LIVER BIOLOGY/PATHOBIOLOGY
Systemic Protection Through Remote Ischemic Preconditioning Is Spread by Platelet-Dependent Signaling in Mice Christian E. Oberkofler,1* Perparim Limani,1* Jae-Hwi Jang,1 Andreas Rickenbacher,1 Kuno Lehmann,1 Dimitri A. Raptis,1 Udo Ungethuem,1 Yinghua Tian,1 Kamile Grabliauskaite,1 Rok Humar,2 Rolf Graf,1 Bostjan Humar,1** and Pierre-Alain Clavien1** Remote ischemic preconditioning (RIPC), the repetitive transient mechanical obstruction of vessels at a limb remote to the operative site, is a novel strategy to mitigate distant organ injury associated with surgery. In the clinic, RIPC has demonstrated efficacy in protecting various organs against ischemia reperfusion (IR), but a common mechanism underlying the systemic protection has not been identified. Here, we reasoned that protection may rely on adaptive physiological reponses toward local stress, as is incurred through RIPC. Standardized mouse models of partial hepatic IR and of RIPC to the femoral vascular bundle were applied. The roles of platelets, peripheral serotonin, and circulating vascular endothelial growth factor (Vegf) were studied in thrombocytopenic mice, Tph12/2 mice, and through neutralizing antibodies, respectively. Models of interleukin-10 (Il10) and matrix metalloproteinase 8 (Mmp8) deficiency were used to assess downstream effectors of organ protection. The protection against hepatic IR through RIPC was dependent on platelet-derived serotonin. Downstream of serotonin, systemic protection was spread through up-regulation of circulating Vegf. Both RIPC and serotonin-Vegf induced differential gene expression in target organs, with Il10 and Mmp8 displaying consistent up-regulation across all organs investigated. Concerted inhibition of both molecules abolished the protective effects of RIPC. RIPC was able to mitigate pancreatitis, indicating that it can protect beyond ischemic insults. Conclusions: We have identified a plateletserotonin-Vegf-Il10/Mmp8 axis that mediates the protective effects of RIPC. The systemic action, the conservation of RIPC effects among mice and humans, and the protection beyond ischemic insults suggest that the platelet-dependent axis has evolved as a preemptive response to local stress, priming the body against impending harm. (HEPATOLOGY 2014;60:1409-1417)
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volution has equipped animals with a range of autonomous mechanisms that protect from imminent harm and increase chances of survival. The “fight-or-flight” or the “freeze” response are well-known examples whereby enemy recognition is translated into a
altered physiological state, increasing performance limits or freezing the body despite fear or pain, respectively.1,2 Unlike the acute responses toward extrinsic stress, the protective reactions of the body toward potential intrinsic harm are less obvious. A fall (or other mechanical insults), for example, bears the risk of blunt trauma, and it is likely that the fall per se will activate a preemptive program to mitigate potential
Abbreviations: 5HTP, 5-hydroxytryptamin; Ab, antibody; ALT, alanine aminotransferase; AST, aspartate transaminase; ECs, endothelial cells; IgG, immunoglobulin G; IHC, immunohistochemistry; Il10, interleukin-10; IP, intraperitoneal; IR, ischemia reperfusion; IRI, ischemia reperfusion injury; Mmp8, matrix metalloproteinase 8; mRNA, messenger RNA; PAF, platelet-activating factor; PBS, phosphate-buffered saline; RIPC, remote ischemic preconditioning; SD, standard deviation; Vegf, vascular endothelial growth factor; WT, wild type. From the 1Laboratory of the Swiss Hepato-Pancreatico-Biliary (HPB) Center, Department of Surgery, University Hospital Zurich, Zurich, Switzerland and 2 Research Unit, Division of Internal Medicine, University Hospital Zurich, Zurich, Switzerland. Received September 4, 2013; accepted February 19, 2014. This work was funded by the Swiss National Foundation (nos. 320030_and 132985; to P.A.C.). *These authors share first authorship. **These authors share senior authorship. 1409
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organ injury resulting from, for example, hemorrhagic shock. Though only vaguely defined, such a protective mechanism exists and is intuitively being exploited in patients. During surgery, blood vessels of the target organ frequently are clamped to prevent blood loss. However, the induced ischemia causes organ injury after reperfusion, significantly increasing postoperative complications. To reduce these risks, surgeons have developed ischemic preconditioning strategies, whereby short periods of preclamping before surgery markedly protect from ischemiareperfusion injury (IRI).3-5 In addition to these strategies, even preclamping of vessels at remote limbs, rather than at the target organ (remote ischemic preconditioning; RIPC), can induce protection. More so, RIPC through mechanical pressure instead of clamping is sufficient to protect various organs against ischemia reperfusion (IR).610 Therefore, RIPC, either by clamping or pressure, is able to afford systemic protection from organ injury.6-15 Animal models of RIPC and IRI have been developed to identify candidate molecules inducing organspecific protection.11-15 However, these animal models also provide a unique opportunity to investigate a conserved biological mechanism that may underlie the transformation of a peripheral signal into systemic protection from an unknown insult. Here, we investigated the role of platelets and downstream signaling events in the spread of systemic protection through RIPC.
Materials and Methods Animals. All animal experiments were in accord with Swiss Federal Animal Regulations and approved by the Veterinary Office of Zurich. Animals 10-12 weeks of age were kept on a 12-hour day/night cycle with free access to food and water. C57Bl/6 mice were obtained from Harlan Laboratories BV (Venray, The Netherlands), and interleukin-10 (Il10)2/2 and Tph12/2 mice were obtained from our in-house captive breeding. Thrombocytopenia was induced by intraperitoneal (IP) injection of 1 mg/kg of anti-CD41 (clone MWReg 30, with immunoglobulin G [IgG]2 as a control; BD Biosciences, Franklin Lakes, NJ) in 200 mL of phosphate-buffered saline (PBS), reducing platelet numbers by >90%.16 Blood cell counts from tail-vein samples collected 24 hours after injection and diluted
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in 20 mL of 12 mM of ethylenediaminetetraacetic acid were assessed using a Coulter AcTdiff counter (Beckman Coulter, Nyon, Switzerland). Animal Surgery. Anesthesia was induced by isoflurane inhalation (2%-4%) by a concentric oronasal mask connected to an anesthetic circuit. Anesthetic depth was monitored by clinical parameters (respiratory rate and depth, color of mucous membranes and inner organs, movement, and reflexes). RIPC was done by clamping the femoral vascular bundle just proximal to its confluence with the femoral nerve (Fig. 1). A protocol of 5 minutes of limb ischemia, followed by 5 minutes of reperfusion for a total of four cycles, was used. Cessation of blood flow was confirmed by the absence of femoral artery pulsation distal from the clamp and a change in foot color. After closure of the femoral wound (approximately 5 minutes after the last RIPC cycle), laparotomy for hepatic ischemia was performed through a midline incision. The membrane connecting the caudate to the left lobe was divided. A microvascular clamp was applied to the portal triad supplying the left and median lobes, rendering approximately 70% of the liver ischemic, while avoiding mesenteric venous congestion by permitting portal decompression through the caudate and right lobes of the liver. Successful occlusion of the portal triad branch was confirmed by a change in color. The abdominal wall was closed by a suture during hepatic ischemia and mice were woken up. After 60 minutes, liver reperfusion was reestablished through the opening of the abdominal cavity, followed by removal of the microvascular clamp. After surgery, animals were allowed to recover on a warming pad in a separate cage until completely conscious. Kidney ischemia was induced by clamping the vessel bundle to both kidneys for 30 minutes through a midline laparotomy under general anesthesia on a warming pad.17 After reperfusion, animals were terminated by exsanguination through blood collection and opening of the diaphragm. Blood was immediately centrifuged at 5,000 rpm for 5 minutes. The serum supernatant was stored at 270 C until assayed for creatinine. Organs were stored in 10% formal saline for histopathology or snap-frozen in liquid nitrogen. Animal Treatment. Serotonin (50 mg/kg) was coinjected IP with either IgG or alpha vascular
Address reprint requests to: Pierre-Alain Clavien, M.D., Ph.D., Department of Surgery, University Hospital Zurich, Raemistrasse 100, CH-8091 Zurich, Switzerland. E-mail: [email protected]; fax: 141 43 255 44 49. C 2014 by the American Association for the Study of Liver Diseases. Copyright V View this article online at wileyonlinelibrary.com. DOI 10.1002/hep.27089 Potential conflict of interest: Nothing to report.
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Fig. 1. (A) Protective effects of RIPC in hepatic IR. Upper panels show serum AST and ALT levels of mice subjected to IR (n 5 6) or to RIPC followed by IR (n 5 6). Lower panels show necrotic injury at 6 hours after IR or IR plus RIPC and corresponding quantification (n 5 6/group) to the left. (B) RIPC activates platelets. The drop in platelet numbers paralleled by an increase in PAF serum levels 1 hour after RIPC is shown (n 5 6/group). *P < 0.05.
endothelial growth factor (aVegf; 4 mg/kg; R&D Systems, Abingdon, UK) one hour before treatment or harvest. Serotonin reloading of platelets from Tph12/2 mice with the precursor, 5-hydroxytryptamin (5HTP), was done as previously described.18 The Mmp8 inhibitor I ((3R)-(1)-[2-(4-methoxybenzenesulfonyl)1,2,3,4-tetrahydroisoquinoline-3-hydroxamate]; Calbiochem, Billerica, MA) was IP injected at 80 nM (100 lL/mouse) 1 hour before IRI, whereas the platelet-activating factor (PAF) receptor antagonist (SM-12502; Sigma-Aldrich, Munich, Germany) was injected at 30 lM. Histological Examination. Hematoxylin and eosine staining was performed on 3-mm archived sections of liver. Immunostainings for Mmp8 (catalog no.: ab53017; Abcam, Cambridge, UK) and Il10 (catalog no.: 505002; Biolegend, Fell, Germany) were done using the Ventana Discovery automated staining system and the iView DAB kit (Ventana Medical Systems, Tucson, AZ). All histological analyses were performed in a blinded fashion. Serum Alanine Aminotransferase/Serum Aspartate Aminotransferase. Serum alanmine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured using a serum multiple biochemical analyzer (Ektachem DTSCII; Johnson & Johnson Inc., Rochester, NY). Creatinine was measured with the
DRI-CHEM 4000i analyzer (Fujifilm, Dielsdorf, Switzerland). Isolation of Mouse Aortic Endothelial Cells. Mouse aortic endothelial cells (ECs) were isolated from aortae of 8- to 10-week-old C57BL/6 mice. Plates (24-well) were coated with fibrin gels, as previously described.19 The excised aorta was cleaned from adjacent tissue, cut in small rings, placed onto the wells, overlaid by fibrin gel, and covered with EC growth media (serum-free Dulbecco’s modified Eagle’s medium), 10% fetal calf serum (Biochrom, Cambridge, UK), 100 lg/mL of endothelial cell growth supplement (Millipore, Zug, Switzerland), and 0.1 lL/ mL of heparin. Every other day, 300 lg/mL of eamino caproic acid (Sigma-Aldrich, St Louis, MO) in PBS were added. After 10 days, outgrowing endothelial sprouts were harvested and the gel-cell mixture was transferred to a six-well plate coated with 0.1% gelatin plus EC growth media. Confluent cells were subcultured and characterized with the endothelial marker, von Willebrand factor (LabForce AG, Nunningen, Switzerland). Quantitative Real-Time Polymerase Chain Reaction. Total RNA was extracted from 50 mg of organ tissue using Trizol reagent (Invitrogen, Basel, Switzerland) following the manufacturer’s instructions. Quantitative real-time polymerase chain reaction was
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performed on complementary DNA (ThermoScript reverse-transcription PCR System; Invitrogen) using an ABI Prism 7500 Sequence Detector System (PE Applied Biosystems, Rotkreuz, Switzerland). TaqMan gene expression assays for Hsp1a (Mm01159846_s1), Gpx1 (Mm00656767_g1), Txn (Mm00726847_s1), Nfkbia (Mm00477798_m1), Mmp8 (Mm00439509_m1), NOS2a (Mm00440485_m1), C3 (Mm00437858_m1), IL1b (Mm00434228_m1), Clec10a (Mm00546124_m1), Il6 (Mm00446190_m1), and Il10 (Mm00439616_m1)
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were from PE Applied Biosystems and normalized to 18S ribosomal RNA (TaqMan control reagents; PE Applied Biosystems). Results represent mean fold induction of messenger RNA (mRNA) expression 6 standard deviation (SD). Statistical Analysis. Differences between groups (n 5 animals) were assessed by a Mann-Whitney’s U test or by one-way analysis of variance, where applicable. Statistical significance was set at P < 0.05, with significant values marked by asterisks. Error bars refer to SDs.
Results RIPC Protects From Hepatic IRI. To gain insight into the chain of events that may constitute the protective mechanisms behind preconditioning, we designed a series of experiments based on hindlimb RIPC and its effects in different organs before or after IR. The initial experiments tested the efficacy of hindlimb RIPC to the femoral vessel bundle in mitigating liver IRI. Blood flow was mechanically interrupted to induce cycles of local ischemia for repetitive periods to the remote limb, followed by 1 hour of organ-specific ischemia to 70% of the murine liver. At 6 hours after reperfusion, we observed significant protection of the liver from IRI, as shown by a marked reduction in
Fig. 2. (A) Dependence of RIPC-induced protection on plateletderived serotonin. Serum ALT levels are shown for control animals (wt) subjected to hepatic IR or to RIPC before IR; for immunothrombocytopenic mice (ITP; depleted of platelets by aCd41 antibodies) subjected to IR, or to RIPC before IR; and for mice carrying platelets without serotonin (Tph12/2) subjected to IR, or to RIPC before IR, or reloaded with serotonin (5HTP; supplementation with the serotonin precursor) and subjected to RIPC before IR, or injected with serotonin before IR. (B) Serum Vegf levels correlate with the protective effect of RIPC. Vegf levels are shown for control mice (wt) either untreated (wt Control) or subjected to RIPC; for serotonin-deficient mice (Tph12/2) either untreated, or subjected to RIPC, or reloaded with serotonin (5HTP), or reloaded with serotonin and subjected to RIPC, or reloaded with serotonin and treated with 200 ng/kg of aVegf 1 hour before RIPC; and for immunothrombocytopenic mice (ITP) either untreated, subjected to RIPC, or injected with 50 mg/kg of serotonin (5HT). Vegf elevation above the detection limit (dotted line; *P < 0.05, relative to untreated controls) was noted only in models that also displayed protection from IRI (wt1RIPC; Tph12/2 15HTP1RIPC; ITP15HT). Treatment with unspecific IgG had no effect on Vegf levels (not shown). Vegf levels were assessed 30 minutes after the last manipulation. (C) Protection through RIPC is dependent on circulating Vegf. Serum ALT levels are shown for control mice (wt) subjected to IR, to RIPC before IR, or treated with 4 lg/kg of Vegf 1 hour before IR (IR 1Vegf wt), or treated with aVegf 1 hour before RIPC; for control mice treated with 4 lg/kg of unspecific IgG 1 hour before IR (wt1IgG); and for serotonindeficient mice (Tph12/2) reloaded with serotonin (5HTP) and treated with aVegf before RIPC and IR, or not reloaded with serotonin but treated with Vegf before IR. *Significant protection (P < 0.05) relative to untreated controls.
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both ALT/AST levels and necrosis by approximately two thirds, compared to controls (Fig. 1A). RIPC Protects Through Platelet-Derived Serotonin. Trying to explore the mechanism that initiates protection from IRI, we reasoned that the stress RIPC puts on vessels may activate platelets. Consistent with this hypothesis, we observed a drop in platelet numbers by 40% after RIPC, along with a concomitant increase in serum levels of PAF and plasma P-selectin (Fig. 1B and Supporting Fig. 1). To substantiate a dependence of RIPC effects on platelets, mice were depleted of platelets by IP injection of Cd41 antibodies (Abs; Supporting Fig. 1). RIPC after immune depletion lost its protective effects, indicating the requirement for platelets (Fig. 2A). Next, we asked whether platelets must be activated to initiate protection. After injection of SM-12502, a PAF receptor (Ptafr) antagonist that inhibits platelet activation, protection through RIPC was again abolished (RIPC control: median, ALT 6,350 U/I; range, 5150-7800; RIPC plus SM-12502: median, ALT 11,200 U/I; range, 8,700-12,800; n 5 5/group). We postulated that molecules released by activated platelets might participate in the protection. Among the numerous factors stored in platelets, we focused on serotonin, because platelets are carriers of essentially all peripheral serotonin.20 Moreover, the molecule can promote tissue repair after ischemia.16 To investigate the role of blood serotonin, we applied RIPC to Tph12/2 mice that lack peripheral serotonin, yet have normal platelet counts.18 RIPC did not protect Tph12/2 mice from hepatic IRI (Fig. 2A), suggesting a key role for plateletderived serotonin in mediating RIPC effects. Serotonin reloading of Tph12/2 mice18 by injecting 50 mg/kg of its precursor, 5HTP (the product of Tph1) restored the protective effect, confirming the requirement of plateletderived serotonin for the RIPC effects (Fig. 2A). To detail this finding, we inhibited platelet activation in wild-type (WT) mice by SM-12502 before serotonin injection, resulting in a partial restoration of protection (RIPC plus SM-12502: median, ALT 11,200 U/I; range, 8,700-12,800; SM-12502 plus serotonin: median, ALT 6,600 U/I; range 4,900-8,850; n55/group). Therefore, serotonin acts downstream of PAF-associated platelet activation, but the incomplete restoration suggests that PAF may incur serotonin-independent protection, perhaps through Ptafr, which is expressed also on nonplatelet cell types. Alternatively, Ptafr inhibition and/or SM12502 might have additional effects that counteract the protective effects of serotonin.21 RIPC Requires Vegf for Its Protective Effects. As a result of its rapid reuptake, serotonin usually acts locally22 and thus may not mediate the systemic RIPC
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effects. To identify potential pathways that may spread systemic protection downstream of serotonin, we investigated Vegf, because Vegf has been implicated in the amelioration of IRI,23 and because serotonin can increase serum Vegf levels.24 Within 30 minutes after RIPC, serum Vegf levels significantly increased in WT mice (Fig. 2B). Injection of recombinant Vegfa into mice before IR conferred similar effects on IRI as RIPC alone (Fig. 2C), indicating that circulating Vegf is sufficient for protection. Vice versa, aVegf Ab neutralized the effect of RIPC (Fig. 2C), indicating that RIPC relies on Vegf for protection. Therefore, both serotonin and Vegf participate in RIPC-induced protection from IRI. Vegf Acts Downstream of Serotonin to Induce RIPC Protection. To investigate the relationship between serotonin and Vegf in RIPC protection, we injected Vegf into serotonin-deficient Tph12/2 mice, which are unresponsive to RIPC. Vegf restored the protective effects of RIPC in Tph12/2 mice (Fig. 2C). Conversely, protection was lost when aVegf Ab was injected concurrently with RIPC into Tph12/2 animals supplemented with 5HTP (i.e., reloading platelets with serotonin; Fig. 2C). We conclude that serotonin mediates RIPC protection through Vegf. In support, RIPC elevated Vegf serum levels above the detection limit in WT mice, in Tph12/2 mice after 5HTP supplementation, and in platelet-depleted animals injected with serotonin, but not in the animal models featuring serotonin deficiency and RIPC resistance (Fig. 2B). Thus, significant Vegf elevation is dependent on the presence of peripheral serotonin and strictly correlates with measures that protect from IRI. Although platelets can store Vegf, serum Vegf after RIPC seems to originate from other cells than platelets, because Vegf levels rise also in platelet-depleted mice injected with serotonin (Fig. 2B). Given the rather local action of serotonin, ECs may be a source of Vegf24 and are also directly affected by RIPC. To address this possibility, we established primary endothelial cultures from mouse aorta and treated the cells with serotonin. Within 60 minutes of treatment, ECs produced Vegf mRNA and secreted Vegf into supernatant (Fig. 3A). Whereas ECs are a likely source of Vegf after RIPC, contribution from other vessel-associated cell types is conceivable. Considering that serotonin injection was protective also in platelet-depleted mice, the amount of Vegf released from ECs (and associated cells) seems to be sufficient for the mediation of RIPC effects. However, in mice with normal platelet counts, release of Vegf from degranulating platelets is likely to contribute to the elevation in circulating Vegf levels.
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Fig. 3. (A) Serotonin induces Vegf secretion from mouse ECs. (B) Serotonin through Vegf protects from renal IRI. Forty-eight hours after IR in the kidney, creatinine levels are markedly elevated (IR), compared to controls (no IR). Serotonin injection (50 mg/kg) together with unspecific IgG 1 hour before renal IR (P < 0.05) reduces creatinine levels both at 24 and 48 hours post-IR, an effect that is abolished by adding aVegf instead of IgG (lower panel). At 48 hours, SDs were not added because only 2 of 5 animals were alive in the unprotected groups. *P < 0.05.
Serotonin-Vegf Mediate Systemic RIPC Effects. RIPC acts systemically, as evinced by the various reports on RIPC-induced protection of liver, heart, stomach, kidney, or lungs.6,11-15 Therefore, we sought to determine whether systemic protection afforded by RIPC is a result of serotonergic release of Vegf into circulation. To assess the effects of serotonin-Vegf in an organ other than the liver, we used a model of 30minute bilateral renal ischemia characterized by 40% survival at 36 hours (2 of 5 mice alive) and creatinine elevations in survivors that persist at 48 hours after reperfusion. Serotonin injection 1 hour before ischemia restored survival to 100% (5 of 5 mice) at 48 hours, along with a significant reduction in creatinine levels (Fig. 3B). In contrast, creatinine levels rose and survival dropped to levels of untreated controls (2 of 5 mice at 36 hours), when animals were pretreated with an aVegf Ab before serotonin injection (Fig. 3B), again indicating that the serotonin-mediated protection from renal IRI is dependent on Vegf. We conclude that the systemic protection from IRI is spread by serotonin-Vegf. Serotonin-Vegf Induce Systemic Protection by Mmp8 and Il10 Up-Regulation in Target Organs. Next, we aimed at identifying what confers protection within specific organs. We measured in liver, kidney, heart, lung, and intestines the expression of a number
of genes previously implicated in preconditioning. Compared to controls, RIPC induced significant expression changes in all investigated organs (Fig. 4A), confirming the systemic action. Intriguingly, only two genes, Mmp8 and Il10, were consistently induced by RIPC across all organs examined (Fig. 4A). Immunohistochemistry (IHC) confirmed the increase of Mmp8 and Il10 in liver after RIPC (Fig. 4B). To examine whether the two genes are associated with protection, we performed RIPC on mice lacking Il10 or pretreated with an Mmp8-specific inhibitor ((3R)-(1)-[2-(4Methoxybenzenesulfonyl)21,2,3,4-tetrahydroisoquinoline-3hydroxamate). RIPC on these mice was efficient in protecting liver from IRI (Fig. 4C). When both molecules were inhibited simultaneously (i.e., by pretreatment of Il102/2 mice with the Mmp8 antagonist), protection was lost, and RIPC even provoked an aggravation of injury (Fig. 4C). Therefore, Il10 and Mmp8 appear to constitute central RIPC effectors that act in concert to induce protection. Similar to RIPC, injection of serotonin alone induced significant Mmp8 and Il10 expression in the target organs, a response that could be inhibited by aVegf pretreatment (Fig. 4D). These findings establish a serotonin-Vegf-Il10/ Mmp8 axis in mediating the systemic organ protection induced by platelet activation.
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Fig. 4. (A) RIPC-induced expression changes across distant organs. In the absence of IR, all investigated organs respond by altered gene expression within 1 hour after RIPC. Note the consistent elevations in Il10 and Mmp8. Fold induction in RIPC-exposed organs was calculated relative to organs from sham-operated controls (n 5 10/goup). (B) IHC for hepatic Il10 and Mmp8. (C) Combined inhibition of Il10 and Mmp8 abolishes the protective effects of RIPC. Serum ALT levels are shown for control mice subjected to IR, for mice subjected to RIPC before IR, for mice treated with an Mmp8 inhibitor before IR, for mice treated with an Mmp8 inhibitor and subjected to RIPC before IR, and for Il10-deficient mice subjected to IR, subjected to RIPC before IR, or treated with an Mmp8 inhibitor and subjected to IR, or to RIPC before IR (n 5 5/group). (D) Serotonin injection (5HT) is sufficient to elevate Il10 and Mmp8 in distant organs. Effects of serotonin were dependent on Vegf (1aVegf). Serotonin (50 mg/kg) was coinjected with either IgG or aVegf (200 ng/kg) 1 hour before harvest. Fold induction was calculated relative to vehicle-treated controls (n 5 5/group).
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Discussion Our experiments support a key role for plateletderived serotonin in systemic tissue protection. Stimuli that activate platelets may converge on the release of serotonin to induce in nearby ECs the secretion of Vegf into circulation, spreading systemic protection to distant organs by up-regulating cytoprotective molecules, such as Il10 and Mmp8. Indeed, serotonin, Vegf, Il10, and Mmp8 have independently been implicated in tissue protection,16,23,25-29 yet their cooperation in priming organs for unknown insults has not been recognized. How preconditioning at a remote limb can spread protection to distant organs has been ill understood thus far. Our findings indicate that the platelet-serotonin-Vegf-Il10/Mmp8 axis is central to the transformation of local stress into systemic protection. The protective effects of RIPC in both hepatic and renal IR were dependent on serotonin and downstream Vegf. Although we have not directly tested additional organs, the RIPC-induced up-regulation of the cytoprotective molecules, Il10 and Mmp8, in liver, kidney, heart, lung, and intestine strongly argues for a common pathway underlying the systemic effects of RIPC. Indeed, Il10/Mmp8 up-regulation could be observed in all examined organs also after injection of serotonin, again in a way that was dependent on Vegf. Likely, other molecules will contribute to the RIPC effects, surely in addition to Il10/Mmp8 at the organ-specific level, but possibly also downstream of serotonin or even in parallel to platelet-dependent signaling. Therefore, we propose that the platelet-serotonin-Vegf-Il10/Mmp8 axis is part of a molecular framework that underlies the systemic effects of remote preconditioning strategies. The knowledge of a universal mechanism behind RIPC may help to optimize its use in the clinic. Il10/ Mmp8 might serve as markers to document RIPC efficacy in the target organ, whereas systemic effects could be monitored by serum Vegf levels. As such, the assessment of the serotonin-VEGF-IL10/MMP8-axis could help to identify clinical situations that may profit from RIPC. Indeed, RIPC might be beneficial in many clinical procedures that involve ischemic insults. Apart from organ resections that require inflow occlusion, RIPC might protect donor organs from ischemic damage during graft preservation and implantation. If so, RIPC might enable the use of marginal grafts that usually would not tolerate ischemic periods, hence expanding the pool of donors. Furthermore, RIPC might provide a safe, physiological way to raise periph-
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eral serotonin levels in patients (i.e., without causing neurological side effects that can result from the injection of serotonin-analogs; Supporting Fig. 1), an effect that might be exploited, for example, to improve the regeneration of old liver.24 Given that RIPC is working in mice and humans, the protective signaling appears to be evolutionarily conserved, potentially acting in many species that harbor platelets, serotonin, and Vegf. In support of a more general biological function, preliminary data (Supporting Fig. 3) indicate that RIPC can mitigate experimental pancreatitis and hepatic acetaminophen toxicity, providing evidence that it protects beyond ischemic insults. RIPC might hence be useful also in clinical situations other than those associated with IR. Considering their recognized function in hemostasis and wound healing, platelets are well designed to transform peripheral stress into a state of intrinsinc alertness. Their activation upon stimuli such as mechanical pressure,30 stasis,31 ischemic insults,32,33 or tissue injury34,35 enables platelets to react in response to a wide range of potentially harmful events. Therefore, we speculate that local stress, such as that incurred by RIPC, may constitute an evolutionary danger signal, activating a preemptive system to protect from potential harm in the proximate future—unlike the acute stress response, where adrenergic signaling provides immediate adaptation to present danger. Given the biological kinship between (nor)epinephrine and serotonin, it remains possible that both systems have coevolved to provide a flexible response toward potential harm. Of note, sympathetic neurons and platelets can communicate and influence one another’s activities.36,37,39 Therefore, the platelet-serotonin-Vegf axis might be part of a larger framework evolved to provide adaptive protection from incoming danger. Acknowledgment: The authors thank Pia Fuchs for her excellent technical assistance.
References 1. Cannon WB. Bodily Changes in Pain, Hunger, Fear and Rage (2nd ed). New York: D. Appleton & Co; 1929. 2. Misslin R. The defense system of fear: behavior and neurocircuitry. Clin Neurophysiol 2003;33:55-66. 3. Lesurtel M, Lehmann K, de Rougemont O, Clavien PA. Clamping techniques and protecting strategies in liver surgery. HPB 2009;11:2905. 4. Jeon J, Watkins A, Wagener G, Samstein B, Guarrera J, Goldstein M, et al. Complex hepatectomy under total vascular exclusion of the liver: impact of ischemic preconditioning on clinical outcomes. W J Surg 2013;37:838-846. 5. Bahde R, Spiegel HU. Hepatic ischaemia-reperfusion injury from bench to bedside. Br J Surg 2010; 97:1461-1475.
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6. R€ udiger HA, Kang KJ, Sindram D, Riehle HM, Clavien PA. Comparison of ischemic preconditioning and intermittent and continuous inflow occlusion in the murine liver. Ann Surg 2002;235:400-407. 7. Clavien PA, Selzner M, R€ udiger HA, Graf R, Kadry Z, Rousson V, Jochum W. A prospective randomized study in 100 consecutive patients undergoing major liver resection with versus without ischemic preconditioning. Ann Surg 2003;238:843-850. 8. Bïtker HE, Kharbanda R, Schmidt MR, Bïttcher M, Kaltoft AK, Terkelsen CJ, et al. Remote ischaemic conditioning before hospital admission, as a complement to angioplasty, and effect on myocardial salvage in patients with acute myocardial infarction: a randomised trial. Lancet 2010;375:727-734. 9. Hausenloy DJ, Mwamure PK, Venugopal V, Harris J, Barnard M, Grundy E, et al. Effect of remote ischaemic preconditioning on myocardial injury in patients undergoing coronary artery bypass graft surgery: a randomised controlled trial. Lancet 2007;370:575-579. 10. Kanoria S, Jalan R, Seifalian AM, Williams R, Davidson BR. Protocols and mechanisms for remote ischemic preconditioning: a novel method for reducing ischemia reperfusion injury. Transplantation 2007;84:445458. 11. Peralta C, Fernandez L, Panes J, Prats N, Sans M, Pique JM, et al. Preconditioning protects against systemic disorders associated with hepatic ischemia-reperfusion through blockade of tumor necrosis factorinduced P-selectin up-regulation in the rat. HEPATOLOGY 2001;33:100113. 12. Oxman T, Arad M, Klein R, Avazov N, Rabinowitz B. Limb ischemia preconditions the heart against reperfusion tachyarrhythmia. Am J Physiol 1997;273:H1707-H1712. 13. Brzozowski T, Konturek PC, Konturek SJ, Pajdo R, Kwiecien S, Pawlik M, et al. Ischemic preconditioning of remote organs attenuates gastric ischemia-reperfusion injury through involvement of prostaglandins and sensory nerves. Eur J Pharmacol 2004;499:201-213. 14. Park KM, Kramers C, Vayssier-Taussat M, Chen A, Bonventre JV. Prevention of kidney ischemia/reperfusion-induced functional injury, MAPK and MAPK kinase activation, and inflammation by remote transient ureteral obstruction. J Biol Chem 2002;277:2040-2049. 15. Xia Z, Herijgers P, Nishida T, Ozaki S, Wouters P, Flameng W. Remote preconditioning lessens the deterioration of pulmonary function after repeated coronary artery occlusion and reperfusion in sheep. Can J Anesth 2003;50:481-488. 16. Nocito A, Georgiev P, Dahm F, Jochum W, Bader M, Graf R, Clavien PA. Platelets and platelet-derived serotonin promote tissue repair after normothermic hepatic ischemia in mice. HEPATOLOGY 2007;45:369-376. 17. Kennedy SE, Erlich JH. Murine renal ischaemia-reperfusion injury. Nephrology 2008;13:390-396. 18. 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. 19. Kiefer FN, Munk VC, Humar R, Dieterle T, Landmann L, Battegay EJ. A versatile in vitro assay for investigating angiogenesis of the heart. Exp Cell Res 2004;300:272-282. 20. Brand T, Anderson GM. The measurement of platelet-poor plasma serotonin: a systematic review of prior reports and recommendations for improved analysis. Clin Chem 2011;57:1376-1386. 21. Penna C, Bassino E, Alloatti G. Platelet activating factor: the good and the bad in the ischemic/reperfused heart. Exp Biol Med 2011;236:390401. 22. Magro A. Serotonin, platelets, and vascular function Central and peripheral mechanisms of cardiovascular regulation, Springer. NATO ASI Series 1986;109:135-161. 23. Faleo G, Neto JS, Kohmoto J, Tomiyama K, Shimizu H, Takahashi T, et al. Carbon monoxide ameliorates renal cold ischemia-reperfusion injury with an upregulation of vascular endothelial growth factor by activation of hypoxia-inducible factor. Transplantation 2008;85:1833-1840.
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24. Furrer K, Rickenbacher A, Tian Y, Jochum W, Bittermann AG, K€ach A, et al. Serotonin reverts age-related capillarization and failure of regeneration in the liver through a VEGF-dependent pathway. Proc Natl Acad Sci U S A 2011;108:2945-2950. 25. Markel TA, Wang Y, Herrmann JL, Crisostomo PR, Wang M, Novotny NM, et al. VEGF is critical for stem cell-mediated cardioprotection and a crucial paracrine factor for defining the age threshold in adult and neonatal stem cell function. Am J Physiol 2008;295:H2308H2314. 26. Nishijima K, Ng YS, Zhong L, Bradley J, Schubert W, Jo N, et al. Vascular endothelial growth factor-A is a survival factor for retinal neurons and a critical neuroprotectant during the adaptive response to ischemic injury. Am J Pathol 2007;171:53-67. 27. Ouyang W, Rutz S, Crellin NK, Valdez PA, Hymowitz SG. Regulation and functions of the IL-10 family of cytokines in inflammation and disease. Ann Rev Immunol 2011;29:71-109. 28. Van Lint P, Libert C. Matrix metalloproteinase-8: cleavage can be decisive. Cytokine Growth Factor Rev 2006;17:217-223. 29. Luo Z, Diaco M, Murohara T, Ferrara N, Isner JM, Symes JF. Vascular endothelial growth factor attenuates myocardial ischemia-reperfusion injury. Ann Thorac Surg 1997;64:993-998. 30. Zhang N, Khachigian LM. Injury-induced platelet-derived growth factor receptor-alpha expression mediated by interleukin-1beta (IL-1beta) release and cooperative transactivation by NF-kappaB and ATF-4: IL1beta facilitates HDAC-1/2 dissociation from promoter. J Biol Chem 2009;284:27933-27943. 31. Harker LA. Role of platelets and thrombosis in mechanisms of acute occlusion and restenosis after angioplasty. Am J Cardiol 1987;60:20B-28B. 32. Hargrave B, Li F. Nanosecond pulse electric field activation of plateletrich plasma reduces myocardial infarct size and improves left ventricular mechanical function in the rabbit heart. J Extra Corpor Technol 2012;44:198-204. 33. Barrabes JA, Garcia-Dorado D, Mirabet M, Inserte J, Agull o L, Soriano B, et al. Antagonism of selectin function attenuates microvascular platelet deposition and platelet-mediated myocardial injury after transient ischemia. J Am Coll Cardiol 2005;45:293-299. 34. Solchaga LA, Bendele A, Shah V, Snel LB, Kestler HK, Dines JS, Hee CK. Comparison of the effect of intra-tendon applications of recombinant human platelet-derived growth factor-BB, platelet-rich plasma, steroids in a rat achilles tendon collagenase model. J Orthop Res 2014; 32:145-150. 35. Aspenberg P. Stimulation of tendon repair: mechanical loading, GDFs and platelets. A mini-review. Int Orthop 2007;3:783-789. 36. Fu LW, Longhurst JC. Activated platelets contribute to stimulation of cardiac afferents during ischaemia in cats: role of 5-HT(3) receptors. J Physiol 2002;544:897-912. 37. Mao HZ, Li Z, Chapleau MW. Platelet activation in carotid sinuses triggers reflex sympathoinhibition and hypotension. Hypertension 1996;27:584-590. 38. Bucher HW, Stucki P. Norepinephrine-induced rise in circulating blood platelets and free fatty acids in arterial and venous blood of patients with ischaemic heart disease and controls. Helv Med Acta 1969;34: 503-509. 39. Francesconi M, Scapin M, Casonato A, Girolami A, Deana R. Adrenaline potentiates type 2B von Willebrand factor-induced activation of human platelets by enhancing both the formation and action of thromboxanes. Thromb Res 2000;100:293-303.
Supporting Information Additional Supporting Information may be found in the online version of this article at the publisher’s website.
Systemic Protection Through Remote Ischemic Preconditioning Is Spread by Platelet-Dependent Signaling in Mice Christian E. Oberkofler,1* Perparim Limani,1* Jae-Hwi Jang,1 Andreas Rickenbacher,1 Kuno Lehmann,1 Dimitri A. Raptis,1 Udo Ungethuem,1 Yinghua Tian,1 Kamile Grabliauskaite,1 Rok Humar,2 Rolf Graf,1 Bostjan Humar,1** and Pierre-Alain Clavien1** Remote ischemic preconditioning (RIPC), the repetitive transient mechanical obstruction of vessels at a limb remote to the operative site, is a novel strategy to mitigate distant organ injury associated with surgery. In the clinic, RIPC has demonstrated efficacy in protecting various organs against ischemia reperfusion (IR), but a common mechanism underlying the systemic protection has not been identified. Here, we reasoned that protection may rely on adaptive physiological reponses toward local stress, as is incurred through RIPC. Standardized mouse models of partial hepatic IR and of RIPC to the femoral vascular bundle were applied. The roles of platelets, peripheral serotonin, and circulating vascular endothelial growth factor (Vegf) were studied in thrombocytopenic mice, Tph12/2 mice, and through neutralizing antibodies, respectively. Models of interleukin-10 (Il10) and matrix metalloproteinase 8 (Mmp8) deficiency were used to assess downstream effectors of organ protection. The protection against hepatic IR through RIPC was dependent on platelet-derived serotonin. Downstream of serotonin, systemic protection was spread through up-regulation of circulating Vegf. Both RIPC and serotonin-Vegf induced differential gene expression in target organs, with Il10 and Mmp8 displaying consistent up-regulation across all organs investigated. Concerted inhibition of both molecules abolished the protective effects of RIPC. RIPC was able to mitigate pancreatitis, indicating that it can protect beyond ischemic insults. Conclusions: We have identified a plateletserotonin-Vegf-Il10/Mmp8 axis that mediates the protective effects of RIPC. The systemic action, the conservation of RIPC effects among mice and humans, and the protection beyond ischemic insults suggest that the platelet-dependent axis has evolved as a preemptive response to local stress, priming the body against impending harm. (HEPATOLOGY 2014;60:1409-1417)
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volution has equipped animals with a range of autonomous mechanisms that protect from imminent harm and increase chances of survival. The “fight-or-flight” or the “freeze” response are well-known examples whereby enemy recognition is translated into a
altered physiological state, increasing performance limits or freezing the body despite fear or pain, respectively.1,2 Unlike the acute responses toward extrinsic stress, the protective reactions of the body toward potential intrinsic harm are less obvious. A fall (or other mechanical insults), for example, bears the risk of blunt trauma, and it is likely that the fall per se will activate a preemptive program to mitigate potential
Abbreviations: 5HTP, 5-hydroxytryptamin; Ab, antibody; ALT, alanine aminotransferase; AST, aspartate transaminase; ECs, endothelial cells; IgG, immunoglobulin G; IHC, immunohistochemistry; Il10, interleukin-10; IP, intraperitoneal; IR, ischemia reperfusion; IRI, ischemia reperfusion injury; Mmp8, matrix metalloproteinase 8; mRNA, messenger RNA; PAF, platelet-activating factor; PBS, phosphate-buffered saline; RIPC, remote ischemic preconditioning; SD, standard deviation; Vegf, vascular endothelial growth factor; WT, wild type. From the 1Laboratory of the Swiss Hepato-Pancreatico-Biliary (HPB) Center, Department of Surgery, University Hospital Zurich, Zurich, Switzerland and 2 Research Unit, Division of Internal Medicine, University Hospital Zurich, Zurich, Switzerland. Received September 4, 2013; accepted February 19, 2014. This work was funded by the Swiss National Foundation (nos. 320030_and 132985; to P.A.C.). *These authors share first authorship. **These authors share senior authorship. 1409
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organ injury resulting from, for example, hemorrhagic shock. Though only vaguely defined, such a protective mechanism exists and is intuitively being exploited in patients. During surgery, blood vessels of the target organ frequently are clamped to prevent blood loss. However, the induced ischemia causes organ injury after reperfusion, significantly increasing postoperative complications. To reduce these risks, surgeons have developed ischemic preconditioning strategies, whereby short periods of preclamping before surgery markedly protect from ischemiareperfusion injury (IRI).3-5 In addition to these strategies, even preclamping of vessels at remote limbs, rather than at the target organ (remote ischemic preconditioning; RIPC), can induce protection. More so, RIPC through mechanical pressure instead of clamping is sufficient to protect various organs against ischemia reperfusion (IR).610 Therefore, RIPC, either by clamping or pressure, is able to afford systemic protection from organ injury.6-15 Animal models of RIPC and IRI have been developed to identify candidate molecules inducing organspecific protection.11-15 However, these animal models also provide a unique opportunity to investigate a conserved biological mechanism that may underlie the transformation of a peripheral signal into systemic protection from an unknown insult. Here, we investigated the role of platelets and downstream signaling events in the spread of systemic protection through RIPC.
Materials and Methods Animals. All animal experiments were in accord with Swiss Federal Animal Regulations and approved by the Veterinary Office of Zurich. Animals 10-12 weeks of age were kept on a 12-hour day/night cycle with free access to food and water. C57Bl/6 mice were obtained from Harlan Laboratories BV (Venray, The Netherlands), and interleukin-10 (Il10)2/2 and Tph12/2 mice were obtained from our in-house captive breeding. Thrombocytopenia was induced by intraperitoneal (IP) injection of 1 mg/kg of anti-CD41 (clone MWReg 30, with immunoglobulin G [IgG]2 as a control; BD Biosciences, Franklin Lakes, NJ) in 200 mL of phosphate-buffered saline (PBS), reducing platelet numbers by >90%.16 Blood cell counts from tail-vein samples collected 24 hours after injection and diluted
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in 20 mL of 12 mM of ethylenediaminetetraacetic acid were assessed using a Coulter AcTdiff counter (Beckman Coulter, Nyon, Switzerland). Animal Surgery. Anesthesia was induced by isoflurane inhalation (2%-4%) by a concentric oronasal mask connected to an anesthetic circuit. Anesthetic depth was monitored by clinical parameters (respiratory rate and depth, color of mucous membranes and inner organs, movement, and reflexes). RIPC was done by clamping the femoral vascular bundle just proximal to its confluence with the femoral nerve (Fig. 1). A protocol of 5 minutes of limb ischemia, followed by 5 minutes of reperfusion for a total of four cycles, was used. Cessation of blood flow was confirmed by the absence of femoral artery pulsation distal from the clamp and a change in foot color. After closure of the femoral wound (approximately 5 minutes after the last RIPC cycle), laparotomy for hepatic ischemia was performed through a midline incision. The membrane connecting the caudate to the left lobe was divided. A microvascular clamp was applied to the portal triad supplying the left and median lobes, rendering approximately 70% of the liver ischemic, while avoiding mesenteric venous congestion by permitting portal decompression through the caudate and right lobes of the liver. Successful occlusion of the portal triad branch was confirmed by a change in color. The abdominal wall was closed by a suture during hepatic ischemia and mice were woken up. After 60 minutes, liver reperfusion was reestablished through the opening of the abdominal cavity, followed by removal of the microvascular clamp. After surgery, animals were allowed to recover on a warming pad in a separate cage until completely conscious. Kidney ischemia was induced by clamping the vessel bundle to both kidneys for 30 minutes through a midline laparotomy under general anesthesia on a warming pad.17 After reperfusion, animals were terminated by exsanguination through blood collection and opening of the diaphragm. Blood was immediately centrifuged at 5,000 rpm for 5 minutes. The serum supernatant was stored at 270 C until assayed for creatinine. Organs were stored in 10% formal saline for histopathology or snap-frozen in liquid nitrogen. Animal Treatment. Serotonin (50 mg/kg) was coinjected IP with either IgG or alpha vascular
Address reprint requests to: Pierre-Alain Clavien, M.D., Ph.D., Department of Surgery, University Hospital Zurich, Raemistrasse 100, CH-8091 Zurich, Switzerland. E-mail: [email protected]; fax: 141 43 255 44 49. C 2014 by the American Association for the Study of Liver Diseases. Copyright V View this article online at wileyonlinelibrary.com. DOI 10.1002/hep.27089 Potential conflict of interest: Nothing to report.
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Fig. 1. (A) Protective effects of RIPC in hepatic IR. Upper panels show serum AST and ALT levels of mice subjected to IR (n 5 6) or to RIPC followed by IR (n 5 6). Lower panels show necrotic injury at 6 hours after IR or IR plus RIPC and corresponding quantification (n 5 6/group) to the left. (B) RIPC activates platelets. The drop in platelet numbers paralleled by an increase in PAF serum levels 1 hour after RIPC is shown (n 5 6/group). *P < 0.05.
endothelial growth factor (aVegf; 4 mg/kg; R&D Systems, Abingdon, UK) one hour before treatment or harvest. Serotonin reloading of platelets from Tph12/2 mice with the precursor, 5-hydroxytryptamin (5HTP), was done as previously described.18 The Mmp8 inhibitor I ((3R)-(1)-[2-(4-methoxybenzenesulfonyl)1,2,3,4-tetrahydroisoquinoline-3-hydroxamate]; Calbiochem, Billerica, MA) was IP injected at 80 nM (100 lL/mouse) 1 hour before IRI, whereas the platelet-activating factor (PAF) receptor antagonist (SM-12502; Sigma-Aldrich, Munich, Germany) was injected at 30 lM. Histological Examination. Hematoxylin and eosine staining was performed on 3-mm archived sections of liver. Immunostainings for Mmp8 (catalog no.: ab53017; Abcam, Cambridge, UK) and Il10 (catalog no.: 505002; Biolegend, Fell, Germany) were done using the Ventana Discovery automated staining system and the iView DAB kit (Ventana Medical Systems, Tucson, AZ). All histological analyses were performed in a blinded fashion. Serum Alanine Aminotransferase/Serum Aspartate Aminotransferase. Serum alanmine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured using a serum multiple biochemical analyzer (Ektachem DTSCII; Johnson & Johnson Inc., Rochester, NY). Creatinine was measured with the
DRI-CHEM 4000i analyzer (Fujifilm, Dielsdorf, Switzerland). Isolation of Mouse Aortic Endothelial Cells. Mouse aortic endothelial cells (ECs) were isolated from aortae of 8- to 10-week-old C57BL/6 mice. Plates (24-well) were coated with fibrin gels, as previously described.19 The excised aorta was cleaned from adjacent tissue, cut in small rings, placed onto the wells, overlaid by fibrin gel, and covered with EC growth media (serum-free Dulbecco’s modified Eagle’s medium), 10% fetal calf serum (Biochrom, Cambridge, UK), 100 lg/mL of endothelial cell growth supplement (Millipore, Zug, Switzerland), and 0.1 lL/ mL of heparin. Every other day, 300 lg/mL of eamino caproic acid (Sigma-Aldrich, St Louis, MO) in PBS were added. After 10 days, outgrowing endothelial sprouts were harvested and the gel-cell mixture was transferred to a six-well plate coated with 0.1% gelatin plus EC growth media. Confluent cells were subcultured and characterized with the endothelial marker, von Willebrand factor (LabForce AG, Nunningen, Switzerland). Quantitative Real-Time Polymerase Chain Reaction. Total RNA was extracted from 50 mg of organ tissue using Trizol reagent (Invitrogen, Basel, Switzerland) following the manufacturer’s instructions. Quantitative real-time polymerase chain reaction was
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performed on complementary DNA (ThermoScript reverse-transcription PCR System; Invitrogen) using an ABI Prism 7500 Sequence Detector System (PE Applied Biosystems, Rotkreuz, Switzerland). TaqMan gene expression assays for Hsp1a (Mm01159846_s1), Gpx1 (Mm00656767_g1), Txn (Mm00726847_s1), Nfkbia (Mm00477798_m1), Mmp8 (Mm00439509_m1), NOS2a (Mm00440485_m1), C3 (Mm00437858_m1), IL1b (Mm00434228_m1), Clec10a (Mm00546124_m1), Il6 (Mm00446190_m1), and Il10 (Mm00439616_m1)
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were from PE Applied Biosystems and normalized to 18S ribosomal RNA (TaqMan control reagents; PE Applied Biosystems). Results represent mean fold induction of messenger RNA (mRNA) expression 6 standard deviation (SD). Statistical Analysis. Differences between groups (n 5 animals) were assessed by a Mann-Whitney’s U test or by one-way analysis of variance, where applicable. Statistical significance was set at P < 0.05, with significant values marked by asterisks. Error bars refer to SDs.
Results RIPC Protects From Hepatic IRI. To gain insight into the chain of events that may constitute the protective mechanisms behind preconditioning, we designed a series of experiments based on hindlimb RIPC and its effects in different organs before or after IR. The initial experiments tested the efficacy of hindlimb RIPC to the femoral vessel bundle in mitigating liver IRI. Blood flow was mechanically interrupted to induce cycles of local ischemia for repetitive periods to the remote limb, followed by 1 hour of organ-specific ischemia to 70% of the murine liver. At 6 hours after reperfusion, we observed significant protection of the liver from IRI, as shown by a marked reduction in
Fig. 2. (A) Dependence of RIPC-induced protection on plateletderived serotonin. Serum ALT levels are shown for control animals (wt) subjected to hepatic IR or to RIPC before IR; for immunothrombocytopenic mice (ITP; depleted of platelets by aCd41 antibodies) subjected to IR, or to RIPC before IR; and for mice carrying platelets without serotonin (Tph12/2) subjected to IR, or to RIPC before IR, or reloaded with serotonin (5HTP; supplementation with the serotonin precursor) and subjected to RIPC before IR, or injected with serotonin before IR. (B) Serum Vegf levels correlate with the protective effect of RIPC. Vegf levels are shown for control mice (wt) either untreated (wt Control) or subjected to RIPC; for serotonin-deficient mice (Tph12/2) either untreated, or subjected to RIPC, or reloaded with serotonin (5HTP), or reloaded with serotonin and subjected to RIPC, or reloaded with serotonin and treated with 200 ng/kg of aVegf 1 hour before RIPC; and for immunothrombocytopenic mice (ITP) either untreated, subjected to RIPC, or injected with 50 mg/kg of serotonin (5HT). Vegf elevation above the detection limit (dotted line; *P < 0.05, relative to untreated controls) was noted only in models that also displayed protection from IRI (wt1RIPC; Tph12/2 15HTP1RIPC; ITP15HT). Treatment with unspecific IgG had no effect on Vegf levels (not shown). Vegf levels were assessed 30 minutes after the last manipulation. (C) Protection through RIPC is dependent on circulating Vegf. Serum ALT levels are shown for control mice (wt) subjected to IR, to RIPC before IR, or treated with 4 lg/kg of Vegf 1 hour before IR (IR 1Vegf wt), or treated with aVegf 1 hour before RIPC; for control mice treated with 4 lg/kg of unspecific IgG 1 hour before IR (wt1IgG); and for serotonindeficient mice (Tph12/2) reloaded with serotonin (5HTP) and treated with aVegf before RIPC and IR, or not reloaded with serotonin but treated with Vegf before IR. *Significant protection (P < 0.05) relative to untreated controls.
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both ALT/AST levels and necrosis by approximately two thirds, compared to controls (Fig. 1A). RIPC Protects Through Platelet-Derived Serotonin. Trying to explore the mechanism that initiates protection from IRI, we reasoned that the stress RIPC puts on vessels may activate platelets. Consistent with this hypothesis, we observed a drop in platelet numbers by 40% after RIPC, along with a concomitant increase in serum levels of PAF and plasma P-selectin (Fig. 1B and Supporting Fig. 1). To substantiate a dependence of RIPC effects on platelets, mice were depleted of platelets by IP injection of Cd41 antibodies (Abs; Supporting Fig. 1). RIPC after immune depletion lost its protective effects, indicating the requirement for platelets (Fig. 2A). Next, we asked whether platelets must be activated to initiate protection. After injection of SM-12502, a PAF receptor (Ptafr) antagonist that inhibits platelet activation, protection through RIPC was again abolished (RIPC control: median, ALT 6,350 U/I; range, 5150-7800; RIPC plus SM-12502: median, ALT 11,200 U/I; range, 8,700-12,800; n 5 5/group). We postulated that molecules released by activated platelets might participate in the protection. Among the numerous factors stored in platelets, we focused on serotonin, because platelets are carriers of essentially all peripheral serotonin.20 Moreover, the molecule can promote tissue repair after ischemia.16 To investigate the role of blood serotonin, we applied RIPC to Tph12/2 mice that lack peripheral serotonin, yet have normal platelet counts.18 RIPC did not protect Tph12/2 mice from hepatic IRI (Fig. 2A), suggesting a key role for plateletderived serotonin in mediating RIPC effects. Serotonin reloading of Tph12/2 mice18 by injecting 50 mg/kg of its precursor, 5HTP (the product of Tph1) restored the protective effect, confirming the requirement of plateletderived serotonin for the RIPC effects (Fig. 2A). To detail this finding, we inhibited platelet activation in wild-type (WT) mice by SM-12502 before serotonin injection, resulting in a partial restoration of protection (RIPC plus SM-12502: median, ALT 11,200 U/I; range, 8,700-12,800; SM-12502 plus serotonin: median, ALT 6,600 U/I; range 4,900-8,850; n55/group). Therefore, serotonin acts downstream of PAF-associated platelet activation, but the incomplete restoration suggests that PAF may incur serotonin-independent protection, perhaps through Ptafr, which is expressed also on nonplatelet cell types. Alternatively, Ptafr inhibition and/or SM12502 might have additional effects that counteract the protective effects of serotonin.21 RIPC Requires Vegf for Its Protective Effects. As a result of its rapid reuptake, serotonin usually acts locally22 and thus may not mediate the systemic RIPC
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effects. To identify potential pathways that may spread systemic protection downstream of serotonin, we investigated Vegf, because Vegf has been implicated in the amelioration of IRI,23 and because serotonin can increase serum Vegf levels.24 Within 30 minutes after RIPC, serum Vegf levels significantly increased in WT mice (Fig. 2B). Injection of recombinant Vegfa into mice before IR conferred similar effects on IRI as RIPC alone (Fig. 2C), indicating that circulating Vegf is sufficient for protection. Vice versa, aVegf Ab neutralized the effect of RIPC (Fig. 2C), indicating that RIPC relies on Vegf for protection. Therefore, both serotonin and Vegf participate in RIPC-induced protection from IRI. Vegf Acts Downstream of Serotonin to Induce RIPC Protection. To investigate the relationship between serotonin and Vegf in RIPC protection, we injected Vegf into serotonin-deficient Tph12/2 mice, which are unresponsive to RIPC. Vegf restored the protective effects of RIPC in Tph12/2 mice (Fig. 2C). Conversely, protection was lost when aVegf Ab was injected concurrently with RIPC into Tph12/2 animals supplemented with 5HTP (i.e., reloading platelets with serotonin; Fig. 2C). We conclude that serotonin mediates RIPC protection through Vegf. In support, RIPC elevated Vegf serum levels above the detection limit in WT mice, in Tph12/2 mice after 5HTP supplementation, and in platelet-depleted animals injected with serotonin, but not in the animal models featuring serotonin deficiency and RIPC resistance (Fig. 2B). Thus, significant Vegf elevation is dependent on the presence of peripheral serotonin and strictly correlates with measures that protect from IRI. Although platelets can store Vegf, serum Vegf after RIPC seems to originate from other cells than platelets, because Vegf levels rise also in platelet-depleted mice injected with serotonin (Fig. 2B). Given the rather local action of serotonin, ECs may be a source of Vegf24 and are also directly affected by RIPC. To address this possibility, we established primary endothelial cultures from mouse aorta and treated the cells with serotonin. Within 60 minutes of treatment, ECs produced Vegf mRNA and secreted Vegf into supernatant (Fig. 3A). Whereas ECs are a likely source of Vegf after RIPC, contribution from other vessel-associated cell types is conceivable. Considering that serotonin injection was protective also in platelet-depleted mice, the amount of Vegf released from ECs (and associated cells) seems to be sufficient for the mediation of RIPC effects. However, in mice with normal platelet counts, release of Vegf from degranulating platelets is likely to contribute to the elevation in circulating Vegf levels.
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Fig. 3. (A) Serotonin induces Vegf secretion from mouse ECs. (B) Serotonin through Vegf protects from renal IRI. Forty-eight hours after IR in the kidney, creatinine levels are markedly elevated (IR), compared to controls (no IR). Serotonin injection (50 mg/kg) together with unspecific IgG 1 hour before renal IR (P < 0.05) reduces creatinine levels both at 24 and 48 hours post-IR, an effect that is abolished by adding aVegf instead of IgG (lower panel). At 48 hours, SDs were not added because only 2 of 5 animals were alive in the unprotected groups. *P < 0.05.
Serotonin-Vegf Mediate Systemic RIPC Effects. RIPC acts systemically, as evinced by the various reports on RIPC-induced protection of liver, heart, stomach, kidney, or lungs.6,11-15 Therefore, we sought to determine whether systemic protection afforded by RIPC is a result of serotonergic release of Vegf into circulation. To assess the effects of serotonin-Vegf in an organ other than the liver, we used a model of 30minute bilateral renal ischemia characterized by 40% survival at 36 hours (2 of 5 mice alive) and creatinine elevations in survivors that persist at 48 hours after reperfusion. Serotonin injection 1 hour before ischemia restored survival to 100% (5 of 5 mice) at 48 hours, along with a significant reduction in creatinine levels (Fig. 3B). In contrast, creatinine levels rose and survival dropped to levels of untreated controls (2 of 5 mice at 36 hours), when animals were pretreated with an aVegf Ab before serotonin injection (Fig. 3B), again indicating that the serotonin-mediated protection from renal IRI is dependent on Vegf. We conclude that the systemic protection from IRI is spread by serotonin-Vegf. Serotonin-Vegf Induce Systemic Protection by Mmp8 and Il10 Up-Regulation in Target Organs. Next, we aimed at identifying what confers protection within specific organs. We measured in liver, kidney, heart, lung, and intestines the expression of a number
of genes previously implicated in preconditioning. Compared to controls, RIPC induced significant expression changes in all investigated organs (Fig. 4A), confirming the systemic action. Intriguingly, only two genes, Mmp8 and Il10, were consistently induced by RIPC across all organs examined (Fig. 4A). Immunohistochemistry (IHC) confirmed the increase of Mmp8 and Il10 in liver after RIPC (Fig. 4B). To examine whether the two genes are associated with protection, we performed RIPC on mice lacking Il10 or pretreated with an Mmp8-specific inhibitor ((3R)-(1)-[2-(4Methoxybenzenesulfonyl)21,2,3,4-tetrahydroisoquinoline-3hydroxamate). RIPC on these mice was efficient in protecting liver from IRI (Fig. 4C). When both molecules were inhibited simultaneously (i.e., by pretreatment of Il102/2 mice with the Mmp8 antagonist), protection was lost, and RIPC even provoked an aggravation of injury (Fig. 4C). Therefore, Il10 and Mmp8 appear to constitute central RIPC effectors that act in concert to induce protection. Similar to RIPC, injection of serotonin alone induced significant Mmp8 and Il10 expression in the target organs, a response that could be inhibited by aVegf pretreatment (Fig. 4D). These findings establish a serotonin-Vegf-Il10/ Mmp8 axis in mediating the systemic organ protection induced by platelet activation.
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Fig. 4. (A) RIPC-induced expression changes across distant organs. In the absence of IR, all investigated organs respond by altered gene expression within 1 hour after RIPC. Note the consistent elevations in Il10 and Mmp8. Fold induction in RIPC-exposed organs was calculated relative to organs from sham-operated controls (n 5 10/goup). (B) IHC for hepatic Il10 and Mmp8. (C) Combined inhibition of Il10 and Mmp8 abolishes the protective effects of RIPC. Serum ALT levels are shown for control mice subjected to IR, for mice subjected to RIPC before IR, for mice treated with an Mmp8 inhibitor before IR, for mice treated with an Mmp8 inhibitor and subjected to RIPC before IR, and for Il10-deficient mice subjected to IR, subjected to RIPC before IR, or treated with an Mmp8 inhibitor and subjected to IR, or to RIPC before IR (n 5 5/group). (D) Serotonin injection (5HT) is sufficient to elevate Il10 and Mmp8 in distant organs. Effects of serotonin were dependent on Vegf (1aVegf). Serotonin (50 mg/kg) was coinjected with either IgG or aVegf (200 ng/kg) 1 hour before harvest. Fold induction was calculated relative to vehicle-treated controls (n 5 5/group).
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Discussion Our experiments support a key role for plateletderived serotonin in systemic tissue protection. Stimuli that activate platelets may converge on the release of serotonin to induce in nearby ECs the secretion of Vegf into circulation, spreading systemic protection to distant organs by up-regulating cytoprotective molecules, such as Il10 and Mmp8. Indeed, serotonin, Vegf, Il10, and Mmp8 have independently been implicated in tissue protection,16,23,25-29 yet their cooperation in priming organs for unknown insults has not been recognized. How preconditioning at a remote limb can spread protection to distant organs has been ill understood thus far. Our findings indicate that the platelet-serotonin-Vegf-Il10/Mmp8 axis is central to the transformation of local stress into systemic protection. The protective effects of RIPC in both hepatic and renal IR were dependent on serotonin and downstream Vegf. Although we have not directly tested additional organs, the RIPC-induced up-regulation of the cytoprotective molecules, Il10 and Mmp8, in liver, kidney, heart, lung, and intestine strongly argues for a common pathway underlying the systemic effects of RIPC. Indeed, Il10/Mmp8 up-regulation could be observed in all examined organs also after injection of serotonin, again in a way that was dependent on Vegf. Likely, other molecules will contribute to the RIPC effects, surely in addition to Il10/Mmp8 at the organ-specific level, but possibly also downstream of serotonin or even in parallel to platelet-dependent signaling. Therefore, we propose that the platelet-serotonin-Vegf-Il10/Mmp8 axis is part of a molecular framework that underlies the systemic effects of remote preconditioning strategies. The knowledge of a universal mechanism behind RIPC may help to optimize its use in the clinic. Il10/ Mmp8 might serve as markers to document RIPC efficacy in the target organ, whereas systemic effects could be monitored by serum Vegf levels. As such, the assessment of the serotonin-VEGF-IL10/MMP8-axis could help to identify clinical situations that may profit from RIPC. Indeed, RIPC might be beneficial in many clinical procedures that involve ischemic insults. Apart from organ resections that require inflow occlusion, RIPC might protect donor organs from ischemic damage during graft preservation and implantation. If so, RIPC might enable the use of marginal grafts that usually would not tolerate ischemic periods, hence expanding the pool of donors. Furthermore, RIPC might provide a safe, physiological way to raise periph-
HEPATOLOGY, October 2014
eral serotonin levels in patients (i.e., without causing neurological side effects that can result from the injection of serotonin-analogs; Supporting Fig. 1), an effect that might be exploited, for example, to improve the regeneration of old liver.24 Given that RIPC is working in mice and humans, the protective signaling appears to be evolutionarily conserved, potentially acting in many species that harbor platelets, serotonin, and Vegf. In support of a more general biological function, preliminary data (Supporting Fig. 3) indicate that RIPC can mitigate experimental pancreatitis and hepatic acetaminophen toxicity, providing evidence that it protects beyond ischemic insults. RIPC might hence be useful also in clinical situations other than those associated with IR. Considering their recognized function in hemostasis and wound healing, platelets are well designed to transform peripheral stress into a state of intrinsinc alertness. Their activation upon stimuli such as mechanical pressure,30 stasis,31 ischemic insults,32,33 or tissue injury34,35 enables platelets to react in response to a wide range of potentially harmful events. Therefore, we speculate that local stress, such as that incurred by RIPC, may constitute an evolutionary danger signal, activating a preemptive system to protect from potential harm in the proximate future—unlike the acute stress response, where adrenergic signaling provides immediate adaptation to present danger. Given the biological kinship between (nor)epinephrine and serotonin, it remains possible that both systems have coevolved to provide a flexible response toward potential harm. Of note, sympathetic neurons and platelets can communicate and influence one another’s activities.36,37,39 Therefore, the platelet-serotonin-Vegf axis might be part of a larger framework evolved to provide adaptive protection from incoming danger. Acknowledgment: The authors thank Pia Fuchs for her excellent technical assistance.
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Supporting Information Additional Supporting Information may be found in the online version of this article at the publisher’s website.
