arachidonic acid induces brain endothelial cell apoptosis via p38-mapk and intracellular calcium...

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Microvascular Research xxx (2014) xxx–xxx

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YMVRE-03424; No. of pages: 15; 4C: 1, 4, 12

Contents lists available at ScienceDirect

Microvascular Research

j ourna l homepage: www.e lsev ie r .com/ locate /ymvre

Arachidonic acid induces brain endothelial cell apoptosis via p38-MAPKand intracellular calcium signaling

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FJustin Evans a, YooSeung Ko a, Wilmer Mata a, Muhammad Saquib a, Joel Eldridge a, Aaron Cohen-Gadol c,H. Anne Leaver d, Shukun Wang a, Maria Teresa Rizzo a,b,⁎a Signal Transduction Laboratory, Methodist Research Institute, Indiana University Health, Indiana University School of Medicine, Indianapolis, IN, USAb Department of Pharmacology, Indiana University School of Medicine, Indianapolis, IN, USAc Department of Neurological Surgery, Indiana University School of Medicine, Indianapolis, IN, USAd Division of Clinical Neuroscience, Edinburgh University, Edinburgh, UK

⁎ Corresponding author at: Signal Transduction LaInstitute, Indiana University Health, 1800 North CapitE504E, Indianapolis, IN 46202, USA. Fax: +1 317 962 936

E-mail address: [email protected] (M.T. Rizzo).

http://dx.doi.org/10.1016/j.mvr.2014.04.0110026-2862/© 2014 Published by Elsevier Inc.

Please cite this article as: Evans, J., et al., Arsignaling, Microvasc. Res. (2014), http://dx.d

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Article history:Accepted 27 April 2014Available online xxxx

Keywords:Arachidonic Acidp38-MAPKCalciumBrain endothelial cellsApoptosis

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RECTED PArachidonic acid (AA), a bioactive fatty acid whose levels increase during neuroinflammation, contributes to

cerebral vascular damage and dysfunction. However, the mode of injury and underlying signaling mechanismsremain unknown. Challenge of primary human brain endothelial cells (HBECs) with AA activated a stressresponse resulting in caspase-3 activation, poly(ADP-ribose) polymerase cleavage, and disruption of monolayerintegrity. AA also induced loss of mitochondrial membrane potential and cytochrome c release consistent withactivation of intrinsic apoptosis. HBEC stimulation with AA resulted in sustained p38-MAPK activation andsubsequent phosphorylation of mitogen-activated protein kinase activated protein-2 (MAPKAP-2) kinase andheat shock protein-27 (Hsp27). Conversely, other unsaturated and saturated fatty acids had no effect. Pharmaco-logical and RNA interference-mediated p38α or p38β suppression abrogated AA signaling to caspase-3 andHsp27, suggesting involvement of both p38 isoforms in AA-induced HBEC apoptosis. Hsp27 silencing alsoblocked caspase-3 activation. AA stimulated intracellular calcium release, which was attenuated by inositol1,4,5-trisphosphate (IP3) receptor antagonists. Blockade of intracellular calcium release decreased caspase-3activation, but had no effect on AA-induced p38-MAPK activation. However, inhibition of p38-MAPK or blockadeof intracellular calcium mobilization abrogated AA-induced cytochrome c release. AA-induced caspase-3 activa-tion was abrogated by pharmacological inhibition of lipooxygenases. These findings support a previously unrec-ognized signaling cooperation between p38-MAPK/MAPKAP-2/Hsp27 and intracellular calcium release inAA-induced HBEC apoptosis and suggest its relevance to neurological disorders associated with vascularinflammation.

© 2014 Published by Elsevier Inc.

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UNCOIntroduction

The ability of brain endothelial cells to survive environmentalstressors is a prerequisite for brain development and homeostasis. Afunctional endothelium is essential to ensure the proper metabolic ex-change between blood and the brain parenchyma, preserve the integri-ty of the blood brain barrier (BBB), and support neuroaxonal growth (Jinet al., 2002; Guo et al., 2008; Weiss et al., 2009). Apoptosis constitutes amode of death in the cerebral endothelium and contributes to disrup-tion of endothelial monolayer integrity and subsequent breakdown ofthe BBB, vasogenic edema, and neuronal damage (Rizzo and Leaver,2010).Moreover, apoptotic endothelial cells are impaired in their abilityto sustain neurogenesis or initiate neovascularization, thus delaying

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achidonic acid induces brainoi.org/10.1016/j.mvr.2014.04

anatomical and functional recovery following insults to the brain. Evi-dence from experimental and clinical studies indicates that injuries tothe cerebral endothelium contribute to the initiation, maintenance,and/or exacerbation of several neurological disorders (Park et al.,2004; Zhou et al., 2004; Rite et al., 2007; Farrall and Wardlaw, 2009;Miyazaki et al., 2011). Thus, targeting the cerebral endothelium mayoffer novel therapeutic strategies to prevent loss of BBB integrity, limitneuronal damage, and enhance brain recovery. Development of suchtherapies, however, awaits a better understanding of the signalingevents that contribute to the destabilization of brain endothelial cellmonolayer integrity and death.

Dysregulated neuroinflammation constitutes a common feature ofseveral neurological disorders (Glass et al., 2010). Arachidonic acid(AA), a biologically active polyunsaturated fatty acid, plays an importantrole in neuroinflammation and vascular dysfunction (Artwohl et al.,2003; Farooqui et al., 2007; Rao et al., 2011). Under resting conditions,AA is esterified at the sn-2 position of membrane phospholipids andexerts structural, signaling, and homeostatic functions (Brash, 2001;

endothelial cell apoptosis via p38-MAPK and intracellular calcium.011

http://dx.doi.org/10.1016/j.mvr.2014.04.011

mailto:[email protected]


http://www.sciencedirect.com/science/journal/00262862


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Fig. 1.AA stimulates intrinsic apoptosis in HBECs. (A). Time course of HBEC detachment inresponse to AA (50 μM). Data are mean ± SD (n = 3). ⁎⁎p b 0.01 and ⁎⁎⁎p b 0.001 vscontrol at each time interval. (B). Representative contrast microscopy images (×20). AA(50 μM; 2 h). (C). Fluorescence microscopy images of Hoechst-stained HBECs. Apoptoticnuclei are shown by arrows (×20). (D). Stimulation of HBECs with AA (50 μM) for the in-dicated times. Western blotting of caspase-3, cleaved caspase-3 fragments and β-actin.Representative blot and quantitative densitometry (n = 3). Data are mean ± SD.⁎p b 0.05 and ⁎⁎⁎p b 0.001 vs control. (E). Stimulation of HBECs with AA at the indi-cated concentrations for 2 h. Western blotting of caspase-3, cleaved caspase-3 frag-ments and β-actin. Representative blot and quantitative densitometry (n = 3). Dataaremean± SD (n=3). ⁎⁎⁎p b 0.001 vs 0. (F). Stimulationwith AA (50 μM) for the indicat-ed times. Western blotting of full-length PARP (116 kDa), cleaved PARP (89 kDa) andβ-actin. Representative blot and quantitative densitometry (n = 3). Data are mean ± SD.⁎⁎p b 0.01 and ⁎⁎⁎p b 0.001 vs control at each time interval. (G). Control or AA-treatedHBECs were stained with MitoCapture and visualized by fluorescence microscopy (×40).Red specks in control cells indicate health mitochondria. Apoptotic cells (diffuse cytosolicgreen-orange fluorescence and intact plasmamembrane) togetherwith necrotic cell debrisare present in AA-treated cells. (H).Western blotting of cytochrome c and β-actin. Stimula-tion with AA (50 μM) for the indicated time intervals. Representative blot and quantitativedensitometry (n = 3). Data are mean ± SD. ⁎⁎⁎p b 0.001 vs control at 120 min. (I). HBECswere stimulated with AA (50 μM) or equimolar concentrations of myristic acid (MA),palmitic acid (PA), stearic acid (SA), oleic acid (OA) or linoleic acid (LA) for 2 h. Extractswere incubatedwith the labeled caspase-3 substrate, DEVD-pNA. Cleaved pNAwas quanti-fied by spectrophotometry. Data are mean ± SD (n = 3).⁎⁎⁎p b 0.001 vs control.

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Rapoport, 2008). During neuroinflammation, levels of unesterified AArapidly increase, mainly as a consequence of phospholipase A2 (PLA2)activation (Cunningham et al., 2006; Nito et al., 2008). Once released,AA acts in a paracrine and/or autocrine manner triggering signalingevents that are ultimately involved in the initiation, maintenance, andexacerbation of vascular inflammation and endothelial cell damage(De Caterina et al., 2000). Early studies suggest that AA and its metabo-lites are major contributing factors to the breakdown of the BBB (Chanand Fishman, 1978; Ohnishi et al., 1992). The pathophysiological rele-vance of AA and its bioactivemetabolites in cerebral endothelium injuryis further supported by the elevated levels of AA detected in patientswith traumatic brain injury, hemorrhagic or ischemic stroke, and neuro-degenerative disorders (Bazan et al., 2002; Pilitsis et al., 2002; Wardet al., 2011). Importantly, inhibition of AA metabolism provides neuro-protection in animalmodels of focal cerebral ischemia and neurodegen-eration (Scali et al., 2003; Gopez et al., 2005). Despite the currentevidence for a role of AA in neuroinflammation and cerebral vascularinjury, the underlying signaling mechanisms are not understood.

Previous studies support the role of p38-MAPK in vascular inflam-mation and endothelial cell apoptosis (Jung et al., 2008; Yang et al.,2010; Wolfson et al., 2011). Interestingly, Nito et al. (2008), using amodel of cerebral ischemia and reperfusion, demonstrated p38-MAPKinvolvement upstream to activation of cytosolic PLA2 (cPLA2) andsubsequent disruption of the BBB. Although these findings suggest theinvolvement of AA downstream to cPLA2 activation, a direct role of thefatty acid in apoptosis of brain endothelial cells has not yet beenestablished.

Fluctuations in intracellular calcium levels regulate cell death in re-sponse to a variety of stimuli, including AA (Penzo et al., 2004; Fanget al., 2008; Giorgi et al., 2008). Moreover, calcium release from theIP3-sensitive intracellular stores can be rapidly taken up by the mito-chondria, causing cytochrome c release and apoptosis (Giorgi et al.,2008). Brain endothelial cells are especially susceptible to mitochondri-al apoptotic insults because of their higher number of mitochondriacompared to peripheral endothelial cells (Oldendorf et al., 1977).While the contribution of AA tomitochondrial dysfunction and apoptosishas been shown in several cellular system (Hillered and Chan, 1988;Takeuchi et al., 1991; Scorrano et al., 2001; Penzo et al., 2002; Penzoet al., 2004), the extent to which AA activates mitochondrial-dependentapoptosis signaling in brain endothelial cells has not yet been investigat-ed. The present study was undertaken to test the hypothesis that AA in-duces apoptosis of brain endothelial cells and elucidate the underlyingsignaling mechanisms. We report that AA induces HBEC mitochondrialapoptosis via a signaling pathway involving p38-MAPK, its downstreamtargets, MAPKAP-2 and Hsp27, and intracellular calcium mobilization.Conversion of AA via the lipooxygenase pathway is required for itsproapoptotic effects on HBECs.

Materials and methods

Hanks' balanced salt solution (HBSS), phosphate buffered saline(PBS), endothelial cell tissue culturemedium(EBM-2) and supplements(EGM-2) were from Lonza (Walkersville, MD). Primary HBECs, attach-ment factor and Bac-Off were from Cell Systems (Kirkland, WA). AA,5,8,11,14-eicosatetraynoic acid (ETYA), palmitic acid, myristic acid,stearic acid, oleic acid, linoleic acid, nordihydroguaiaretic acid (NDGA),staurosporine, β-actin antibodies (clone AC-15), and phospho-specificp38-MAPK antibodies (diphosphorylated p38-MAPK; clone p38-TY)were from Sigma (Saint Louis, MO). Tris-glycine gels, See Blue Plus-2protein standard, and Lipofectamine RNAiMAX were from Invitrogen(Carlsbad, CA). Caspase-3 inhibitor II, SB203580, SB202474, andacetoxymethylester of Fura-2 (Fura-2 AM) were from EMB Bioscience(La Jolla, CA). Aminoethyldiphenylborinate (2-APB), SC-560, andXestospongin C were from Cayman (Ann Arbor, MI). RWJ67657 wasfrom Tocris (Ellisville, MO). Caspase-3/CPP32 colorimetric assay andMitoCapture assay kits were from Biovision Inc. (Mountain View, CA).

Please cite this article as: Evans, J., et al., Arachidonic acid induces brainsignaling, Microvasc. Res. (2014), http://dx.doi.org/10.1016/j.mvr.2014.04

SignalSilence siRNA p38α MAPK RNAII, SignalSilence siRNA p38βMAPK RNAII, Signal Silence Hsp27 RNAII, SignalSilence siRNA control,caspase-3, phospho-Hsp27, phospho-MAPKAP-2 (clone 27B7), cyto-chrome c (clone 136F3), poly(ADP-ribose) polymerase (PARP), andGAPDH antibodies were from Cell Signaling Technology (Danvers,MA). Hsp27 and p38-MAPK antibodies and horseradish peroxidase-linked secondary antibodies were from Santa Cruz (Santa Cruz, CA).



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UNCOMAPKAP-2 antibodies were from Novus Biologicals (Littleton, CO).

Hyperfilms were from Phenix Research Products (Candler, NC).

Cell culture

Primary HBECs were cultured in EBM-2 tissue culture medium con-taining EGM-2 supplements, 10% Bac-Off and 2% fetal bovine serum at37 °C in a humidified atmosphere of 95% air and 5% CO2 (Rush et al.,2007). Confluent cells exhibited a cobblestone appearance. Experimentswere performed using cells at passages 3–12. Cells did not show changesin morphology over this passage range.

Induction of apoptosis

HBECs were seeded onto attachment factor-coated 6-well plates incomplete EBM-2 medium at 5 × 105/well. The following day, cellswere stimulated with AA or vehicle in serum-free and growth factor-free EBM-2 medium, unless otherwise indicated. AA was dissolved inethanol and presented to cells in a final concentration of 0.2% ethanol.Stock solutions of inhibitors were prepared in dimethyl sulfoxide(DMSO). Control HBECs received equal volumes of ethanol or DMSO.


Quantification of cell detachment

HBECs were washed in PBS to remove loosely attached cells,fixed, and stained with Diff-Quick (Fisher Scientific, Kalamazoo,MI). Stained HBECs were washed in PBS, air dried prior to additionof 1 ml lysing solution (PBS containing 1% sodium dodecyl sulfate)and kept overnight at −80 °C. Following incubation for 30 min at37 °C, the staining intensity was measured using a Bio Tek Uquantmicroplate reader at 620 nm. The absorbance of lysing solution from atissue culture well containing no cells and stained exactly as describedabove, was used as background reading and subtracted from the absor-bance of each sample.

Proliferation assay

HBECs were plated in 24-well plates (5 × 104/well) in EBM-2medium and incubated at 37 °C and 5% CO2. The following day, cellswere stimulated with AA in complete EBM-2 medium for 48 h at 37 °Cand 5% CO2. 3H-thymidine (2.5 μCi) was added to each well during thelast 8 h of incubation. 3H-thymidine incorporation was assayed aspreviously described (Payner et al., 2006).



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Caspase-3/CPP32 colorimetric assay

Caspase-3 activity was measured by quantification of p-nitroanilide(pNA) released from the labeled caspase-3 substrate DEVD-pNA (Asp-Glu-Val-Asp-pNA) following the manufacturer's instructions. Briefly,HBECs were plated in 6-well plates (5 × 105/well) the day prior to theexperiments and stimulated as indicated. Attached and suspendedHBECs were pooled, washed in PBS, lysed and subjected to centrifuga-tion (10,000×g; 1 min). Lysates (25–50 μg) were incubated in a buffercontaining DEVD-pNA (200 μM) and 10 mM DTT (37 °C; 2 h). pNA re-lease was quantified by spectrophotometry at 405 nm. The absorbanceof lysis buffer containing identical amounts of the DEVD-pNA wassubtracted from the absorbance of each sample.

Morphological detection of apoptosis

To detect apoptotic nuclei, HBECs grown on attachment factor-coated glass coverslips, were stimulated as indicated, fixed with 4%paraformaldehyde for 15 min at room temperature, and stained withHoechst 33342 (10 μg/ml) for 30 min at room temperature. HBECswere washed in PBS, air dried, and visualized by a Leica DM RB fluores-cence microscope. Mitochondrial apoptosis was detected with the


MitoCapture assay kit following the manufacturer's instructions.HBECs were visualized by fluorescence microscopy using a band-passfilter that detects FICT and rhodamine. Images were collected usingthe SPOT Advanced version 4.7 Software (Sterling Heights, MI).

RNAi transfection

HBECs (~40–50% confluent) were transfected under serum-freeconditions with p38α (100 nM), p38β (100 nM), Hsp27 (200 nM) orcontrol siRNA sequences using Lipofectamine RNAiMAX. Six hourslater, EBM-2 medium containing growth factors and serum was added,and incubation was continued for 48 h. Cells were stimulated and sub-jected to Western blotting as indicated.

Western blotting

HBECs were plated in 6-well plates the day prior to stimulation. Fol-lowing stimulation, detached and attached cells were collected togetherby centrifugation at 4 °C on ice-cold PBS containing 5mMNaF and 1mMNa3VO4. Proteinswere extracted in lysis buffer (50mMTris–HCl pH 8.0,150mMNaCl, 5mMEDTA, 10mMNaF, 10mMdisodiumpyrophosphate,1 mM Na3VO4, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 10 μg/ml



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Fig. 2. Caspase-3 activation partially mediates the antiproliferative effects of AA. (A).HBECs were pretreated with Z-DEVD-FMK or vehicle prior to stimulation with AA(50 μM; 2 h). Extracts were incubated with the pNA-labeled caspase-3 substrate, DEVD-pNA. Cleaved pNA was quantified by spectrophotometry. Data are mean ± SD (n = 3).⁎⁎⁎p b 0.001 AA vs control; ⁎⁎⁎p b 0.00 AA + Z-DEVD-FMK vs AA; ⁎⁎p b 0.01 Z-DEVD-FMK control vs vehicle control. (B). 3H-thymidine incorporation following 48 h stimula-tion with AA (50 μM) or ceramide (50 μM) in the absence or presence of Z-DEVD-FMK.Data are mean ± SD of quadruplicates determinations of a representative experiment.⁎p b 0.05 and ⁎⁎⁎p b 0.001 vs control. (C). 3H-thymidine incorporation in HBECs treatedwith AA (50 μM; 48 h) without or with apocynin. Data are mean ± SD of triplicate deter-minations of a representative experiment. ⁎⁎⁎p b 0.001 vs control.


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aprotinin, 10 μg/ml leupeptin, 1 mM phenylmethylsulphonylfluoride),resolved by gel electrophoresis and transferred to nitrocellulose mem-branes. Immunoblotting was performed using the following primary an-tibodies: phospho- p38-MAPK (1:2000); p38-MAPK (1:500); phospho-Hsp27 (1:2000); phospho-MAPKAP-2 (1:1000); Hsp27 (1:200);cytochrome c (1:800); PARP (1:1000); caspase-3 (1:1000); β-actin(1:2500); GAPDH (1:1000). After incubation with the appropriatehorseradish-peroxidase-linked secondary antibodies, immunoreac-tive bands were visualized by enhanced chemiluminescence (ECL).Densitometry analyses of immunoreactive bands were performedusing the UN-Scan-IT Software Program (Silk Scientific, Orem, UT).

Intracellular calcium measurement

Changes in intracellular calcium concentrations were measuredusing Fura-2 AM (Rizzo et al., 1999). HBECs were grown overnight onattachment factor-coated glass coverslips (22 × 9 mm). The followingday, cells were loaded for 30 min in phenol-free HBSS containing 1 mMCaCl2 and 5 μM Fura-2 AM at 37 °C and 5% CO2. Coverslips were washedwith HBSS and equilibrated for 10 min to allow de-esterification ofFura-2 AM. Calcium signals were detected via continuous recording at37 °C in a PerkinElmer Luminescence Spectrometer LS50B at alternatingexcitation wavelengths of 340/380 nm. Emission fluorescence intensitywas collected at 510 nm. Data are expressed as 340/380 Fura-2 fluores-cence ratio.

Statistical analyses

Data are means ± SD. Differences between groups were evaluatedby ANOVA and Tukey post hoc (GraphPad Software). Differences be-tween two groups were analyzed using the two-tailed t test. p b 0.05was considered statistically significant.

Results

AA induces HBEC mitochondrial apoptosis

To determine whether AA stimulates HBEC apoptosis, cells wereseeded onto 6-well plates in complete EBM-2 medium at 5 × 105/well.HBECs formed a confluent monolayer 24 h after plating and werechallengedwith 50 μMAA in serum- and growth factor-free EBM-2me-dium. HBECsweremonitoredmicroscopically for several time intervals.Treatment with AA significantly decreased HBEC attachment to the cul-ture wells in a time-dependent manner (24.2% ± 8.0, 74.7% ± 13.5,84.3%±5.4, and 81.8% ± 0.15 at 60, 90, 120, and 180min, respectively)compared with control (Fig. 1A). Representative photographs of HBECsfollowing 2 h exposure to vehicle or AA are shown (Fig. 1B). AA-treatedHBECs also displayed morphological features of apoptosis, includingcondensed and fragmented nuclei (Fig. 1C). Thesemorphologic changeswere accompanied bymarked caspase-3 activation. As shown in Fig. 1D,AA stimulated cleavage of the inactive caspase-3 precursor into theactive caspase-3 fragments in a time-dependent manner. Levels of ac-tive caspase-7 also increasedwith identical kinetics following treatmentwith 50 μM AA (not shown). AA stimulated caspase-3 activation in adose-dependent manner. Caspase-3 activation was detected followingstimulation with 35 μM and 50 μM AA, while 10 μM or 25 μM AA hadno effect (Fig. 1E). However, in subconfluent HBECs (2 × 105/well),25 μM AA activated caspase-3. In contrast, 5–10 μM AA had no effect(Fig. S1A). Based on these observations, all subsequent experimentswere performed in confluent HBECs stimulated with AA (50 μM; 2 h),unless otherwise indicated.

Next, cleavage of PARP, a nuclear protein cleaved by caspase-3, wasexamined. AA stimulated PARP cleavage into the 89 kDa active fragmentwith kinetics identical to those of caspase-3 (Fig. 1F).

To determine whether AA induced caspase-3 activation via theintrinsic mitochondrial pathway of apoptosis, we assessed loss of


mitochondrial membrane potential, an intracellular event that leads tothe release of cytochrome c frommitochondria into the cytosol and sub-sequent caspase-9 and caspase-3 activation (Galluzzi et al., 2012). Weused MitoCapture, a cationic dye that, in healthy cells, accumulates inmitochondria emitting red punctuated fluorescence, whereas, in cellsin which themitochondrialmembrane potential is lost, the dye remainsin the cytosol and emits green fluorescence. HBECs showed a diffusecytosolic green-orange fluorescence in response to treatment with AA(50 μM; 2 h), whereas control cells showed intracellular red inclusions,indicating intact mitochondria (Fig. 1G).



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To confirm activation of intrinsic apoptosis, we measured cyto-chrome c release following treatment with 50 μM AA for varioustime intervals (0.5, 1, 2 h). Cytochrome c release significantly in-creased following 2 h stimulation with AA (Fig. 1H). Taken together,these findings implicate the intrinsic pathway of apoptosis in AA-induced HBEC death.

Next, the effect of AA on caspase-3 activity was compared to that ofother fatty acids with various chain length and degree of unsaturation.AA (50 μM; 2 h) stimulated caspase-3 activity, as shown by increasedpNa release from the labeled caspase-3 selective substrate (pNa-DEVD), compared to control (Fig. 1I). In contrast, equimolar concentra-tions of saturated and unsaturated fatty acids, including myristic acid(C14:0), palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1),and linoleic acid (C18:2) were ineffective.

AA-induced inhibition of HBEC proliferation is partially rescued byinhibition of caspase-3

To establish the functional consequences of AA-induced apoptosis,we measured HBEC proliferation in the presence or absence of Z-DEVD-FMK, a cell-permeable irreversible inhibitor of caspase-3. Pre-treatment with Z-DEVD-FMK blocked basal and AA-induced caspase-3activity (Fig. 2A). Moreover, AA-induced inhibition of HBEC prolifera-tion was partially rescued by Z-DEVD-FMK (57.8% ± 8.5 compared tocells treated with AA only). In contrast Z-DEVD-FMK failed to block

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Fig. 3. AA stimulated p38-MAPK, MAPKAP-2 and Hsp27 phosphorylation. (A, B). AA-induced tibody that recognizes p38-MAPK phosphorylated at Tyr-182/Thr-180 (phospho-p38) followed bblot and quantitative densitometry (n=3). Data aremean± SD. ⁎⁎p b 0.01 and ⁎⁎⁎p b 0.001 vsor equimolar concentrations of myristic acid (MA), palmitic acid (PA), stearic acid (SA), oleic acShown is a representative blot and quantitative densitometry (n = 6). Data are mean ± SD. ⁎

stimulation with AA (50 μM; 2 h). Immunoblotting with antibodies against p-MAPKAP-2mean ± SD. ⁎⁎⁎p b 0.001 AA vs control; and ⁎⁎⁎p b 0.001 AA + SB203580 vs AA. (E). HBERepresentative blot and quantitative densitometry (n = 3). Data are mean ± SD. ⁎⁎⁎p b 0


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the antiproliferative effect of C2-ceramide (Fig. 2B). The antioxidantapocynin also failed to prevent AA-induced inhibition of HBEC prolifer-ation (Fig. 2C).

AA stimulates p38-MAPK, MAPKAP-2, and Hsp27 phosphorylation

To test the hypothesis that p38-MAPK contributes to AA-inducedHBEC apoptosis, cells were exposed to 50 μM AA or vehicle, and activa-tion of p38-MAPK was assessed by immunoblotting using an antibodythat recognizes activated p38-MAPK (phosphorylated at Tyr182 andThr180 residues within its activation loop) (Cuadrado and Nebreda,2010). As shown in Fig. 3A, p38-MAPK phosphorylation significantlyincreased in a time-dependent manner reaching maximal levels at 2 hstimulation with AA and declined thereafter. AA stimulated p38-MAPKphosphorylation at concentrations of 35–50 μM, whereas lower concen-trations (10–25 μM) had no effect (Fig. 3B). However, similar to caspase-3 activation, 25 μM AA stimulated p38-MAPK phosphorylation insubconfluent HBECs, whereas 5–10 μM AA had no effect (Fig. S1B).Myristic acid, stearic acid, palmitic acid, oleic acid or linoleic acid, each50 μM concentrations, failed to stimulate p38-MAPK phosphorylation(Fig. 3C). An immunoreactive band migrating at approximately 50 kDawas consistently detected with the phospho-p38-MAPK antibody (Figs.3A,B,C). The identity of this band remains to be determined.

Next,we examined the ability of AA to activateMAPKAP-2 andHsp27,two downstream targets of p38-MAPK (Cuadrado and Nebreda, 2010).

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me- and dose-dependent p38-MAPK activation detected by immunoblotting with an anti-y stripping and reprobingwith an antibody against total p38-MAPK (p38). Representativecontrol at each time point; ⁎⁎⁎p b 0.001 vs 0. (C). HBECswere stimulatedwith AA (50 μM)id (OA) or linoleic acid (LA) for 2 h. p38-MAPK activation was detected as described in A,B.⁎⁎p b 0.001 vs control. (D). Pretreatment of HBECs with SB203580 or SB202474 prior toor MAPKAP-2. Representative blot and quantitative densitometry (n = 4). Data areCs were treated as in D. Immunoblotting with antibodies against p-Hsp27 or Hsp27..001 vs control; and ⁎⁎⁎p b 0.001 AA + SB203580 vs AA.



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Fig. 3 (continued).


Please cite this article as: Evans, J., et al., Arachidonic acid induces brain endothelial cell apoptosis via p38-MAPK and intracellular calciumsignaling, Microvasc. Res. (2014), http://dx.doi.org/10.1016/j.mvr.2014.04.011


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AA significantly increasedMAPKAP-2 phosphorylation at the p38-MAPK-dependent site, Thr334 (Fig. 3D). Hsp27phosphorylation at Ser82 also in-creased after AA stimulation (Fig. 3E). Pretreatment with SB203580, ap38-MAPK inhibitor, attenuated AA-induced MAPKAP-2 and Hsp27phosphorylation (70.2% ± 11.3 and 76.3% ± 18.3, respectively). Incontrast, SB202474, the inactive SB203580 analogue, had no effect(Figs. 3D,E).

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Pharmacological inhibition of p38-MAPK and Hsp27 knockdown abrogateAA-induced apoptosis

To establish the contribution of p38-MAPK to AA-induced apoptosis,HBECs were pretreated with SB203580 or SB202474 prior to measure-ment of AA-induced caspase-3 activation. SB203580 abrogated AA-induced cleavage of caspase-3 and release of the labeled caspase-3

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s with 25 μM SB203580 or SB202474 prior to stimulation with AA (50 μM; 2 h). Westernata are mean± SD (n= 3). ⁎⁎⁎p b 0.001 AA vs control; and ⁎⁎p b 0.01 AA+ SB203580 vsb 0.001 AA vs control; ⁎⁎⁎p b 0.001 AA+ SB203580 vs AA. (C,D). Pretreatment of HBECsEC detachment wasmeasured at 620 nm. Data aremean± SD (n=3). ⁎⁎⁎p b 0.001 AA vss were transfected as indicated and stimulated with AA (50 μM; 2 h) or vehicle. Western= 3). Data are mean ± SD. Upper panel: ⁎⁎⁎p b 0.001 control (siRNA Hsp27) vs controld ⁎⁎⁎p b 0.001 AA (siRNA Hsp27) vs AA (siRNA control). (F). Cell detachment in HBECsA (siRNA control) vs control (siRNA control); and ⁎⁎⁎p b 0.001 AA (siRNA Hsp27) vs AA



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substrate, whereas SB202474 had no effect (Figs. 4A,B). SB203580or RWJ67657, another selective p38-MAPK inhibitor, significantly de-creased AA-induced HBEC detachment (78.9% ± 1.9 and 67.2% ± 22respectively), whereas SB202474 did not (Figs. 4C,D).

Next, we determined the consequence of Hsp27 knockdown onAA-induced caspase-3 activation. Hsp27 protein expression significantly


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decreased following transfection with Hsp27 targeting siRNA se-quences compared to control siRNA-transfected cells (76.4% ± 6.6 and78.6% ± 3.0 control and AA-treated cells, respectively) (Fig. 4E). More-over, Hsp27 knockdown markedly attenuated AA-induced caspase-3activation (Fig. 4E) and cell detachment (Fig. 4F). These findings sup-port the involvement of the p38-MAPK/MAPKAP-2/Hsp27 cascade inAA-induced HBEC apoptosis.

Requirement of p38α and p38β for AA-induced caspase-3 activation andHsp27 phosphorylation

Because SB203580 and RWJ67657 selectively inhibit p38α and p38βisoforms of p38-MAPK (Lee et al., 1994;Wadsworth et al., 1999), the re-sults shown above suggested that both or either p38α or p38βmediat-ed AA-dependent apoptotic signaling. To distinguish between thesepossibilities, HBECs were transfected with siRNA sequences targetingp38α or p38β. Non-targeting siRNA sequences were used as controls.The degree of knockdown was evaluated 48 h later by immunoblottingusing selective p38α or p38β antibodies. Caspase-3 activation andHsp27 phosphorylation were monitored as readouts of p38α andp38β silencing after AA stimulation (50 μM; 2 h). As shown in Figs.5A,B, p38α or p38β protein expression significantly decreased fol-lowing transfection with p38α or p38β targeting siRNA comparedto control siRNA-transfected cells (p38α: 78.4% ± 11.6 control;76.6% ± 8.7 AA-treated cells. p38β: 74.6% ± 7.8 control; 77.9% ±14.7 AA-treated cells). Knockdown of either p38 isoform markedlydecreased AA-induced caspase-3 activation and Hsp27 phosphoryla-tion. Importantly, the siRNA sequences used here selectively silencedp38α or p38β protein expression (Fig. 5C). Thus, p38α and p38β areboth involved in transmitting AA-induced apoptotic signaling inHBECs.

AA-induced intracellular calciummobilization does notmediate p38-MAPKphosphorylation but is involved in caspase-3 activation

To understand early signaling events involved in activation of p38-MAPK byAA, we focused on calciumdynamics because intracellular cal-cium mobilization is required for AA-induced activation of the c-junamino-terminal kinase (JNK) (Rizzo et al., 1999). Moreover, increasesin intracellular calcium are implicated in regulation of apoptotic eventsin response to a variety of agonists, including AA (Penzo et al., 2004;Giorgi et al., 2008). Thus, we asked whether AA stimulated intracellularcalcium release in HBECs. AA (50 μM) added to Fura 2-AM-labeledHBECs induced a rapid release of intracellular calcium (Fig. 6A). This re-lease resulted from depletion of intracellular calcium stores and wasfollowed by calcium influx upon addition of calcium in the extracellularmedium (Fig. 6A). 2-APB, a membrane-permeable IP3 receptor antago-nist (Maruyama et al., 1997), attenuated AA-dependent depletion of in-tracellular calcium stores and subsequent calcium entry (Fig. 6A).However, 2-APB did not attenuate AA-induced p38-MAPK activation, al-though abrogated AA-induced caspase-3 activation (Figs. 6B,C). Similar-ly, the IP3 receptor-selective antagonist, Xestospongin C (Gafni et al.,1997), failed to block p38-MAPK activation but significantly attenuatedAA-induced caspase-3 activation (Figs. 6D,E). Thus, AA-induced p38-MAPK activation occurs independently of intracellular calcium release.However, intracellular calcium release contributes to AA-inducedcaspase-3 activation.

AA-dependent p38-MAPK activation and intracellular calciummobilizationconverge to stimulate mitochondrial apoptosis

The findings that AA-induced caspase-3 activationwas abrogated byinhibiting either intracellular calcium release or p38-MAPK activationsuggest that both signaling events contribute to AA-dependent mito-chondrial apoptosis. To test this possibility, we measured cytochromec release in HBECs pretreated with 2-APB, SB203580, or RWJ67657.



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AA-induced cytochrome c release was abrogated by 2-APB, SB203580,or RWJ67657 (Figs. 7A,B,C,D). Thus, intracellular calcium mobilizationand p38-MAPK activation converge to stimulate AA-dependent apopto-sis via the release of cytochrome c.

AA metabolism is required for AA-induced apoptosis of HBECs

To determine whether AA itself or its conversion into eicosanoidswas required for HBEC apoptosis, HBECs were stimulated for 2 h withAA (50 μM) or ETYA (50 μM), a non-metabolizable AA analogue thatmimics the effects of AA itself in many cellular systems (Rizzo et al.,1999; Fang et al., 2008). As shown in Fig. S2A, AA increased caspase-3activity compared to control cells, while ETYA had no effect (Fig. S2A).Similarly, ETYA failed to disrupt HBEC monolayer integrity or activatep38-MAPK (not shown).

To gain insights into the requirement of eicosanoids for AA-inducedapoptosis, HBECswere preincubatedwith SC-560 (1–10 μM), a selectivecyclooxygenase-1 (COX-1) inhibitor, prior to measuring AA-dependentcaspase-3 activity. SC-560 did not prevent AA-induced caspase-3 activa-tion (Fig. S2B). In contrast, pretreatmentwithNDGA, an antioxidant andpan-lipooxygenase inhibitor that targets 5, 12 and 15 lipooxygenases,rescued AA-induced caspase-3 activation in a dose-dependent manner(Fig. S2C). NDGA (25 μM), however, had no effect on staurosporine-induced caspase-3 activation (not shown). Similarly, the antioxidant

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Fig. 5. p38α and p38βmediate AA-induced caspase-3 activation and phosphorylation ofHsp27.against p38α, caspase-3, p-Hsp27, or β-actin. Representative blots and quantitative densitomettrol (siRNA control); and ⁎p b 0.05 AA (siRNA p38α) vs AA (siRNA control). Lower left panel: ⁎⁎

AA (siRNA control). Lower right panel: ⁎⁎p b 0.01 AA (siRNA control) vs control (siRNA controindicated and subjected to immunoblotting with antibodies against p38β, caspase-3, p-Hare mean ± SD. Upper right panel: ⁎p b 0.05 control (siRNAp38β) vs control (siRNA conAA (siRNA control) vs control (siRNA control); ⁎p b 0.05 AA (siRNA p38β) vs AA (siRNA cand ⁎p b 0.05 AA (siRNA p38β) vs AA (siRNA control). (C). Membranes were stripped and


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apocynin did not prevent AA-induced apoptosis (not shown). These re-sults suggest that lipooxygenasemetabolitesmediate AA-inducedHBECapoptosis.

Discussion

The present study provides mechanistic insights into the previouslyreported effects of AA on breakdown of the BBB and, as summarized inFig. 8, emphasizes a previously unrecognized signaling cooperation be-tween p38-MAPK and intracellular calcium mobilization in mediatingAA-induced brain endothelial cell apoptosis.

While previous studies have highlighted the critical role of AA inactivation of p38-MAPK in several cell types (Hii et al., 1998; Rizzoet al., 2002), to our knowledge this is the first study to demonstrateactivation of p38-MAPK by AA in brain endothelial cells and its contri-bution to apoptosis and disruption of brain endothelial monolayerintegrity. Our findings also support the involvement of MAPKAP-2 andHsp27, two downstream p38-MAPK substrates, which have been impli-cated in p38-MAPK-induced apoptosis and disruption of monolayerintegrity in pulmonary endothelial cells and human umbilical vein en-dothelial cells (Yang et al., 2010; Wolfson et al., 2011). Consistent withthis possibility, pharmacological inhibition or siRNA-mediated knock-down of p38-MAPK attenuated AA-induced MAPKAK-2 and Hsp27phosphorylation. The involvement of p38-MAPK/MAPKAP-2/Hsp27

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trol); ⁎p b 0.05 AA (siRNA p38β) vs AA (siRNA control). Lower left panel: ⁎⁎p b 0.01ontrol). Lower right panel: ⁎⁎p b 0.01 AA (siRNA control) vs control (siRNA control);reprobed with antibodies against p38α (upper), p38β (lower) or β-actin.



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in AA-induced apoptosis is further supported by abrogation of AA-induced caspase-3 activation and cell detachment following Hsp27 si-lencing. Thesefindings support the role of Hsp27 as a crucial downstreamcomponent of the p38-MAPK apoptotic signaling cascade activated byAA. It should be emphasized that while Hsp27 phosphorylation contrib-utes to p38-MAPK-dependent apoptosis and antiproliferative signalingin endothelial cells (Trott et al., 2009), in other cell types Hsp27 phos-phorylation results in cytoprotection (Garrido et al., 2006; Stetler et al.,2012). The reasons for these divergent cellular outcomes are presentlyunclear, but Hsp27 interaction with distinct effectors, substrates, orbinding partners among different cell types, could explain the opposingresponses on cell survival.

The p38-MAPK family includes four isoforms, p38α, p38β, p38γ,p38δ, which differ in tissue distribution profiles, substrate specificityand cellular outcomes (Cuadrado and Nebreda, 2010). p38α and p38βare the main p38-MAPK isoforms expressed in the brain (Jiang et al.,1996). Their contribution to brain endothelial cell apoptosis is notknown. Our findings indicate that both isoforms contribute to AA-induced brain endothelial cell apoptosis. Consistentwith this possibility,pharmacologic inhibitors of both p38α and p38β abrogated AA-inducedphosphorylation of MAPKAP-2 and Hsp27, and caspase-3 activation.Moreover, silencing of either p38 isoform blocked AA-induced caspase-


3 activation. The marked reduction of caspase-3 activation, followingsilencing of either p38α or p38β, further suggests a redundant andnoncompensatory role of both isoforms in mediating AA-induced brainendothelial cell apoptosis.

Our findings are also consistent with AA-induced mitochondrialapoptosis as shown by loss of mitochondrial membrane potentialand release of cytochrome c. Inhibition of p38-MAPK abrogated AA-induced cytochrome c release, suggesting the involvement of p38-MAPK in regulation of the intrinsic apoptotic pathway (Owens et al.,2009). However, we observed that AA induced caspase-3 and PARPcleavage as early as 1 h, whereas cytochrome c release was detectedat 2 h. These findings indicate that AA-induced mitochondrial apo-ptosis accounts only in part for caspase-3 activation and suggest thecoparticipation of the extrinsic apoptotic pathway.

Perturbation of intracellular calcium levels has been reported tome-diate AA-dependent apoptotic signals (Penzo et al., 2004; Fang et al.,2008).Moreover, we previously demonstrated that AA-induced calciumrelease from the endoplasmic reticulumwas a critical component of thesignaling pathway leading to JNK activation in bone marrow stromalcells (Rizzo et al., 1999). These observations led us to postulate theparticipation of intracellular calcium in the upstream signaling eventsleading to AA-induced p38-MAPK activation and subsequent apoptosis.



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Pharmacological inhibition of IP3R-dependent intracellular calciumattenuated AA-dependent calcium signals, cytochrome c release andcaspase-3 activation, but failed to prevent p38-MAPK activation. Thus,

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while intracellular calcium mobilization is an important upstream sig-nal for AA-induced mitochondrial apoptosis, is not required for p38-MAPK activation.

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Fig. 7. Requirement of p38-MAPK and intracellular calcium for AA-induced cytochrome crelease. (A). HBECs were preincubated with 2-APB prior to stimulation with AA (50 μM).Immunoblotting of cytochrome c and β-actin. Shown is a representative blot andquantitative densitometry (n = 3). Data are mean ± SD. ⁎⁎⁎p b 0.001 AA vs control;and ⁎⁎⁎p b 0.001 AA +2-APB vs AA. (B). HBECs were preincubated with SB203580prior to stimulation with AA (50 μM). Immunoblotting of cytochrome c and β-actin.Shown is a representative blot and quantitative densitometry (n = 3). ⁎⁎⁎p b 0.001 AAvs control; and ⁎⁎p b 0.01 AA + SB203580 vs AA. (C). HBECs were preincubated withRWJ67657 prior to stimulation with AA (50 μM). Immunoblotting of cytochrome c andβ-actin. Shown is a representative blot and quantitative densitometry (n = 3). ⁎p b 0.01AA vs control; and ⁎p b 0.01 AA + RWJ67657 vs AA.


UNIn the brain, as in other organs, levels of unesterified AA are in

submicromolar concentrations during normal physiological conditions(Richieri and Kleinfeld, 1995) but significantly increase reachingmicro-molar concentrations in response to experimental andpathological con-ditions (Rehncrona et al., 1979; Yoshida et al., 1980; Siesjo et al., 1982;Pilitsis et al., 2002; Artwohl et al., 2003; Farias et al., 2011). Importantlylevels of AA in the CSF of patients with ischemic or hemorrhagic stroke

Fig. 6. Intracellular calcium is not involved in AA-induced p38-MAPK phosphorylation butmedi2-APB or vehicle prior to AA (50 μM)stimulation (solid arrow) in the absence of extracellular Ca2

Fura-2 fluorescence ratio. Representative recording (n = 3). (B). Preincubation with 2-APB orMAPK. Representative blot and quantitative densitometry (n=3). Data aremean± SD (n=3)itored by the release of pNA. Data are mean± SD (n= 3). ⁎⁎⁎p b 0.001 AA vs control; and ⁎⁎⁎pstimulation with 50 μMAA. Immunoblotting of phospho-p38-MAPK and total p38-MAPK. ReprAA vs control. (E). Cells were treated as in D. Immunoblotting of caspase-3 and β-actin. Represevs control; ⁎p b 0.05 AA + Xestospongin vs AA.


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correlated with worse clinical outcome (Pilitsis et al., 2003). Thus, theconcentrations of AA, negatively affecting HBEC survival in the presentstudy, is within the range of those detected in vivo during brain injuriesand vascular inflammation. Intriguingly, 25 μMAA stimulated caspase-3and p38-MAPK activation in subconfluent HBECs but had no effect onconfluent HBECs. These findings underscore the complex interactionsbetween AA concentrations and endothelial monolayer density inregulating AA-dependent apoptotic responses. Because subconfluentendothelial cells release higher amounts of AA compared to confluentendothelial cells (Whatley et al., 1994), it is possible that the endoge-nous release of AA from subconfluent HBECs contributes to theproapoptotic effects of 25 μM AA added exogenously. Our studies alsoindicate that the proapoptotic effects of 50 μM AA are unlikely a resultof a nonspecific lipid or detergent effect because the other fatty acidstested failed to stimulate p38-MAPK and caspase-3 activation whenused at concentrations of 50 μM.

Cultured brain endothelial cells largely produce PGE2 andmonohydroxyeicosatetraenoic acids (HETEs) when exposed to highconcentrations of AA (Moore et al., 1988).Moreover, lipooxygenaseme-tabolites are implicated in mitochondrial apoptosis (Nazarewicz et al.,2007). In here we observed that ETYA failed to stimulate caspase-3 ac-tivity, while NDGA, at concentrations previously reported to suppresslipooxygenase activity and HETE production in cultured brain endothe-lial cells (Moore et al., 1988), blocked AA-induced brain endothelial cellapoptosis. The antioxidant properties of NDGA are not likely responsi-ble for its rescue effects because the antioxidant apocynin did not pre-vent AA-induced inhibition of brain endothelial proliferation anddisruption of monolayer integrity. On the other hand, SC-560, at con-centrations reported to suppress prostaglandin production (Smithet al., 1988), had no effect on AA-induced caspase-3 activation. Collec-tively, thesefindings support the requirement of AAmetabolic transfor-mation via the lipooxygenase pathway for its proapoptotic effects onbrain endothelial cells. A comprehensive assessment of the involvedlipooxygenase metabolites and their contributions to each of the twosignaling components of the apoptotic pathway activated by AA re-quires further study.

In summary, the present study provides novel insights into themechanisms of human brain endothelial cell injury following expo-sure to exogenous AA, a lipid mediator known to critically contrib-ute to neuroinflammation and BBB destabilization. Our findings areconsistent with a cooperative role of p38-MAPK/MAPKAP-2/Hsp27activation and intracellular calcium mobilization in activating amitochondrial apoptotic pathway responsible for AA-induced endo-thelial cell injury and death. Although additional studies are re-quired to characterize further the upstream events leading to AA-induced p38-MAPK activation and intracellular calcium mobiliza-tion, as well as the involvement of additional members of theMAPK family, the signaling network elucidated in here may haveimportant mechanistic implications in neurological disorders initi-ated, maintained, or exacerbated by inflammation of the cerebralendothelium.

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.mvr.2014.04.011.

Conflicts of interest

None.

ates AA-induced caspase-3 activation. (A). Fura-2 AM-labeled HBECswere pretreatedwith+. Ca2+ added to the extracellularmedium (dashed arrow).Data are expressed as 340/380vehicle and stimulation with AA. Immunoblotting of phospho-p38-MAPK and total p38-. ⁎⁎p b 0.01 AA vs control. (C). HBECswere stimulated as in B. Caspase-3 activity wasmon-b 0.001 AA+ 2-APB AA vs AA. (D). Preincubation with Xestospongin C or vehicle prior to

esentative blots and quantitative densitometry (n= 3). Data aremean± SD. ⁎⁎⁎p b 0.001ntative blots and quantitative densitometry (n= 4). Data aremean± SD. ⁎⁎⁎p b 0.001 AA





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Apoptosis

Disruption of monolayer integrity

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Arachidonic Acid

Cytochrome cHsp-27

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Fig. 8. Proposed signaling pathwaymediating AA-inducedHBEC apoptosis. Stimulation of HBECswith AA triggers signaling events characterized by intracellular calciummobilization andphosphorylation of p38-MAPK, MAPKAP-2 and Hsp27. Intracellular calcium and activated p38-MAPK/MAPKAP-2/Hsp27-dependent signaling converge into the mitochondria to inducecaspase-3-dependent apoptosis and cell detachment. Conversion of AA by lipooxygenases (LOXs) is required for AA-dependent apoptotic signaling. The solid and dashed arrows indicatedirect and indirect experimental evidence in support of the proposed targets/pathways, respectively. Pharmacological or siRNA-mediatedmanipulation of the proposed targets/pathwaysis indicated. Arrowheads: ►activation: │ inhibition.


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Acknowledgments

We thank Dr. Dino Rotondo for the critical suggestions, Dr. RafatSiddiqui for the use of the spectrometer, Elaine Bammerline for theillustrations, Marilyn Michael Yurk for the editorial assistance, JamieHeitzenrater and Andrea Doell for the technical assistance. This workwas supported by Institutional Funds to M.T.R.

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