chemotac-tic agents as well as surface expression of adhesionmolecules.24 Indeed, this mechanism is supported by ourfinding of decreased postischemic PMN infiltration inplatelet-depleted animals, whereas the mere inhibition ofplatelet aggregation did not have such an effect.

Another study, which showed reduced postischemicliver injury in mice deficient in P-selectin,15 must also beinterpreted with caution, because these animals displayreduced endothelial–leukocyte adhesion in addition totheir platelet phenotype. Our results imply that the ob-served protection is related to an inhibition of leukocyteinfiltration rather than a specific platelet-mediated effect.Taken together, the presence or activation of plateletsdoes not contribute to tissue injury and necrosis followinghepatic ischemia.

In our study, platelet depletion reduced the hepaticexpression and release of proinflammatory mediators aswell as the recruitment of neutrophils. This effect couldbe attributed to a disturbed interaction of platelets andneutrophils at the endothelial level. However, in the set-ting of inhibited platelet aggregation, neutrophil infiltra-tion remained unaffected. This hints against apredominant role of secondary capture of neutrophils byplatelets in this setting. These results suggest that theseblood constituents play an important role in chemoattrac-

Fig. 8. Effects of platelet-derived serotonin on postischemic liver regeneration and tissue repair. (A) Morphometric analysis of the extent of livernecrosis 7 days after the initial ischemic insult in wild-type and Tph1�/� animals. (B) Number of PCNA-positive (*P � 0.008) and (C) Ki67-positive(*P � 0.007) hepatocytes in tissue specimen after 60 minutes of ischemia and 48 hours of reperfusion.

374 NOCITO ET AL. HEPATOLOGY, February 2007

tion to the postischemic liver. Although the reduced ex-pression and release of these mediators in platelet-depleted animals can already be found at 4 hours ofreperfusion, this partial attenuation of the inflammatoryreaction is apparently insufficient to reduce the ensuinginjury. Because a depletion of neutrophils has been con-vincingly shown to protect the liver from reperfusion in-jury,25 our data point to the possibility that the amount ofneutrophils infiltrating the postischemic liver indepen-dently of platelet function are sufficient to exert theiradverse effects, resulting in extensive liver damage. Thisinterpretation is also supported by the finding that IL-1receptor–deficient mice display reduced neutrophil re-cruitment without effects on the extent of liver injury.21

Recovery from liver damage has been termed the liverwound healing response.26 This requires distinct mecha-nisms such as inflammation, tissue resorption, and re-modeling, as well as hepatocyte proliferation. We haverecently shown platelet-derived serotonin to mediate liverregeneration in a mouse model of partial hepatectomy.17

Consistent with these findings, both thrombopenic ani-mals and mice lacking peripheral serotonin exhibited re-duced hepatocyte proliferation 48 hours after the initialischemic insult. Thus, the effect on liver regeneration inplatelet-depleted mice could also be ascribed to the di-minished expression of TNF-� or IL-6, both of whichhave been identified as key mediators of regeneration.27-29

However, in our experiments, the expression levels ofTNF-� and IL-6 did not differ in postischemic livers ofTph1�/� and wild-type mice. Yet there was a significantreduction in regeneration in these animals. This observa-tion points to two independent pathways that act syner-gistically on hepatocyte proliferation.

The role of TNF-� and IL-6 in liver regeneration hasbeen extensively discussed.27-29 They appear to initiate thehepatocyte cell cycle entry into the G1 phase. These stud-ies were predominantly performed in models of partialhepatectomy, where the loss of liver mass is instantaneous,triggering a synchronous regenerative response in theremnant liver. In contrast, after ischemia and reperfusionthe injury is focal and less synchronized, leading to a re-generative activity mainly at the site of damage. This mayexplain the only partial induction of hepatocyte prolifer-ation. In the serotonin-dependent model, the postisch-emic regenerative response was more severely impaired,suggesting that serotonin is required to secure hepatocyteproliferation.

Although hepatic inflammation is detrimental, it isalso a part of the normal wound healing process. Plateletscontain an array of preformed peptide mediators andgrowth factors, including platelet factor-4, platelet-de-rived growth factor, vascular endothelial growth factor,

and many others. These mediators are rapidly releasedupon tissue injury and play critical roles in the inductionof inflammation and tissue repair. Vascular endothelialgrowth factor promotes migration of endothelial cells,thus initiating angiogenesis at the tissue repair stage.30 Inturn, platelet factor-4 and platelet-derived growth factorare chemotactic for neutrophils, monocytes, and fibro-blasts,31,32 all of which are active players in the resolutionof liver injury. A reduction of neutrophil recruitment hasindeed been reported to impair wound closure in theskin.33 Consistent with these findings, the reduced neu-trophil infiltration in platelet-depleted animals was asso-ciated with a prolonged persistence of necrotic tissue.This effect was caused by the absence of platelets andplatelet-derived factors, resulting in a reduced expressionof proinflammatory molecules in the liver.

The fact that mice lacking peripheral serotonin dis-played reduced hepatocellular proliferation in spite ofnormal injury, inflammation, and resolution of necrotictissue suggests that other platelet-derived factors are in-volved at different steps during postischemic tissue repairand regeneration.

In conclusion, our results convincingly acquit plateletsof causing postischemic liver injury, but highlight for thefirst time their importance in different steps of postisch-emic tissue repair through the modulation of inflamma-tion and the release of serotonin. Taken together, thesefindings point to a novel role of platelets in hepatic woundhealing.

Acknowledgment: We thank Udo Ungethum andMarion Bawohl for technical assistance. We are also grate-ful to Bernhard Odermatt for help in establishing theimmune thrombocytopenia model.

References1. Xu Y, Huo Y, Toufektsian MC, Ramos SI, Ma Y, Tejani AD, et al. Acti-

vated platelets contribute importantly to myocardial reperfusion injury.Am J Physiol Heart Circ Physiol 2006;290:H692-H699.

2. Okada Y, Marchevsky AM, Zuo XJ, Pass JA, Kass RM, Matloff JM, et al.Accumulation of platelets in rat syngeneic lung transplants: a potentialfactor responsible for preservation-reperfusion injury. Transplantation1997;64:801-806.

3. Kuroda T, Shiohara E, Homma T, Furukawa Y, Chiba S. Effects of leu-kocyte and platelet depletion on ischemia-reperfusion injury to dog pan-creas. Gastroenterology 1994;107:1125-1134.

4. Caldwell-Kenkel JC, Thurman RG, Lemasters JJ. Selective loss of non-parenchymal cell viability after cold ischemic storage of rat livers. Trans-plantation 1988;45:834-837.

5. McKeown CM, Edwards V, Phillips MJ, Harvey PR, Petrunka CN, Stras-berg SM. Sinusoidal lining cell damage: the critical injury in cold preser-vation of liver allografts in the rat. Transplantation 1988;46:178-191.

6. Momii S, Koga A. Time-related morphological changes in cold-stored ratlivers. A comparison of Euro-Collins solution with UW solution. Trans-plantation 1990;50:745-750.

HEPATOLOGY, Vol. 45, No. 2, 2007 NOCITO ET AL. 375

7. Cywes R, Packham MA, Tietze L, Sanabria JR, Harvey PR, Phillips MJ, etal. Role of platelets in hepatic allograft preservation injury in the rat.HEPATOLOGY 1993;18:635-647.

8. Gao W, Bentley RC, Madden JF, Clavien PA. Apoptosis of sinusoidalendothelial cells is a critical mechanism of preservation injury in rat livertransplantation. HEPATOLOGY 1998;27:1652-1660.

9. Sindram D, Porte RJ, Hoffman MR, Bentley RC, Clavien PA. Plateletsinduce sinusoidal endothelial cell apoptosis upon reperfusion of the coldischemic rat liver. Gastroenterology 2000;118:183-191.

10. Selzner N, Rudiger H, Graf R, Clavien PA. Protective strategies againstischemic injury of the liver. Gastroenterology 2003;125:917-936.

11. Clavien PA, Harvey PR, Strasberg SM. Preservation and reperfusion inju-ries in liver allografts. An overview and synthesis of current studies. Trans-plantation 1992;53:957-978.

12. Sindram D, Porte RJ, Hoffman MR, Bentley RC, Clavien PA. Synergismbetween platelets and leukocytes in inducing endothelial cell apoptosis inthe cold ischemic rat liver: a Kupffer cell-mediated injury. FASEB J 2001;15:1230-1232.

13. Gujral JS, Bucci TJ, Farhood A, Jaeschke H. Mechanism of cell deathduring warm hepatic ischemia-reperfusion in rats: apoptosis or necrosis?HEPATOLOGY 2001;33:397-405.

14. Khandoga A, Biberthaler P, Enders G, Teupser D, Axmann S, Luchting B,et al. P-selectin mediates platelet-endothelial cell interactions and reperfu-sion injury in the mouse liver in vivo. Shock 2002;18:529-535.

15. Yadav SS, Howell DN, Steeber DA, Harland RC, Tedder TF, Clavien PA.P-selectin mediates reperfusion injury through neutrophil and platelet se-questration in the warm ischemic mouse liver. HEPATOLOGY 1999;29:1494-1502.

16. Khandoga A, Biberthaler P, Enders G, Axmann S, Hutter J, Messmer K, etal. Platelet adhesion mediated by fibrinogen-intercelllular adhesion mole-cule-1 binding induces tissue injury in the postischemic liver in vivo.Transplantation 2002;74:681-688.

17. Lesurtel M, Graf R, Aleil B, Walther DJ, Tian Y, Jochum W, et al. Platelet-derived serotonin mediates liver regeneration. Science 2006;312:104-107.

18. Walther DJ, Peter JU, Bashammakh S, Hortnagl H, Voits M, Fink H, et al.Synthesis of serotonin by a second tryptophan hydroxylase isoform. Sci-ence 2003;299:76.

19. Yadav SS, Sindram D, Perry DK, Clavien PA. Ischemic preconditioningprotects the mouse liver by inhibition of apoptosis through a caspase-dependent pathway. HEPATOLOGY 1999;30:1223-1231.

20. Lentsch AB, Yoshidome H, Cheadle WG, Miller FN, Edwards MJ. Che-mokine involvement in hepatic ischemia/reperfusion injury in mice: roles

for macrophage inflammatory protein-2 and Kupffer cells. HEPATOLOGY

1998;27:507-512.21. Kato A, Gabay C, Okaya T, Lentsch AB. Specific role of interleukin-1 in

hepatic neutrophil recruitment after ischemia/reperfusion. Am J Pathol2002;161:1797-1803.

22. Gachet C. ADP receptors of platelets and their inhibition. Thromb Hae-most 2001;86:222-232.

23. Evensen SA, Jeremic M, Hjort PF. Experimental thrombocytopenia in-duced by Busulphan (Myleran) in rabbits: extremely low platelet levels andintact plasma clotting system. Thromb Diath Haemorrh 1968;19:570-577.

24. Gawaz M, Neumann FJ, Dickfeld T, Koch W, Laugwitz KL, AdelsbergerH, et al. Activated platelets induce monocyte chemotactic protein-1 secre-tion and surface expression of intercellular adhesion molecule-1 on endo-thelial cells. Circulation 1998;98:1164-1171.

25. Jaeschke H, Farhood A, Smith CW. Neutrophils contribute to ischemia/reperfusion injury in rat liver in vivo. FASEB J 1990;4:3355-3359.

26. Friedman SL, Yamasaki G, Wong L. Modulation of transforming growthfactor beta receptors of rat lipocytes during the hepatic wound healingresponse. Enhanced binding and reduced gene expression accompany cel-lular activation in culture and in vivo. J Biol Chem 1994;269:10551-10558.

27. Cressman DE, Greenbaum LE, DeAngelis RA, Ciliberto G, Furth EE, PoliV, et al. Liver failure and defective hepatocyte regeneration in interleukin-6-deficient mice. Science 1996;274:1379-1383.

28. Sakamoto T, Liu Z, Murase N, Ezure T, Yokomuro S, Poli V, et al. Mitosisand apoptosis in the liver of interleukin-6-deficient mice after partial hep-atectomy. HEPATOLOGY 1999;29:403-411.

29. Yamada Y, Kirillova I, Peschon JJ, Fausto N. Initiation of liver growth bytumor necrosis factor: deficient liver regeneration in mice lacking type Itumor necrosis factor receptor. Proc Natl Acad Sci U S A 1997;94:1441-1446.

30. Liekens S, De Clercq E, Neyts J. Angiogenesis: regulators and clinicalapplications. Biochem Pharmacol 2001;61:253-270.

31. Deuel TF, Senior RM, Chang D, Griffin GL, Heinrikson RL, Kaiser ET.Platelet factor 4 is chemotactic for neutrophils and monocytes. Proc NatlAcad Sci U S A 1981;78:4584-4587.

32. Deuel TF, Senior RM, Huang JS, Griffin GL. Chemotaxis of monocytesand neutrophils to platelet-derived growth factor. J Clin Invest 1982;69:1046-1049.

33. Subramaniam M, Saffaripour S, Van De Water L, Frenette PS, MayadasTN, Hynes RO, et al. Role of endothelial selectins in wound repair. Am JPathol 1997;150:1701-1709.

376 NOCITO ET AL. HEPATOLOGY, February 2007