ly2228820dimesylate,aselectiveinhibitorofp38 mitogen ... · cytokine signaling, and pericytes play...

LY2228820 Dimesylate, a Selective Inhibitor of p38Mitogen-activated Protein Kinase, Reduces AngiogenicEndothelial Cord Formation in Vitro and in Vivo*□S

Received for publication, October 4, 2012, and in revised form, January 11, 2013 Published, JBC Papers in Press, January 18, 2013, DOI 10.1074/jbc.M112.425553

Courtney M. Tate‡, Wayne Blosser‡, Lisa Wyss§, Glenn Evans‡, Qi Xue¶, Yong Pan¶, and Louis Stancato‡1

From ‡Oncology, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana 46285, §Discovery Research, AdvancedTesting Laboratory, Cincinnati, Ohio 45242, and ¶ImClone Systems, New York, New York 10016

Background: Angiogenesis is a critical process for tumor growth and survival.Results: LY2228820 dimesylate, a p38 MAPK-specific inhibitor, or shRNA knockdown of p38�, MK2, or HSP27 reducedangiogenic cord formation.Conclusion: p38 MAPK modulated soluble factors released from stromal and tumor cells and reduced their downstreamsignaling in endothelial cells.Significance: Antiangiogenic activity of LY2228820 dimesylate may lead to anti-tumor growth effects.

LY2228820 dimesylate is a highly selective small moleculeinhibitor of p38� and p38� mitogen-activated protein kinases(MAPKs) that is currently under clinical investigation forhumanmalignancies. p38MAPK is implicated in awide range ofbiological processes, in particular those that support tumori-genesis. One such process, angiogenesis, is required for tumorgrowth and metastasis, and many new cancer therapies aretherefore directed against the tumor vasculature. Using an invitro co-culture endothelial cord formation assay, a surrogate ofangiogenesis, we investigated the role of p38 MAPK in growthfactor- and tumor-driven angiogenesis using LY2228820 dime-sylate treatment and by shRNA gene knockdown. p38 MAPKwas activated in endothelial cells upon growth factor stimula-tion, with inhibition by LY2228820 dimesylate treatment caus-ing a significant decrease in VEGF-, bFGF-, EGF-, and IL-6-in-duced endothelial cord formation and an even more dramaticdecrease in tumor-driven cord formation. In addition toinvolvement in downstream cytokine signaling, p38MAPK wasimportant for VEGF, bFGF, EGF, IL-6, and other proangiogeniccytokine secretion in stromal and tumor cells. LY2228820dime-sylate results were substantiated using p38� MAPK-specificshRNA and shRNA against the downstream p38 MAPK effec-tors MAPKAPK-2 and HSP27. Using in vivo models of func-tional neoangiogenesis, LY2228820 dimesylate treatmentreduced hemoglobin content in a plug assay and decreasedVEGF-A-stimulated vascularization in a mouse ear model.Thus, p38� MAPK is implicated in tumor angiogenesis throughdirect tumoral effects and through reduction of proangiogeniccytokine secretion via the microenvironment.

The p38 mitogen-activated protein kinases (MAPKs) arestrongly activated by stress and inflammatory cytokines, lead-

ing to modulation of many cellular functions, including prolif-eration, differentiation, and survival (1). Four different p38MAPK isoforms have been identified, p38�, -�, -�, and -�,which may have both overlapping and specific functions (1, 2).p38� MAPK (p38�) and p38� MAPK (p38�) are ubiquitouslyexpressed, whereas p38� MAPK and p38� MAPK demonstratespecific tissue expression. p38�, the most abundant isoform, ispresent in most cells and is exclusively critical for mouse devel-opment (3–5). Upstream p38 MAPK kinases (MKKs),2 such asMKK3 and MKK6, can differentially regulate p38 isoforms, asevidenced by the inability of MKK3 to effectively activate p38�(6). A major substrate for p38 MAPK is MAPK-activated pro-tein kinase-2 (MAPKAPK-2; MK2), a serine threonine kinasethat directly phosphorylates the ubiquitously expressed heatshock protein 27 (HSP27). HSP27 is activated in response toosmotic stress, reactive oxygen species, and inflammatory cyto-kines andmay play a role in cell migration, apoptosis, and actincytoskeleton organization (7).Previous reports indicate a role for p38 MAPK in a wide

range of biological processes, in particular tumor cell prolifer-ation in vitro and in vivo (8, 9). Angiogenesis is required fortumor growth and metastasis; therefore, many new potentialcancer therapies are directed against the tumor vasculature.Angiogenesis is the formation of vascular tubes composed of aninner lining of endothelial cells, and, as they mature, vesselsacquire a coating of perivascular cells (referred to as pericytes,smooth muscle cells, or mural cells) that envelop the surface ofthe vascular tube and are critical for the development andmaintenance of the vasculature (10–11). Angiogenesis is stim-ulated by a variety of soluble factors, including vascular endo-thelial growth factor (VEGF), basic fibroblast growth factor(bFGF), endothelial growth factor (EGF), and interleukin 6(IL-6) (12, 13). Endothelial cells and pericytes communicate via

* C. T., W. B., G. E., Q. X., Y. P., and L. S. have Eli Lilly shares received via 401(k)and bonus plans.

□S This article contains supplemental Figs. 1–7.1 To whom correspondence should be addressed: Oncology Research, Eli Lilly

and Company, Lilly Corporate Center, Indianapolis, IN 46285. Tel.: 317-655-6910; Fax: 317-276-1414; E-mail: [email protected].

2 The abbreviations used are: MKK, MAPK kinase; MK2, MAPK-activated pro-tein kinase 2; bFGF, basic fibroblast growth factor; HSP27, heat shock pro-tein 27; CD31, cluster of differentiation 31; ADSC, adipose-derived stemcell; ECFC, endothelial colony-forming cell; p-p38, p-HSP27, and p-MK2,phosphorylated p38, HSP27, and MK2, respectively.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. 9, pp. 6743–6753, March 1, 2013© 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

MARCH 1, 2013 • VOLUME 288 • NUMBER 9 JOURNAL OF BIOLOGICAL CHEMISTRY 6743

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cytokine signaling, and pericytes play a role in maintaining theintegrity of endothelial cells by serving as support structures(14). In addition to vascular stabilization, pericytes are impor-tant for modulation of endothelial cell migration, proliferation,and survival (11, 15). Previous findings suggest a role for p38MAPK inmodulating tumor angiogenesis in tumor cells and/orhost endothelial cells (7, 16–20), but this potential role is notwell defined.We investigated the role of p38MAPK in individual cytokine

and tumor-driven angiogenesis through pharmacological inhi-bition of p38 MAPK using LY2228820 dimesylate treatmentand by shRNA gene knockdown. LY2228820 dimesylate (Fig.1A) is a potent small molecule ATP-competitive inhibitor ofp38 MAPK that is highly selective for the p38� and p38� iso-forms and is currently under clinical investigation for humanmalignancies. We report that the p38 MAPK pathway is acti-vated in endothelial cells in response to VEGF, bFGF, EGF, andIL-6 stimulation and is involved in individual cytokine-drivenand tumor-driven cord formation. Inhibition of p38 MAPK intumor cells also led to decreased secretion of VEGF, bFGF,EGF, IL-6, IL-8, and other proangiogenic factors. Small mole-cule inhibitor results were substantiated by shRNA knockdownof p38� and downstream p38MAPK effectorsMK2 andHSP27but not p38�. Consequently, p38� plays a role in endothelialcell angiogenesis along with stromal and tumor cell cytokinesecretion. LY2228820 dimesylate treatment yielded antiangio-genic effects in vivo via decreased hemoglobin content in aMatrigelTM plug assay, a measure of functional neoangiogen-esis, and decreased VEGF-A-stimulated vascularization in amouse earmodel. p38� and its downstream effectors,MK2 andHSP27, are therefore implicated in tumor angiogenesis, andp38� plays an integral role in key proangiogenic cytokinesecretion.

EXPERIMENTAL PROCEDURES

Cell Culture—U-87-MG, MDA-MB-231, SK-OV-3, A-2780,NCI-H1650, and PC-3 cells were grown according to theAmer-ican Type Culture Collection (ATCC, Manassas, VA) guide-lines. LXFA-629 non-small cell lung adenocarcinoma cells(Oncotest, Freiburg, Germany) were maintained in RPMI 1640medium supplemented with 10% heat-inactivated FBS and 1%glutamine (all from Invitrogen). All cells were grown andtreated in uncoated tissue culture-treated flasks in a humidifiedatmosphere at 37 °C and 5% CO2.shRNA Knockdown—U-87-MG and MDA-MB-231 cells

were transduced (multiplicity of infection 9) with MISSION�shRNA lentiviral transduction particles (Sigma-Aldrich) (non-target control, SCH202V; p38�, NM_001315; p38�, NM_002751), selected with 5 �g/ml puromycin, and screened forprotein knockdown by Western blot analysis as describedbelow. Adipose-derived stem cell (ADSC)/endothelial colony-forming cell (ECFC) co-cultures were transduced followingECFC plating in cord formation as described below with 30 �lof MISSION� shRNA lentiviral transduction particles (Sigma-Aldrich) (non-target control, SCH202V; p38�, NM_001315;p38�, NM_002751; MK2, NM_032960; HSP27, NM_001540)for 72 h prior to analysis for Western blot, cord formation,

cytokine secretion, or phosphoprotein immunoassay asdescribed below.In Vitro Cord Formation Assay—ADSCs (Zen-Bio, Research

Triangle Park, NC) were plated at 75,000 cells/well into 96-wellHTS Transwell� (Corning Inc.) receiver plates (tumor-driven)or 50,000 cells/well (growth factor-driven) into 96-well blackpoly-D-lysine-coated plates, and tumor cells were plated at25,000 cells/well in 96-well HTS Transwell� (Corning Inc.)plates in co-culture medium (MCDB-131medium (Invitrogen)supplemented with L-ascorbic acid 2-phosphate, dexametha-sone, tobramycin, insulin (all from Sigma-Aldrich), and Cell-Prime rTransferrin AF (Millipore, Ballerica, MA)) for 24 h.ADSC medium was removed, and 6,000 (tumor-driven) or5,000 (growth factor-driven) human ECFCs (Lonza, Basel,Switzerland) per well were overseeded. Treatment with 10ng/ml VEGF, bFGF, or EGF or 100 ng/ml IL-6 (all from Invit-rogen) and DMSO or 1 �M LY2228820 dimesylate treatmentoccurred 4 h following ECFC plating and continued for 96 h.Cells were directly fixed for 10 min with 3.7% formaldehyde(Sigma-Aldrich) followed by ice-cold 70% ethanol for 20min at25 °C. Cells were rinsed once with PBS, blocked for 30min with1% BSA, and immunostained for 1 h with antiserum directedagainst cluster of differentiation 31 (CD31) (R&D Systems,Minneapolis, MN) diluted to 1 �g/ml in 1% BSA. Cells werewashed three times with PBS and incubated for 1 h with 5�g/ml donkey �-sheep-Alexa-488 (Invitrogen), �-smoothmuscle actin Cy3 conjugate (1:200; Sigma-Aldrich), and 200ng/mlHoechst 33342 (Invitrogen) in 1%BSA,washedwith PBS,and then imaged using the cord formation algorithm on theCellomics� ArrayScan� VTI at an image magnification of �5(Thermo Fisher Scientific). For assessment of proliferation andapoptosis, cells were plated, treated, and fixed for cord forma-tion as mentioned above and then immunostained with Ki67(1:100; Millipore), 5 �g/ml goat �-rabbit-Alexa-647 (Invitro-gen), and 200 ng/ml Hoechst 33342 (Invitrogen) or the In SituCell Death Detection Kit (Roche Applied Science) according tothe manufacturer’s recommendations and then imaged usingthe target activation algorithm on the Cellomics� ArrayScan�VTI at an image magnification of �20 (Thermo Fisher Scien-tific). Cell motility was analyzed using the Cellomics� cellmotility kit (Thermo Fisher Scientific) by plating 500 ADSC orECFC cells on prepared blue fluorescent microsphere platesaccording to themanufacturer’s recommendations. Treatmentwith DMSO or 1 �M LY2228820 dimesylate and 10 ng/mlVEGF, bFGF, or EGF or 100 ng/ml IL-6 occurred 24 h followingcell plating and continued for 18 h. Cells were then fixed,stained, and imaged using the cell motility algorithm on theCellomics� ArrayScan� VTI at an image magnification of �20(Thermo Fisher Scientific) according to the manufacturer’srecommendations.Western Blot—Whole cell protein extracts were isolated by

cell lysis with 1% SDS and brief sonication, and protein concen-tration was quantified using the Bradford method. Thirtymicrograms of protein were subjected to electrophoresis on4–20% precast Tris-glycine gradient gels (Invitrogen), trans-ferred to nitrocellulose (Invitrogen), blocked with 5% blottinggrade blocker (Bio-Rad) in Tris-buffered saline containing 0.1%Tween (TBST), probed with primary antiserum, washed with

p38� MAPK Promotes Angiogenesis

6744 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 288 • NUMBER 9 • MARCH 1, 2013


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TBST, and incubated with an appropriate horseradish peroxi-dase-labeled secondary antibody. Membranes were washedwith TBST, and signal was detected by ECL (Thermo FisherScientific). Antisera directed against p38�, p38�, and totalHSP27 (all from Cell Signaling Technology, Danvers, MA);phospho-p38 MAPK (Thr-180/Tyr-182), phospho-HSP27(Ser-15), phospho-MAPKAPK2 (Thr-334), and total MAP-KAPK2 (all from Epitomics, Burlingame, CA); and �-actin(Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were dilutedwith 5% blotting grade blocker (Bio-Rad) in TBST. Densitom-etry was performed using ImageJ analysis software (NationalInstitutes ofHealth) as per the request of the ImageJ developers.Phosphoprotein Immunoassays—ADSCs/ECFCs were plated

following the cord formation protocol above, or ECFCs wereplated in normal growth medium (2 � 104) in 96-well tissueculture dishes for 24 h prior to replacement of medium withco-culturemedium for 2 h. ADSCs/ECFCs or ECFCs were thenstimulated for 15 min with 10 ng/ml VEGF, bFGF, EGF, 100ng/ml IL-6, or conditioned medium from U-87-MG or MDA-MB-231 cells (described below) and analyzed with the phos-pho-p38 (Thr-180, Tyr-182) or phospho-MAPKAPK2 (Thr-334) whole cell kit according to the manufacturer’srecommendations (Meso Scale Discovery, Rockville, MD).Cytokine Analysis—ADSCs or ADSCs/ECFCs were plated in

co-culture medium following the cord formation protocolabove. Tumor cells (2� 105) were plated in co-culturemediumin 6-well tissue culture dishes. Four hours after ECFC plating or24 h after tumor cell plating, medium was replaced, and treat-ments were added for 72 h prior to medium collection and cellnumber counts. Cell debris was removed from conditionedmedium by centrifugation, and samples were analyzed fresh orwere frozen at �20 °C until analysis. Samples were analyzedwith Quantikine� Colorimetric Sandwich ELISAs (R&D Sys-tems) according to the manufacturer’s recommendations.In Vivo MatrigelTM Plug Angiogenesis Assay—ADSCs (0.5 �

106) and ECFCs (2 � 106) were mixed with 200 �l of growthfactor-reducedMatrigelTM (BD Biosciences) on ice and subcu-taneously injected into the flanks of athymic nude female mice(Harlan, Indianapolis, IN), one implant per animal. Mice weredosed orally three times daily with LY2228820 dimesylate (20and 40 mg/kg) or twice daily with sunitinib (25 mg/kg), whichwere prepared internally, beginning 4 h prior to cell implanta-tion. After 5 days of dosing, implants were removed and flash-frozen in liquid nitrogen, and hemoglobin was quantified usingtheQuantiChromTMhemoglobin assay kit (Bioassay, Hayward,CA) as described previously (21).Angiogenesis Ear Assay—Animal protocols were approved by

the Imclone System Inc. andMispro Biotech Services Corpora-tionAnimal Care andUseCommittee. A nonreplicating adeno-viral vector engineered to express the predominant (164-aminoacid) murine isoform of VEGF-A (Ad-VEGF-A164; 1 � 108plaque-forming units), as described previously (22), wasinjected intradermally into the dorsal ears of 8-week-old athy-mic nu/nu mice (Charles River, Wilmington, MA) as describedpreviously (23).Mice (n� 5) were dosed orally with sunitinib at40 mg/kg daily, LY2228820 dimesylate at 30 mg/kg twice a day,or vehicle (HEC-Tween) twice a day starting 1 day before injec-tion of adenovirus VEGF-A and harvested at day 5 after adeno-

virus injection. Ears were mounted flat under a glass slide withimmersion oil and photographed using a Leica M80 photomi-croscope as described previously (23). The images of ear vascu-lature were quantified by Image-Pro Analyzer version 7.0(MediaCybernetics, Bethesda, MD).Statistical Analysis—Statistical significance of data were

assessed by a two-tailed Student’s t test with equal variancecompared with data obtained for DMSO or non-target shRNAcontrols (in vitro) or vehicle controls (in vivo). Statistical signif-icance was assigned to p values of �0.05.

RESULTS

VEGF, bFGF, EGF, and IL-6 Activate p38 MAPK Signaling—LY2228820 dimesylate (Fig. 1A) is a highly selective ATP-com-petitive inhibitor of p38� and p38� that does not alter p38MAPK activation but reduces downstream p38 MAPK signal-ing (24). The human kinome map of LY2228820 dimesylateactivity indicates the specificity of the inhibitor for p38 MAPKcompared with that of sunitinib, a multitargeted receptor tyro-sine kinase inhibitor with an antiangiogenic mechanism ofaction (25) (supplemental Fig. 1). Smooth muscle cells andendothelial cells are exposed to many growth factors and pro-inflammatory cytokines that contribute to angiogenesis, andp38MAPK is implicated in downstream cytokine signaling (18,26); therefore, p38 MAPK signaling was analyzed in ECFCs, asubtype of umbilical cord blood-derived endothelial cells thatcan form intrinsic in vivo vessels upon transplantation intoimmunodeficient mice (27), and ADSCs, which are similar tomesenchymal stem cells, which can give rise to cells with peri-cytic properties that can stabilize vascular assembly in vitro(28). In ECFCs, there was a robust increase in VEGF-dependentphosphorylated p38 MAPK (p-p38) expression (p � 0.05),whereas bFGF, EGF, and IL-6 showed a modest (�25%)increase in p-p38 by a highly sensitive immunoassay (Fig. 1C).Importantly, ligand-induced up-regulation of p-p38 in ECFCsresulted in increased expression of the downstream effectorsp-MK2 and p-HSP27 byWestern blot (Fig. 1B) and a significantincrease (p � 0.05) in p-MK2 by immunoassay (Fig. 1C). Acti-vation of these effectors was significantly impaired followinginhibition of p38 MAPK by LY2228820 dimesylate treatment(Fig. 1B). Furthermore, LY2228820 dimesylate treatment abro-gated basal p38 pathway activity in ECFCs andADSCs (Fig. 1B).LY2228820 Dimesylate Treatment Reduced VEGF-, bFGF-,

EGF-, and IL-6-driven Cord Formation—Because p38 pathwayactivation occurs following treatment with proangiogenic fac-tors, a surrogate cord formation assay to model key morpho-genic features of blood vessel formation (29) was used to ana-lyze the effect of LY2228820 dimesylate treatment on in vitroendothelial cord formation. The ECFC and ADSC co-culturecan establish cord networks in vitro and functional blood ves-sels in vivo (21). ADSCs, which serve as the feeder layer for theECFCs, can differentiate into pericyte-like cells that express�-smooth muscle actin, a contractile filament expressed inpericytes. Formation of vascular networks by ECFCs can bevisualized by CD31 immunostaining (30), accompanied byADSC migration and increased density near the cords alongwith increased �-smooth muscle actin expression. LY2228820dimesylate treatment significantly reduced (p� 0.05) basal and




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VEGF-, bFGF-, EGF-, and IL-6-driven cord formation alongwith �-smooth muscle actin expression (Fig. 1D). In addition,cords that were established without stimulation (basal) or withVEGF, bFGF, EGF, and IL-6 for 4 days prior to LY2228820dimesylate treatment showed significant regression (p � 0.05)in both cord formation and �-smooth muscle actin expression(supplemental Fig. 2). Cell viability of the ADSC/ECFC co-cul-turewas not altered by LY2228820 dimesylate treatment, which

ensured that the observed effects on cord formation were notdue to cell toxicity (data not shown).Endothelial cells require cytokine stimulation for survival,

proliferation, andmigration, all of which are essential for angio-genesis, and the paracrine signaling between endothelial cellsand pericytes is important for cord formation. In addition,endothelial cells secrete factors, partly to attract pericytes toenvelop the vessel wall and promote vessel maturation (31);

FIGURE 1. LY2228820 dimesylate treatment reduced VEGF-, bFGF-, EGF-, and IL-6-driven cord formation. A, chemical structure of LY2228820 dimesylate(LY). B, whole cell protein extracts were isolated from ECFCs or ADSCs following pretreatment with DMSO (�) or 1 �M LY2228820 dimesylate (�) for 30 min priorto the addition of 10 ng/ml VEGF, bFGF, EGF, or 100 ng/ml IL-6, and then the extracts were subjected to Western blot analysis using antisera directed againstp-p38, p38�, p38�, p-MK2, total MK2, p-HSP27, total HSP27, and �-actin as a loading control. C, whole cell protein extracts were isolated from ECFCs following15-min PBS (basal), 10 ng/ml VEGF, bFGF, EGF, or 100 ng/ml IL-6 treatment, and then extracts were subjected to p-p38 and p-MK2 analysis by a phosphoproteinimmunoassay. Graphs represent means � S.E. (error bars) from three independent experiments, and asterisks denote statistically significant (*, p � 0.05)differences compared with basal controls. D, the ADSC/ECFC co-cultures were treated with DMSO or 1 �M LY2228820 dimesylate simultaneously with PBS(basal) or 10 ng/ml VEGF, bFGF, EGF or 100 ng/ml IL-6 for 96 h prior to immunohistochemistry for CD31 (green), �-smooth muscle actin (red), and Hoechst 33342to stain all nuclei (blue). Representative images (�5 magnification) are shown; graphs represent means � S.E. after basal cord formation data were subtractedfrom VEGF, bFGF, EGF, and IL-6 data from three independent experiments; and asterisks denote statistically significant (*, p � 0.05) differences compared withDMSO controls. E, conditioned medium was collected from the ADSC/ECFC co-cultures or ADSCs alone treated with DMSO or 1 �M LY2228820 dimesylate for72 h and subjected to ELISA analysis for VEGF, bFGF, EGF, and IL-6. Graphs represent means � S.E. from three independent experiments, and asterisks denotestatistically significant (*, p � 0.05) differences compared with DMSO controls.




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therefore, a role for p38 MAPK on ADSC/ECFC co-culture,ADSC, and ECFC cytokine secretion was analyzed. The ADSC/ECFC co-culture along with ADSCs alone showed a significantreduction (p � 0.05) in VEGF, bFGF, EGF, and IL-6 secretionupon LY2228820 dimesylate treatment (Fig. 1E). ECFCssecreted undetectable to minimal amounts (�8 pg/ml) ofVEGF, bFGF, EGF, and IL-6 (data not shown). However, theamount of VEGF, bFGF, EGF, and IL-6 was slightly enhanced(�10–30%) in the ADSC/ECFC co-cultures compared withADSCs alone, indicating that cell contact between ADSCs andECFCs may be important for cytokine secretion. These dataindicate that p38 MAPK is involved both in downstream cyto-kine signaling and cytokine release from stromal cells. Statisti-cally significant decreases (p � 0.05) in ECFC proliferation,assessed by expression of the proliferation marker Ki67, ECFCmotility, or ECFC apoptosis, assessed by TUNEL analysis, werenot observed with LY2228820 dimesylate treatment uponVEGF, bFGF, EGF, or IL-6 stimulation (supplemental Fig. 3).Knockdown of p38�, MK2, or HSP27 Reduced VEGF-, bFGF-,

EGF-, and IL-6-driven Cord Formation—The use of a p38�/�-selective inhibitor, such as LY2228820 dimesylate, is critical inestablishing a role for p38 MAPK function in angiogenesis butdoes not distinguish between the functions of p38� and p38�.To further investigate a role for the p38 MAPK pathway inangiogenesis, gene knockdown using shRNA targeted againstp38� and p38� in ADSC/ECFC co-cultures were generated.Knockdown of p38� but not p38� in ADSCs/ECFCs was effec-tive in blocking p38 MAPK signaling, as evidenced by reducedlevels of p-p38, p-MK2, and p-HSP27 byWestern blot (Fig. 2B)and by immunoassay (Fig. 2D), which led to a significant reduc-tion (p � 0.05) in VEGF-, bFGF-, EGF-, and IL-6-driven(knockdown of p38� also significantly reduced IL-6-drivencord formation but to a lesser extent than p38�) cord formation(Fig. 2A). These results are similar to those of LY2228820 dime-sylate treatment, which strengthens the notion that compoundeffects are specific to inhibition of p38�. To further support arole for p38MAPK and the p38MAPK pathway in cord forma-tion, knockdown of downstream signaling effectors MK2 andHSP27 in the ADSC/ECFC co-culture also significantlyreduced (p � 0.05) VEGF-, bFGF-, EGF-, and IL-6-driven cordformation (Fig. 2A). Knockdown of p38�, MK2, or HSP27 butnot p38� also significantly reduced (p � 0.05) cytokine secre-tion of VEGF, bFGF, and IL-6 from ADSC/ECFC co-cultures(Fig. 2C). Secretion of EGF from ADSC/ECFC co-cultures wassignificantly reduced (p� 0.05) with p38� knockdown and wasonly slightly reducedwithMK2 orHSP27 knockdown (Fig. 2C).Knockdown of p38� significantly inhibited (p � 0.05) activa-tion of p-p38 and p-MK2 followingVEGF stimulation (Fig. 2D).Knockdown of MK2 also significantly reduced (p � 0.05) basaland VEGF-induced p-MK2 expression (Fig. 2D). In contrast,knockdown of p38�, MK2, or HSP27 did not alter VEGF-in-duced activation of p-p38 by immunoassay analysis (Fig. 2D).This indicates that p38� and not p38� is the main mediator ofVEGF, EGF, bFGF, and IL-6 cytokine secretion and down-stream signaling through MK2 and HSP27 in our proangio-genic co-culture system. As observed with LY2228820 dimesy-late treatment, cell viability ofADSC/ECFCco-cultureswas notaltered upon shRNA treatment; therefore, the anti-cord form-

ing effects did not stem from a cytotoxic event (data notshown). Similar results were obtained with two additionalshRNAclones targeting differentmRNAregions of p38�, p38�,MK2, andHSP27, indicating that the effects observed are prob-ably due to reduced expression of the intended target genes(data not shown).LY2228820 Dimesylate Treatment Reduced Tumor-driven

Cord Formation—To more closely represent tumor angiogen-esis, instead of individual cytokines stimulating cord formation,LY2228820 dimesylate effects on tumor-conditioned medium-driven and tumor cell-driven cord formation were analyzed.Conditioned media from commonly used, well characterizedU-87-MG glioblastoma and MDA-MB-231 breast cancer cellsincreased protein expression of p-p38, p-MK2, and p-HSP27 byWestern blot and significantly increased (p � 0.05) p-p38 andp-MK2 by immunoassay (supplemental Fig. 4, A and B). Thisindicates that cytokines secreted from U-87-MG and MDA-MB-231 tumor cells activate p38 MAPK signaling. LY2228820dimesylate treatment significantly reduced (p � 0.05)U-87-MG and MDA-MB-231 tumor-conditioned medium-driven cord formation, indicating a function for p38 MAPK instromal cells downstream of tumor-secreted cytokines (supple-mental Fig. 4C). LY2228820 dimesylate also significantlyreduced (p � 0.05) tumor-driven cord formation and smoothmuscle actin expression from a range of tumor histologies,including U-87-MG, MDA-MB-231, ovarian (A-2780 and SK-OV-3), lung (LXFA-629 and NCI-H1650), and prostate (PC-3)(Fig. 3A). LY2228820 dimesylate treatment inhibited down-stream p38 MAPK signaling (p-MK2 and p-HSP27) inU-87-MG and MDA-MB-231 cells (Fig. 3B) along with eachtumor cell line analyzed (data not shown). In tumor-drivencord formation, LY2228820 dimesylate effects on the tumorcells and downstream cytokine signaling could not be separatedbecause all three cell lines (tumor, ADSC, and ECFC)were con-currently treated with LY2228820 dimesylate, necessitatingpretreatment of U-87-MG or MDA-MB-231 tumor cells withLY2228820 dimesylate prior to cell plating. Pretreatment oftumor cells with LY2228820 dimesylate significantly reducedcord formation (supplemental Fig. 5) and secretion (p� 0.05) ofVEGF, bFGF, EGF, and IL-6 from U-87-MG (Fig. 3C) alongwith A-2780, SK-OV-3, and PC-3 tumor cell lines (data notshown) and VEGF, bFGF, and IL-6 fromMDA-MB-231 tumorcells (Fig. 3C; basal EGF is below the limit of detection inMDA-MB-231 cells). LY2228820 dimesylate treatment also reducedsecretion of IL-8 and other proangiogenic cytokines (angioge-nin, HGF, PlGF, PDGF-AA) secreted from U-87-MG, MDA-MB-231, SK-OV-3, and A-2780 tumor cells (data not shown).Pretreatment of tumor cells with LY2228820 dimesylate andthe addition of compound into the cord formation assay led tothe greatest inhibition of cord formation (supplemental Fig. 5),further supporting the notion that LY2228820 dimesylate treat-ment has a direct effect on tumor cell cytokine secretion andcytokine signaling in ADSC/ECFC cells, especially because cellviability of tumor cells and ADSC/ECFC co-cultures wereunchanged (data not shown).Knockdown of p38� in Tumor Cells Reduced Tumor-driven

Cord Formation—To further investigate the role of p38� andp38� in tumor cell cytokine secretion and tumor-induced cord




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formation, stable knockdown of p38� or p38� was assessed inU-87-MG andMDA-MB-231 cells. It is important to note thatcomplete protein knockdown was not achieved for either iso-form, which may have affected results on cord formation.Knockdown of p38� but not p38� in both cell lines reducedexpression of p-p38, p-MK2, and p-HSP27 (Fig. 4B and supple-mental Fig. 6B), and significantly reduced (p � 0.05) tumor-

driven cord formation (Fig. 4A and supplemental Fig. 6A) alongwith VEGF, bFGF, EGF (in U-87-MG, undetectable in MDA-MB-231), and IL-6 secretion (Fig. 4C and supplemental Fig.6C). Similar results were obtained with additional U-87-MGandMDA-MB-231 stable shRNAknockdown lines, which con-firmed the importance of p38� in controlling tumor cell cyto-kine secretion and cord formation.

FIGURE 2. Knockdown of p38�, MK2, or HSP27 reduced VEGF-, bFGF-, EGF-, and IL-6-driven cord formation. A, the ADSC/ECFC co-cultures were treatedwith non-targeting (control), p38�, p38�, MK2, or HSP27 shRNA for 72 h prior to induction of cord formation without (PBS, basal) or with 10 ng/ml VEGF, bFGF,EGF, or 100 ng/ml IL-6 for 96 h before immunohistochemistry for CD31 (green), �-smooth muscle actin (red), and Hoechst 33342 to stain all nuclei (blue).Representative images (�5 magnification) are shown; graphs represent means � S.E. (error bars) from three independent experiments after basal cordformation data were subtracted from VEGF, bFGF, EGF, and IL-6 data; and asterisks denote statistically significant differences (*, p � 0.05) compared withnon-targeting shRNA controls. B, whole cell protein extracts were isolated from the ADSC/ECFC co-cultures following 72-h shRNA treatment for the indicatedgene and subjected to Western blot analysis using antisera directed against p38�, p38�, p-p38, p-MK2, total MK2, p-HSP27, total HSP27, and �-actin as aloading control, and protein quantification was determined with densitometry. C, conditioned medium was collected from the ADSC/ECFC co-culturesfollowing 72 h shRNA treatment for the indicated gene and subjected to ELISA analysis for VEGF, bFGF, EGF, and IL-6. Graphs represent means � S.E. from threeindependent experiments, and asterisks denote statistically significant (*, p � 0.05) differences compared with DMSO controls. D, whole cell protein extractswere isolated from ECFCs following 72-h shRNA treatment for the indicated gene following by 15 min of PBS (basal) or 10 ng/ml VEGF treatment, and thenextracts were subjected to p-p38 and p-MK2 analysis by a phosphoprotein immunoassay. Graphs represent means � S.E. from three independent experiments,and asterisks denote statistically significant (* and #, p � 0.05) differences compared with respective shRNA control PBS-treated (*) or shRNA control VEGF-treated (#) samples.




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Cord Formation Rescue with the Addition of VEGF, bFGF,EGF, and IL-6 in Conditioned Media from Tumor Cells withStable p38� Knockdown—To determine if the reduction inVEGF, bFGF, EGF (U-87-MG), and IL-6 is contributing to thereduction in cord formation observed with stable knockdownof p38� in U-87-MG and MDA-MB-231 tumor cells, we per-formed an add back experiment. Conditionedmedia from con-trol shRNA cells or p38� stable knockdown cells for U-87-MGandMDA-MB-231 cells were collected and analyzed for VEGF,bFGF, EGF (U-87-MG cells only; MDA-MB-231 cells hadundetectable amounts of EGF), and IL-6 cytokine levels. Theaddition of 1� or 2� amounts (compared with the controlshRNA conditioned medium) of the individual deficient cyto-kines or a mixture of the deficient cytokines to the p38� stableknockdown conditioned medium was used to assess cord for-mation. Importantly, the addition of a 1� or 2� mixture ofVEGF, bFGF, EGF (U-87-MG only), and IL-6 significantlyincreased (p � 0.05) cord formation compared with theU-87-MG or MDA-MB-231 p38� knockdown tumor condi-tioned medium (supplemental Fig. 7). This indicates thatreduction in VEGF, bFGF, and, to a lesser extent, EGF and IL-6upon p38� knockdown in tumor cells contributes to the reduc-tion in cord formation observed.

LY2228820 Dimesylate Treatment Reduced HemoglobinContent and Ear Angiogenesis in Vivo—To extend a role for thep38 MAPK pathway in angiogenesis in vivo, we testedLY2228820 dimesylate in a neoangiogenesis MatrigelTM plugmodel consisting of ADSCs/ECFCs that form blood vessels fol-lowing co-implantation into the flank of a nude mouse (28).Five days after implantation, ADSC/ECFC cells formed exten-sive networks of blood vessels whose functionality was assessedby measuring hemoglobin content. LY2228820 dimesylate andsunitinib, an approved angiogenesis inhibitor used as a positivecontrol, were given at clinically relevant doses, and both causeda significant reduction (p � 0.05) in hemoglobin content (Fig.5A).To further analyze the relevant effects of p38 MAPK on

angiogenesis in vivo, we tested LY2228820 dimesylate in an earangiogenesis model consisting of intradermal injection of Ad-VEGF-A164 into nude mouse ears that induces a robust angio-genic response (23). Similar to the MatrigelTM plug assay, bothLY2228820 dimesylate and sunitinib treatment caused a signif-icant reduction (p � 0.05) in ear vascularity (Fig. 5B), whichindicates that LY2228820 dimesylate treatment impaired neo-angiogenesis in vivo. All in vitro and in vivo experiments wereconfirmed with a second generation p38�- and p38�-specific

FIGURE 3. LY2228820 dimesylate treatment reduced tumor-driven cord formation. A, ADSC/ECFC co-cultures with permeable transwells containingmedium (no cells) or the indicated tumor cells (U-87-MG, MDA-MB-231, A-2780, SK-OV-3, LXFA-629, NCI-H1650, and PC-3) were treated with DMSO or 1 �M

LY2228820 dimesylate (LY) for 96 h prior to immunohistochemistry for CD31 (green), �-smooth muscle actin (red), and Hoechst 33342 to stain all nuclei (blue).Representative images (�5 magnification) are shown; graphs represent means � S.E. (error bars) from three independent experiments after no cells (basal)data were subtracted; and asterisks denote statistically significant (*, p � 0.05) differences compared with DMSO controls. B, whole cell protein extracts wereisolated from the indicated tumor cells following treatment with DMSO (�) or 1 �M LY2228820 dimesylate (�) for 4 h and subjected to Western blot analysisusing antisera directed against p-p38, p38�, p38�, p-MK2, total MK2, p-HSP27, total HSP27, and �-actin as a loading control. C, conditioned medium wascollected from U-87-MG or MDA-MB-231 tumor cells treated with DMSO or 1 �M LY2228820 dimesylate for 72 h and subjected to ELISA analysis for VEGF, bFGF,EGF, and IL-6. Graphs represent means � S.E. from three independent experiments, and asterisks denote statistically significant (*, p � 0.05) differencescompared with DMSO controls.




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ATP-competitive inhibitor, further indicating that the resultsobtained in these studies are due to specific inhibition of p38MAPK signaling (data not shown).

DISCUSSION

Our work examined the role of p38 MAPK signaling in cordformation in vitro and neoangiogenesis in vivo, and our dataimplicate p38 MAPK both in the release of cytokines fromtumor and stromal cells and in mediating their downstreameffects on endothelial cells. Previous studies using p38 MAPKinhibitors were limited by an inability to distinguish tumor ver-sus stromal cell effects and delineate p38 MAPK isoform-spe-cific effects. Our data support a positive role for p38 MAPK

activation in angiogenesis, similar towhatwas reported by Jack-son et al. (33), where small molecule inhibitors of p38 MAPKkinase reduced angiogenesis in an inflammatory angiogenesismodel. Others have also reported a role for p38 MAPK in pro-inflammatory angiogenesis in vivo in a prostate cancer ratmodel (16), in shear stress-mediated angiogenesis (34), inhuman coronary artery endothelial cell tube formation (17),and in vitro in basal and tumor conditionedmedia capillary-likestructure formation in a co-culture system (35). Furtherstrengthening a potential role for p38 MAPK in angiogenesis,lack of the p38 MAPK upstream activator, MKK3, causes defi-ciency in both primary and placental blood vessel development(36).In this report, VEGF, bFGF, EGF, and IL-6 were shown to

activate the p38 MAPK pathway in endothelial cells.

FIGURE 4. Knockdown of p38� MAPK in U-87-MG tumor cells reducedtumor-driven cord formation. A, stable U-87-MG shRNA cell lines for a non-targeting shRNA (control), p38�, p38�, or medium (no cells) were plated inpermeable transwells with ADSC/ECFC co-cultures for 96 h and then sub-jected to immunohistochemistry for CD31 (green), �-smooth muscle actin(red), and Hoechst 33342 to stain all nuclei (blue). Representative images (�5magnification) are shown, graphs represent means � S.E. (error bars) fromthree independent experiments after no cells data (basal cord formation)were subtracted, and asterisks denote statistically significant (*, p � 0.05)differences compared with the non-targeting shRNA control. B, whole cellprotein extracts were isolated from the indicated cell lines and subjected toWestern blot analysis using antisera directed against p38�, p38�, p-p38,p-MK2, total MK2, and �-actin as a loading control. C, conditioned mediumwas collected from stable U-87-MG shRNA cell lines and subjected to ELISAanalysis for VEGF, bFGF, EGF, and IL-6. Graphs represent means � S.E. fromthree independent experiments, and asterisks denote statistically significant(*, p � 0.05) differences compared with non-targeting shRNA controls.

FIGURE 5. LY2228820 dimesylate treatment reduced hemoglobin con-tent and ear vascularization in vivo. A, an ADSC/ECFC cell mixture was co-implanted subcutaneously into the flanks of athymic nude mice (8 mice/treatment group). Oral dosing of mice began 4 h prior to cell implantation andoccurred three times daily with LY2228820 dimesylate (LY) (20 and 40 mg/kg)or twice daily with sunitinib (25 mg/kg). After 5 days of dosing, MatrigelTM plugswere removed, and hemoglobin was quantified. The graph is representativeof three independent experiments and indicates means � S.E. (error bars)from one experiment. Asterisks denote statistically significant (*, p � 0.05)differences compared with vehicle controls. B, mice were dosed orally withvehicle (HEC-Tween), LY2228820 dimesylate at 30 mg/kg twice a day, orsunitinib at 40 mg/kg daily, starting 1 day before injection of adenovirusVEGF-A (Ad-VEGF-A164). Ears were harvested 5 days after adenovirus injectionand imaged (representative images from two independent experiments areshown; �8 magnification), and vasculature was quantified. The graph is rep-resentative of two independent experiments, indicates means � S.E., andasterisks denote statistically significant (*, p � 0.05) differences comparedwith vehicle controls.




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LY2228820 dimesylate treatment reduced VEGF-, bFGF-,EGF-, and IL-6-driven cord formation and �-smooth muscleactin expression (pericyte marker) along with the secretion ofthose soluble factors from ADSCs and a proangiogenic ADSC/ECFC co-culture. Our results are consistent with previous find-ings that p38 MAPK mediates both signaling downstream ofcytokine receptors and cytokine release (33, 34, 37). Thus, p38MAPK-dependent production of VEGF, bFGF, EGF, and IL-6may be critical for angiogenesis. VEGF was reported to induceactivation of p38MAPKandHSP27 in endothelial cells througha p38MAPK-dependent pathway (7, 18, 26). p38MAPK signal-ing was also shown to be responsible for activation of HSP27and mediate smooth muscle cell migration upon PDGF, IL-1�,andTGF� stimulation (38). In endothelial cells, p38MAPKwasalso activated by bFGF, and inhibition of p38MAPK abrogatedbFGF-mediated tube formation of MSS31 stromal cells andendothelial cell migration (39). Therefore, p38 MAPK is acti-vated by many proangiogenic factors and probably mediatesendothelial cell function.p38 MAPK signaling has also been implicated in the regula-

tion of endothelial and mural cell migration (37, 38) andrecruitment during angiogenesis (11). Inhibition of p38MAPKled to reduced HSP27 phosphorylation, actin reorganization,and endothelial cell migration (18), which was dependent onMK2 following VEGF stimulation (26). Furthermore, overex-pression ofMKK6, an upstream activator of p38MAPK kinase,enhanced HSP27 phosphorylation and migration in humanumbilical vein endothelial cells (40), whereas siRNA-mediatedMK2 knockdown in MSS31 spleen endothelial cells inhibitedVEGF-induced cell migration (26). It remains to be determinedwhether p38MAPKmediates ECFC or ADSCmigration in ourcord formation system, but we did not observe significantreduction in migration with cytokine-stimulated ECFCs aloneor ADSCs alone upon LY2228820 dimesylate treatment.In addition to reducing individual cytokine-induced cord

formation, LY2228820 dimesylate treatment displayed a morepronounced reduction in tumor-conditioned medium-drivenand tumor cell-driven cord formation. The fact that p38MAPKwas shown to be activated downstream of many proangiogeniccytokine receptors suggests that LY2228820 dimesylate treat-mentmay have an additive effect when amultitude of cytokinesare being released from tumor cells and/or that LY2228820dimesylate treatment has an effect on tumor cell cytokinesecretion and signaling downstream of cytokine receptors instromal cells. Previous reports also indicate that p38MAPK canregulate cytokine secretion from tumor cells, including VEGFsecretion in malignant gliomas (41) and other tumor cells (19).Pretreatment of tumor cells with LY2228820 dimesylate, whichdoes not affect tumor cell viability, reduced cord formationwhich suggests that LY2228820 dimesylate treatment alterscytokine secretion from tumor cells. Indeed, LY2228820 dime-sylate treatment significantly reduced secretion of VEGF,bFGF, EGF, IL-6, and other proangiogenic (IL-8 and angioge-nin) cytokines from a variety of tumor cell lines. Although IL-8has been shown to play an important role in tumor growth,angiogenesis, andmetastasis (42), it was not a potent inducer ofcord formation in our in vitro system (data not shown). Pre-treatment of tumor cells with LY2228820 dimesylate alongwith

the addition of LY2228820 dimesylate into the cord formationsystem led to a greater inhibition of cord formation than justpretreatment of tumor cells with LY2228820 dimesylate or theaddition of LY2228820 dimesylate into the cord formationassay. This suggests that LY2228820 dimesylate treatmentreduced tumor-driven cord formation in part by decreasingcytokine secretion from tumor and/or stromal cells and byaffecting the stromal cell response to cytokine stimulation.In addition to small molecule inhibition of p38 MAPK, pro-

tein knockdown of p38� in stromal or tumor cells reduced cordformation. Protein knockdown of p38� only reduced IL-6-driven cord formation in stromal cells, indicating a role for bothp38� and p38� in IL-6-mediated cord formation. Targetedinactivation of themouse p38� gene results in embryonic deathdue to a placental defect (3–5), and angiogenesis was abnormalin the yolk sac and the embryo itself, resulting in immaturenetworks of vessels (4). In contrast, single knock-out of p38�,p38�, or p38�, or both p38� and � result in viable, healthy mice(43, 44). In addition, MK2 activity is abolished in p38� knock-out mice (3), whereas p38� knock-out mice exhibit normalMK2 activity (43), which highlights diverse downstream path-way activation among p38 MAPK isoforms. One study usingSB203580, a small molecule that completely inhibits p38� andonly partially inhibits p38�, suggests that p38� is the principalisoform controlling proliferation and migration of endothelialcells (40). Similarly, our results indicate that downstream p38MAPK signaling throughMK2 andHSP27 is mediated by p38�and not p38� in stromal and tumor cells, which suggests inde-pendent, non-redundant functions between the p38 MAPKisoforms.In our experiments, knockdown of p38� but not p38�

reduced secretion of VEGF, bFGF, EGF, and IL-6 productionfrom stromal and tumor cells, implicating p38� as a criticalmediator of proangiogenic cytokine secretion. Importantly, theaddition of the reduced VEGF, bFGF, EGF (U-87-MG only),and IL-6 in stable U-87-MG or MDA-MB-231 p38� knock-down tumor cells was able to rescue cord formation, indicatingthat these cytokines appear to be key players involved in p38MAPK signaling and cord formation on our co-culture system.Additional studies indicate that p38� is the predominant iso-form involved in cytokine production in vivo following lipopo-lysaccharide (LPS) stimulation (43), and gene transfer of p38�and the upstream activator MKK3 significantly increasedexpression of bFGF and PDGF-A in the normal heart (45), fur-ther supporting the concept of independent, non-redundantfunctions of p38 MAPK isoforms. In addition to p38�, knock-down of the p38 MAPK pathway protein MK2 or HSP27 instromal cells also reduced cord formation along with VEGF,bFGF, and IL-6 secretion, a result substantiated inMK2 knock-out mice, where reduced angiogenesis in wound healing is inconcert with reduced expression of several cytokines (GM-CSF, VEGF, IFN�, MCP1, TNF, IL-6, and IL-1�) comparedwith wild-type mice (46). In addition, targeted deletion of MK2in macrophages led to decreased production of LPS-inducedtumor necrosis factor (TNF), IL-6, and other cytokines (47).Taken together, these results reveal a role for the p38 MAPKpathway components MK2 and HSP27 in cytokine productionand angiogenesis.




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In addition to endothelial cells, pericytes play an importantrole in endothelial cell function and vascular formation (10, 11).Pericytes have been associated mainly with stabilization ofblood vessels but also can sense angiogenic stimuli, guidesprouting tubes, elicit endothelial survival, and exhibit mac-rophage-like activities (31). Several molecules regulate pericytecontractile tone and function as paracrine signals that reveal aninteraction between endothelial cells and pericytes in the regu-lation of blood flow (31). Tumors usually contain a small num-ber of functional pericytes important for vessel stability, func-tion, and endothelial cell survival (31). Highlighting theimportance of pericyte vessel stability function, glioblastomasfrequently contain vessels that are not covered by pericytes, andthese vessels are more dependent on VEGF as an endothelialcell survival factor (48). Furthermore, blocking PDGF receptorsignaling resulted in detachment of pericytes from tumor ves-sels and restricted tumor growth (49). Inhibition of p38MAPKsignaling with LY2228820 dimesylate treatment or knockdownof p38� significantly reduced �-smooth muscle actin expres-sion from pericyte-like cells in our cord formation assay. Thissuggests that p38 MAPK may play an important role in para-crine signaling between endothelial cells and pericytes, but itremains unknown whether p38 MAPK signaling is importantfor pericyte function or recruitment in vivo. Tumor vesselswithout pericytes appear more vulnerable and may be moreresponsive to anti-endothelial cell drugs (31). Multiple humanrenal tumor models treated with a combination of LY2228820dimesylate and sunitinib show potentiation of sunitinib activ-ity,3 which may be due in part to decreased numbers of peri-cytes interacting with the vessels, causing the endothelial cellsto be more susceptible to the antiangiogenic therapy.MKKs are crucial enzymes involved in several biological

pathways that control cell differentiation, proliferation, andsurvival (50). In response to extracellular stimuli, MKKsbecome activated and phosphorylate MAPKs, including extra-cellular signal-regulated protein kinase (ERK), c-Jun NH2-ter-minal kinase (JNK), and p38 MAPK. Others report importantroles for the otherMAPK signaling pathways (ERK and JNK) intumor angiogenesis (13, 32). A variety of small molecule inhib-itors that target MEK, ERK, and JNK were observed to be anti-angiogenic in our in vitro co-culture system (data not shown),further evidence indicating the importance of MAPK signalingin angiogenesis. Our results indicate a positive role for p38MAPK signaling, in particular p38� MAPK, MK2, and HSP27in angiogenesis and p38� MAPK in tumor and stromal cellcytokine release.

Acknowledgments—We thankMarkUhlik,Michelle Swearingen, andSimon Chen for in vitro cord formation assay development, D’ArcyBrewer for in vivo MatrigelTM plug assistance, and Susan Pratt forhelpful discussions.

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Louis StancatoCourtney M. Tate, Wayne Blosser, Lisa Wyss, Glenn Evans, Qi Xue, Yong Pan and

in Vivo and in VitroKinase, Reduces Angiogenic Endothelial Cord Formation LY2228820 Dimesylate, a Selective Inhibitor of p38 Mitogen-activated Protein

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