Original Communications

Accepte

ReprintVan Nu46202. E

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� 2012

doi:10.1

Pretreating mesenchymal stem cellswith interleukin-1b and transforminggrowth factor-b synergistically increasesvascular endothelial growth factorproduction and improvesmesenchymal stem cell–mediatedmyocardial protection after acuteischemiaYong Luo, PhD,a Yue Wang, PhD,a Jeffrey A. Poynter, MD,a Mariuxi C. Manukyan, MD,a

Jeremy L. Herrmann, MD,a Aaron M. Abarbanell, MD,a Brent R. Weil, MD,a andDaniel R. Meldrum, MD,a,b,c,d Indianapolis, IN

Background. Mesenchymal stem cells (MSCs) improve postischemic myocardial function in part throughtheir secretion of growth factors such as vascular endothelial growth factor (VEGF). Pretreating MSCswith various cytokines or small molecules can improve VEGF secretion and MSC-mediatedcardioprotection. However, whether 1 cytokine can potentiate the effect of another cytokine in MSCpretreatment to achieve a synergistic effect on VEGF production and cardioprotection is poorly studied.Methods. MSCs were treated with interleukin (IL)-1b and/or transforming growth factor (TGF)-b1 for24 hours before experiments. VEGF production was determined by enzyme-linked immunosorbent assay.Isolated hearts from adult male Sprague-Dawley rats were subjected to 15 minutes of equilibration, 25minutes of ischemia, and 40 minutes reperfusion. Hearts (n = 5–7 per group) were randomly infusedwith vehicle, untreated MSCs, or MSCs pretreated with IL-1b and/or TGF-b1. Specific inhibitors wereused to delineate the roles of p38 mitogen-activated protein kinase (MAPK) and SMAD3 in IL-1b– andTGF-b1–mediated stimulation of MSCs.Results. MSCs cotreated with IL-1b and TGF-b1 exhibited synergistically increased VEGF secretion, andthey greatly improved postischemic myocardial functional recovery. Ablation of p38 MAPK and SMAD3activation with specific inhibitors negated both IL-1b– and TGF-b1–mediated VEGF production inMSCs and the ability of these pretreated MSCs to improve myocardial recovery after ischemia.Conclusion. Pretreating MSCs with 2 cytokines may be useful to fully realize the potential of cell-basedtherapies for ischemic tissues. (Surgery 2012;151:353-63.)

From the Department of Surgery,a Indiana University School of Medicine; the Department of CardiovascularSurgery,b Methodist Hospital; the Department of Cellular and Integrative Physiology,c and the Center forImmunobiology,d Indiana University School of Medicine, Indianapolis, IN

d for publication September 22, 2011.

requests: Daniel R. Meldrum, MD, 635 Barnhill Drive,ys Medical Science Bldg Rm. #2017, Indianapolis, IN-mail: dmeldrum@iupui.edu.

60/$ - see front matter

Mosby, Inc. All rights reserved.

016/j.surg.2011.09.033

ACCUMULATING EVIDENCE has shown that mesenchy-mal stem cells (MSCs) can protect the myocardiumfrom ischemic injury.1 These MSCs mediate theirtherapeutic effects largely through the productionof paracrine growth factors.1 These growth factorspromote angiogenesis, reduce apoptosis, increasecell survival and ultimately improve postischemic

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myocardial function.2 One of these growth factors,vascular endothelial growth factor (VEGF), plays acritical role in mediating the beneficial effects ofMSCs. Increased VEGF has been shown to mediateMSC-induced protection in the ischemic heart.3 Inaddition, VEGF overexpression in MSCs incremen-tally improves myocardial protection.4 Therefore,pretreatment of MSCs with cytokines or small mol-ecules to increase MSC VEGF production may aug-ment their cardioprotective effects following acuteischemia/reperfusion. Whether these cytokinescan enhance each other’s function in VEGF pro-duction and MSC mediated myocardial protectionto produce a synergistic effect is largely unknown.

Interleukin (IL)-1b increases the expression ofVEGF transcripts in several cell lines includingbone marrow mesenchymal stem cells and cardiacmyocytes.5 Two IL-1b receptors on the cell mem-brane have been identified. The type I IL-1 receptor(IL-1RI) mediates signal transduction upon bind-ing IL-1b while the type II receptor (IL-1RII) servesas a decoy receptor without the initiation of down-stream signal transduction, therefore inhibitingIL-1b signal transduction through IL-1RI by trap-ping IL-1b and reducing available ligand for inter-action.6 Upon IL-1b binding of IL-1RI, a series ofdownstream phosphorylation events leads to theactivation of transcription factors including nuclearfactor-kB, c-jun, Sp1, and ATF2.5 These activatedtranscription factors orchestrate the expression oftarget genes such as VEGF. Among the kinasesdownstream of IL-1RI, p38 mitogen-activated pro-tein kinase (MAPK) is preferentially activated byIL-1b during heart ischemia. Global ischemia acti-vates p38 MAPK and the activation is sustained dur-ing reperfusion in the isolated perfused rat heart.5

IL-1b–activated p38 MAPK increases the transcrip-tion of VEGF, whereas inhibition of p38 MAPKablates IL-1b–induced VEGF expression in cardiacmyocytes.5

Several years have passed since initial evidenceshowed that transforming growth factor (TGF)-b1can induce VEGF secretion.7 TGF-b1 is a 25-kDahomodimeric protein that belongs to the TGF-b su-perfamily consisting of >30 members, includingTGF-b, activins, and bone morphogenetic proteins.The initiation of TGF-b1 signaling occurs throughinteraction with the TGF-b type II receptor(TbR-II), a constitutively active kinase. ThisTGF-bI–TbR-II complex is then recognized byTGF-b type I receptor (TbR-I). After the recruit-ment of TbR-I, TbR-II transphosphorylates TbR-Iat serine and threonine residues which activatesTbR-I, propagating downstream signal transduc-tion through the recruitment and phosphorylation

of downstream proteins, most importantly the tran-scription factor SMADs (Sma and Mad related pro-teins).8 Upon activation through the disruption ofautoinhibition by phosphorylation, the SMADstranslocate to the nucleus from cytoplasm and in as-sociation with other transcription factors activatetarget gene transcription including VEGF.8 Resultsof several studies have shown that TGF-b1 treatmentincreases the expression of VEGF in a variety of celltypes, including lung fibroblasts, macrophages, andmesenchymal stem cells through SMAD3 andERK1/2.9,10 TGF-b1 does not stimulate VEGFproduction in SMAD3 knockout cells or cellspretreated with SMAD3 siRNA.11 Two Smad3/4-binding elements in the VEGF promoter regionare critical for TGF-b1–induced VEGF secretion.9

A large body of evidence has shown that 1 cyto-kine may potentiate the effect of another toachieve synergistic effects. For instance, studieshave demonstrated that TNF-b and interferon havesynergistic effects on IL-8 secretion.12 AlthoughIL-1b and TGF-b1 activate the expression ofVEGF through different pathways, it is unknownwhether preconditioning MSCs using IL-1b andTGF-b1 cotreatment can synergistically increaseVEGF production in MSCs and maximize theircardioprotective effects after acute ischemia/reperfusion injury. Therefore, we hypothesized inthis study that; (1) preconditioning MSCs withIL-1b and TGF-b1 will provide a synergistic effecton VEGF secretion; and (2) myocardial functionalrecovery after acute ischemia/reperfusion injuryafter intracoronary infusion of MSCs precondi-tioned with both IL-1b and TGF-b1 will be im-proved to a greater degree compared withnonpreconditioned MSCs and MSCs precondi-tioned by IL-1b or TGF-b1 individually; and (3)the synergistic effect on VEGF secretion by the com-binational treatment of IL-1b and TGF-b1 is medi-ated by the activation of p38 MAPK signal andSMAD3.

MATERIALS AND METHODS

Animals. Normal, adult, male Sprague-Dawleyrats were obtained fromHarlan (250–330 g; Indian-apolis, IN). Adult male C57BL/6J mice wereobtained from Jackson Laboratory (Bar Harbor,ME). Animals were fed a standard diet and accli-mated in a quiet quarantine room for 1 week beforethe experiments. The animal protocol was reviewedand approved by the Indiana Animal Care and UseCommittee of Indiana University. All animals re-ceived humane care in compliance with the Guidefor the Care and Use of Laboratory Animals (NIHpublication No. 85-23, revised 1996).

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Preparation of mouse bone marrow MSCs. Asingle-step stem cell purification method usingadhesion to cell culture flask plastic was used, aspreviously described.13 Briefly, mouse bonemarrowcells were collected disaggregated, and washed.The cell pellet was then resuspended and culturedin 75-cm2 culture flasks with complete media at378C, 5% CO2. MSCs between passages 3 and 8were used for experimentation.

Isolated heart perfusion (Langendorff). Heartswere isolated as previously described.13 Rats wereanesthetized (sodium pentobarbital, 60 mg kg�1,intraperitoneally [IP]) and heparinized (500 U,IP). Hearts were rapidly excised, cannulated, andperfused in isovolumetric mode (75 mmHg) witha modified Krebs-Henseleit solution at 378C andbubbled with 95% O2. A water-filled latex balloonwas inserted into the left ventricle. The balloonpressure was adjusted to a mean left ventricularend-diastolic pressure (EDP) of 10 mmHg (range,6–16) during equilibration. The preload volumewas held constant during the entire experimentto allow continuous recording of the left ventricu-lar developed pressure (LVDP). Hearts wereallowed to equilibrate for 15 minutes before vehi-cle or MSCs infusion and paced at 350 beats/minto ensure a standard heart rate between groups.For the cytokines pretreating experiment, MSCswere pretreated with indicated cytokines for 24hours before infusion. Immediately, 1 mL vehicleor MSCs (1 3 106 cells/mL) were infused before is-chemia according to the following groups (n = 5–7per group): (1) vehicle control; (2) MSCs; (3) IL-1b–pretreated MSCs; (4) TGF-b1–pretreated MSCs;and (5) IL-1b– and TGF-b1–pretreated MSCs. Forthe inhibitors pretreating experiment, 1 mL MSCs(13 106 cells/mL) were infused immediately beforeischemia according to the following groups (n = 5per group): (1) MSCs; (2) IL-1b– and TGF-b1–pretreated MSCs; and (3) IL-1b– and TGF-b1–pre-treated MSCs with p38 MAPK inhibitor (SB203580,EMD Chemicals, Gibbstown, NJ) and SMAD3 inhib-itor (SIS3, EMD Chemicals). Before infusion, MSCswere washed with phosphate-buffered saline, resus-pended in 1 mL warm (378C) oxygenated modifiedKrebs-Henseleit solution, then infused into the cor-onary arteries 1 minute immediately before ische-mia. A 3-way stopcock above the aortic root wasused to create global ischemia, during which theheart was placed in a 378C degassed organ bathfor 25 minutes. Hearts were then reperfused for40 minutes after ischemia. The LVDP and EDPwere continuously recorded using a PowerLab 8preamplifier/digitizer (AD Instruments Inc, Mil-ford, MA) and an Apple iMac computer (Apple

Computer Inc, Cupertino, CA). Themaximum pos-itive and negative values of the first derivative ofpressure (+dP/dt and �dP/dt) were calculated us-ing PowerLab software.

Enzyme-linked immunosorbent assay. MSCswere plated on 12-well cell culture dishes in trip-licates or quadruplicates for each treatment groupat the density of 5 3 104/well/mL the day beforetreatment. MSCs were treated with indicated cyto-kines and/or inhibitors for 24 hours. Supernatantswere then collected and VEGF production wasdetermined by the enzyme-linked immunosorbentassay kit according to the manufacturer’s instruc-tions (R&D Systems Inc, Minneapolis, MN). Allsamples and standards were measured in dupli-cate. Values were normalized by cell number(pg/105 cells).

Presentation of data and statistical analysis.Data were plotted using GraphPad Prism software(GraphPad Software, Inc., La Jolla, CA). Allreported values are mean ± standard error of themean. Statistical differences between control andtreatment groups were determined using theStudent t test or 1-way ANOVA with Bonferronipost hoc analysis.

RESULTS

Cotreatment with IL-1b and TGF-b1 synergisti-cally increased MSCs VEGF secretion. MSCstreated with IL-1b exhibited a dose-dependentincrease in VEGF production compared with thecontrol group (Fig 1, A). MSCs treated withTGF-b1 also displayed a dose-dependent increasein VEGF production compared with the controlgroup (Fig 1, B). We chose 12.5 ng/mL as ourtreatment dose for our later experiments becausethis was a low concentration, but increased VEGFproduction effectively compared with the controlgroup (775.2 ± 18.35 vs 1310 ± 23.63 pg/105 cellsin IL-1b–treated MSCs; P < .0001; Fig 1, A) and689.7 ± 20.05 vs 1062 ± 29.70 pg/105 cells inTGF-b1–treated MSCs; P < .0005; Fig 1, B). Thecombination of IL-1b and TGF-b1 treatments re-sulted in greater production of VEGF than thecontrol group (472.3 ± 17.06 vs 1470 ± 26.15 pg/105 cells; P < .0001; Fig 1, C). Interestingly, com-pared with IL-1b or TGF-b1 treatment alone,cotreatment with IL-1b and TGF-b1 furtherincreased the production of VEGF (IL-1b vs IL-1b+ TGF-b1: 641.1 ± 8.867 vs 1470 ± 26.15 pg/105

cells; P < .0001; TGF-b1 vs IL-1b + TGF-b1: 910.5± 14.51 vs 1470 ± 26.15 pg/105 cells; P < .0001;Fig 1, C). Compared with controls, IL-1b treatmentgave a 36% increase in VEGF production, whereasTGF-b1 treatment gave a 93% increase in VEGF

Fig 1. Effect of interleukin (IL)-1b and transforminggrowth factor (TGF)-b1 on murine mesenchymal stemcell (MSC) vascular endothelial growth factor (VEGF)production in vitro. A, MSC VEGF production by IL-1btreatment using concentration as labeled. B, MSCVEGF production by TGF-b1 treatment using concen-tration as labeled. C, MSC VEGF production by IL-1b,or/and TGF-b1 treatment at 12.5 ng/mL each. Resultsare mean values ± standard error of the mean; n =3–4 per group. *P < .05 vs control; #P < .05 vs IL-1bstimulation; &P < .05 vs TGF-b1 stimulation as deter-mined by 1-way analysis of variance with Bonferronipost hoc analysis.

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production (Fig 1, C). The cotreatment with these 2cytokines gave a 211% increase in VEGF produc-tion, a number greater than the addition of the in-creases by the treatment with IL-1b and TGF-b1individually, which was 129%, indicating that co-treatment with IL-1b and TGF-b1 produced a syner-gistic effect on MSCs VEGF secretion.

Effect of IL-1b and TGF-b1 pretreated MSCs onpostischemic myocardial function. Postischemicrecovery of LVDP (expressed as the percentage ofbaseline function during equilibration) was signif-icantly greater in hearts infused with MSCs com-pared with vehicle controls (48.0 ± 4.3% vs 33.3 ±2.1%; P< .05; Fig 2, A and B). Cotreatment of MSCswith both IL-1b and TGF-b1 greatly enhanced theirabilities in the postischemic recovery of LVDP com-pared with MSCs (71.9 ± 6.0% vs 48.0 ± 4.3%; P <.05; Fig 2, A and B). In addition, the postischemicrecovery of LVDP in hearts infused with MSCs co-treated with both IL-1b and TGF-b1 was greaterthan those infused with MSCs pretreated with ei-ther IL-1b or TGF-b1 (71.9 ± 6.0% vs 58.4 ± 2.1%for IL-1b + TGF-b1 vs IL-1b; P < .05 and 71.9 ±6.0% vs 54.6 ± 4.0% for IL-1b + TGF-b1 vs TGF-b1; P < .05; Fig 2, A and B). LVDP recovery at theend of reperfusion increased from 48.0 ± 4.3% to71.9 ± 6.0% when MSCs were cotreated with IL-1band TGF-b1, a difference of 23.9%. LVDP recoveryat the end of reperfusion increased from48.0 ± 4.3% to 58.4 ± 2.1% when MSCs were pre-treated with IL-1b and to 54.6 ± 4.0% when MSCswere pretreated with TGF-b1, differences of10.4% and 6.6% respectively. Therefore, the differ-ence in LVDP recovery in MSCs cotreated with IL-1b and TGF-b1 was greater than the addition ofthe differences in LVDP recovery in those treatedwith cytokines individually. The recovery of +dP/dt, which represents myocardial contractility, wasalso greatly enhanced in hearts infused with MSCscotreated with IL-1b and TGF-b1 compared withthose infused with MSCs, MSCs pretreated withIL-1b, or MSCs pretreated with TGF-b1(71.5 ± 7.7% vs 45.7 ± 4.4% for IL-1b + TGF-b1 vsMSCs; P < .05; 71.5 ± 7.7% vs 59.0 ± 1.8% for IL-1b + TGF-b1 vs IL-1b; P < .05; and 71.5 ± 7.7% vs50.0 ± 3.7% for IL-1b + TGF-b1 vs TGF-b1; P <.05; Fig 2, C and D). Like the recovery in +dP/dt,the recovery of �dP/dt, which represents myocar-dial relaxation, was also greatly increased in heartsinfused with MSCs cotreated with IL-1b and TGF-b1compared with those infused with MSCs, MSCs pre-treated with IL-1b, or MSCs pretreated with TGF-b1(72.5 ± 10.0% vs 41.3 ± 3.7% for IL-1b + TGF-b1 vsMSCs; P< .05; 72.5 ± 10.0% vs 54.1 ± 2.7% for IL-1b+ TGF-b1 vs IL-1b; P < .05; and 72.5 ± 10.0% vs 48.6

Fig 2. Myocardial function after acute ischemia/reperfusion in hearts pre-ischemic infusion with vehicle, mesenchy-mal stem cells (MSCs), interleukin (IL)-1b–pretreated MSCs (12.5 ng/mL), transforming growth factor (TGF)-b1–pre-treated MSCs (12.5 ng/mL), or IL-1b– and TGF-b1–pretreated MSCs (12.5 ng/mL each). Left ventricular developedpressure (% of equilibration) over time (A) and at end reperfusion (B); +dP/dt (% of equilibration) over time (C)and at end reperfusion (D); �dP/dt (% of equilibration) over time (E) and at end reperfusion (F); end-diastolicpressure (mmHg) over time (G) and at end reperfusion (H). Results are mean values ± standard error of themean. IL-1b– and TGF-b1–pretreated MSCs vs MSCs (*P < .05); vs IL-1b–pretreated MSCs (#P < .05); vs TGF-b1–pre-treated MSCs (&P < .05) as determined by 1-way analysis of variance with Bonferroni post hoc analysis at individualtime points.

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± 3.4% for IL-1b + TGF-b1 vs TGF-b1; P < .05, Fig 2,E and F). Hearts infused with MSCs cotreated withIL-1b and TGF-b1 showed a significantly lower EDPat end-reperfusion compared with those infusedwith MSCs and MSCs pretreated with TGF-b1(49.4 ± 11.0% vs 65.6 ± 8.4%; for IL-1b + TGF-b1 vsMSCs; P < .05; and 49.4 ± 11.0% vs 67.8 ± 6.7% forIL-1b + TGF-b1 vs TGF-b1; P < .05; Fig 2, G and H).However, there is no significant EDP differences atend-reperfusion between IL-1b and TGF-b1 cotreat-ment and IL-1b treatment (49.4 ± 11.0% vs 53.2 ±3.6%; P > .05; Fig 2, G and H).

Effects of p38 MAPK inhibitor and SMAD3inhibitor on IL-1b– and TGF-b1–mediated VEGFproduction. To test whether IL-1b and TGF-b1increase VEGF secretion in MSCs through p38MAPK pathway and SMAD3, respectively, we treatedMSCs with specific p38 MAPK inhibitor SB203580and SMAD3 inhibitor SIS3. Inhibition of p38 MAPKabolished IL-1b–mediated VEGF secretion at a con-centrationof8mmol/L(Fig3,A). Similarly, 20mmol/L SIS3 treatment inhibited TGF-b1–mediated VEGFsecretion (Fig 3, B). On the other hand, treatmentwith SIS3 did not affect IL-1b–mediated VEGFsecretion (Fig 3, C), whereas treatment withSB203580 only gave a minimal effect on TGF-b1–mediated VEGF secretion (Fig 3, D), suggestingIL-1b and TGF-b1 mediate VEGF secretion mainlythrough p38 MAPK pathway and SMAD3, respec-tively and specifically. Cotreatment with SB203580and SIS3 together totally ablated IL-1b– and TGF-b1–mediated VEGF production while individualtreatment by SB203580 or SIS3 only partially in-hibited IL-1b– and TGF-b1–mediated VEGF pro-duction (Fig 3, E).

Effects of p38 MAPK inhibitor and SMAD3inhibitor on MSC-mediated myocardial functionalrecovery. The improved recovery of myocardialfunction observed after infusion of MSCs cotreatedwith IL-1b and TGF-b1 was suppressed by p38MAPK and SMAD3 inhibition (Fig 4). Comparedwith the LVDP recovery in IL-1b– and TGF-b1–pre-treated group, LVDP recovery at end-reperfusionwas significantly inhibited down to a level compara-ble with the MSC group when hearts were infusedwith IL-1b– and TGF-b1–pretreated MSCs treatedwith SB203580 and SIS3. No difference was ob-served in LVDP recovery after infusion with MSCsor IL-1b– and TGF-b1–pretreated MSCs treatedwith SB203580 and SIS3 (50.6 ± 2.8% vs47.8 ± 3.1%; P > .05; Fig 4, A and B). The sametrend and facts also applied to +dP/dt and �dP/dt recoveries (51.2 ± 3.1% vs 51.5 ± 4.4% for+dP/dt; 48.8 ± 4.2% vs 48.6 ± 4.3% for �dP/dt;P > .05; Fig 4, C–F). EDP was significantly elevated

when hearts were infused with IL-1b– andTGF-b1–pretreated MSCs treated with SB203580and SIS3 compared with those infused with IL-1b–and TGF-b1–pretreated MSCs (57.6 ± 5.3% vs43.2 ± 9.3%; P < .05; Fig 4, G and H), indicatingthat the beneficial effect on myocardial functionalrecovery by infusion of MSCs cotreated with IL-1band TGF-b1 was suppressed by inhibiting p38MAPK and SMAD3.

DISCUSSION

In the present study, we demonstrated thatcotreatment of MSCs with IL-1b and TGF-b1synergistically increases VEGF secretion. Further-more, infusing hearts with IL-1b– and TGF-b1–pre-treated MSCs greatly improved the recovery ofmyocardial function after acute ischemia/reperfu-sion injury in the Langendorff model. The IL-1b–and TGF-b1–induced VEGF production in MSCs ismediated through p38 MAPK and SMAD3. Ablationof p38 MAPK and SMAD3 activation with specificinhibitors negated both IL-1b– and TGF-b1–medi-ated VEGF production in MSCs and the ability ofthese pretreated MSCs to improve myocardialrecovery after ischemia.

The Langendorff is an ex vivo model used tostudy cardiac physiology, acute global ischemia/reperfusion, and the efficacy of drug or cell ther-apy on the heart. One advantage of the Langen-dorff is that the global ischemia/reperfusion inthe Langendorff model can mimic the effects ofischemia/reperfusion on the heart during cardiacsurgery when the heart is arrested and restarted.Another advantage of the Langendorff model is itsability to generate reproducible data regardingex vivo heart function. Because of these advan-tages, the Langendorff model has providedvaluable data on numerous researches on ischemicheart diseases.

Accumulated evidence has shown that one ofthe primary mechanisms of MSC-mediated myo-cardial protection from ischemia is through theparacrine effects of growth factors released by MSCthat promote cell proliferation and survival, inhibitapoptosis and enhance angiogenesis.14 VEGF hasbeen shown to enhance myocardial recoveryfrom acute ischemia/reperfusion injury by sup-pressing apoptosis of myocytes and promoting ne-ovascularization. Injection of MSCs overexpressingVEGF into the ischemic penumbra decreased in-farct size, increased angiogenesis in the survivingmyocardium, and improved functional recoveryin coronary artery ligation studies.15 In addition,infusion of exogenous VEGF immediately beforeischemia improved postischemic myocardial

Fig 3. Effect of p38 mitogen-activated protein kinase (MAPK) inhibition or/and SMAD3 inhibition on interleukin (IL)-1b– or/and transforming growth factor (TGF)-b1–stimulated mesenchymal stem cell (MSC) vascular endothelialgrowth factor (VEGF) production in vitro. (A) VEGF production by MSCs treated with IL-1b (12.5 ng/mL) with orwithout p38 MAPK inhibitor SB203580 (p38i) at indicated concentration. (B) VEGF production by MSCs treatedwith TGF-b1 (12.5 ng/mL) with or without SMAD3 inhibitor SIS3 at indicated concentration. (C) VEGF productionby MSCs treated with IL-1b (12.5 ng/mL) with or without 8 mmol/L p38 MAPK inhibitor SB203580 (p38i) or 20mmol/L SMAD3 inhibitor SIS3. (D) VEGF production by MSCs treated with TGF-b1 (12.5 ng/mL) with or withoutSMAD3 inhibitor 20 mmol/L SIS3 or 8 mmol/L p38 MAPK inhibitor SB203580 (p38i). (E) VEGF production byMSCs treated with IL-1b (12.5 ng/mL) or/and TGF-b1 (12.5 ng/mL) with or without 8 mmol/L p38 MAPK inhibitorSB203580 (p38i) or/and 20 mmol/L SMAD3 inhibitor SIS3. Results are mean values ± SE, n = 3–6 per group. *P < .05,increased vs control; &P < .05, suppressed vs control; @P < .05 difference between TGF-b1 and TGF-b1 + SIS3; ^P < .05difference vs IL-1b + TGF-b1 + P38I + SIS3 as determined by 1-way analysis of variance followed by a Bonferroni post hocanalysis.

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functional recovery.16 On the other hand, theknockdown of VEGF expression in MSCs by siRNAsignificantly decreased VEGF production, and thepretreatment of these cells impaired stem

cell–mediated myocardial function.17 Consistentwith these data, our work indicates that when theVEGF production by IL-1b and TGF-b1 in MSCswere inhibited by p38 MAPK inhibitor and

Fig 4. Myocardial function after acute ischemia/reperfusion in hearts given pre-ischemic infusions of mesenchymalstem cells (MSCs), interleukin (IL)-1b, and transforming growth factor (TGF)-b1 (12.5 ng/mL each) pretreatedMSCs, or IL-1b and TGF-b1 (12.5 ng/mL each) pretreated MSCs treated with p38 mitogen-activated protein kinase in-hibitor SB203580 (8 mmol/L) and SMAD3 inhibitor SIS3 (20 mmol/L). LVDP (% of equilibration) over time (A) and atend reperfusion (B); +dP/dt (% of equilibration) over time (C) and at end reperfusion (D); �dP/dt (% of equilibra-tion) over time (E) and at end reperfusion (F); end-diastolic pressure (mmHg) over time (G) and at end-reperfusion(H). Results are mean values ± standard error of the mean; n = 5 per group. IL-1b– and TGF-b1–pretreated MSCs vsMSCs (*P < .05); vs IL-1b– and TGF-b1–pretreated MSCs treated with SB203580 and SIS3 (#P < .05) as determinedby 1-way analysis of variance with Bonferroni post hoc analysis at individual time points.

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Fig 5. Putative interactions at the mesenchymal stem cell (MSC) vascular endothelial growth factor (VEGF) promoterregion in response to interleukin (IL)-1b and transforming growth factor (TGF)-b1 cotreatment. IL-1b activates thetranscription factors ATF2 and Sp1 through p38 mitogen-activated protein kinase signaling and TGF-b1 activatesSMAD3 through phosphorylation. Cotreating MSCs with IL-1b and TGF-b1 may lead to the activation and the cooper-ative interactions of transcription factors ATF2, Sp1, and SMAD3 at VEGF promoter region, thus inducing the synergis-tic expression of VEGF through the formation of a transcription complex.

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SMAD3 inhibitor, the improved myocardial func-tional recovery was also inhibited in hearts infusedwith these MSCs, suggesting that VEGF played acritical role in MSC-mediated myocardial functionrecovery.

However, our data cannot exclude the impor-tant roles other growth factors may play inMSC-mediated myocardial function recovery. Pre-treating MSCs with various cytokines or smallmolecules can improve VEGF secretion and fur-ther enhance MSC-mediated myocardial func-tional recovery. Whether pretreating MSCs with>1 cytokine provides a synergistic, beneficial effecton MSC-mediated myocardial recovery is stillpoorly studied. Recently, the study from our labo-ratory showed that IL-6 and TGF-a costimulatedVEGF production in MSCs.18 The study reportedherein provides additional evidence that infusionof MSCs cotreated with 2 cytokines can synergisti-cally enhance myocardial protection from ische-mia. Beside providing a synergistic increase inVEGF production and beneficial myocardial pro-tection effect, cotreating MSCs with 2 cytokinesmay lower the treatment dose for each cytokineto minimize adverse effects without sacrificing

the beneficial cardioprotective effect of VEGF. Ithas been shown that IL-1b increased proinflamma-tory response by increasing the expression of mul-tiple proinflammatory mediators,19 which creates ahostile environment and provides adversary effectson MSC-mediated myocardial function recovery.On the other hand, IL-1b promotes MSCs’ produc-tion of VEGF, through which MSC infusion pro-motes neo-angiogenesis, limits infarct size, andimproves postischemic cardiac function in modelsof myocardial infarction.20 Therefore, IL-1b exe-cutes both beneficial and potentially deleteriousfunctions during the postischemic period. In fact,there are controversial reports on the role of IL-1b in postinfarction remodeling. One reportshowed that disruption of IL-1b signaling byknocking out IL-1RI protected against the develop-ment of adverse remodeling after myocardial in-farction, suggesting a deleterious role of IL-1b inmyocardial recovery.21 A prior report demon-strated that injection with IL-1b neutralizing anti-body resulted in delayed wound healing andworse remodeling of the infarcted heart, indicat-ing that IL-1b may play a protective and beneficialrole in myocardial recovery.22 Therefore, it is of best

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interest to lower the dosage of IL-1b and at the sametime maintain a high level of VEGF production inMSC pretreatment. In our study, cotreating MSCswith IL-1b and TGF-b1 at 12.5 ng/mL each provideda greater VEGF secretion (by 3.11-fold) comparedwith those pretreated with either IL-1b or TGF-b1 at100 ng/mL individually (by 2.87-fold and 1.77-fold,respectively), suggesting cotreatment with a lowdose of 2 cytokines together may achieve great bene-ficial effect while keeping the unwanted adversary ef-fect at a low level. Furthermore, this strategy avoidsany direct administration of IL-1b to the heart.

The activation of gene transcription involvesmultiple interactions between transcription factorsand activators with basic transcriptional machineryaround the promoter region. To achieve synergis-tic VEGF expression, we treated MSCs with bothIL-1b, which activates transcription factors ATF2and Sp1 through the p38 MAPK pathway,5 andTGF-b1, which activates SMAD3.8 ATF2 is a keysubunit of transcription factor AP-1. An AP-1 bind-ing site has been found around �1168 bp and mul-tiple Sp1 binding sites have been identified tolocate around �73 bp near the VEGF promoter.23

In addition, SMAD3 binding sites have been dis-covered at �933 bp and �746 bp near the VEGFpromoter.9 Furthermore, SMAD 3 has been shownto bind ATF2 directly and cooperate with ATF2 toactivate gene expression.24 Other reports havedemonstrated the interactions and cooperation be-tween SMAD3 and Sp1 are important for the in-duction of optimum expression of certaingenes.25 In our study, the activation of p38 MAPKsignal and SMAD3 by cotreating MSCs with IL-1band TGF-b1 may lead to cooperative interactionsbetween transcription factors ATF2, Sp1, andSMAD3 at the VEGF promoter region and, there-fore, induce the synergistic expression of VEGFthrough the formation of transcription complexes(Fig 5). Further research is necessary to character-ize the precise mechanisms responsible for thissynergistic effect on VEGF production and myocar-dial protection.

Yong Luo and Yue Wang contribute equally to thiswork.

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