YMVRE-03424; No. of pages: 15; 4C: 1, 4, 12 Microvascular Research xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Microvascular Research journal homepage: www.elsevier.com/locate/ymvre
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Article history: Accepted 27 April 2014 Available online xxxx
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Keywords: Arachidonic Acid p38-MAPK Calcium Brain endothelial cells Apoptosis
Signal Transduction Laboratory, Methodist Research Institute, Indiana University Health, Indiana University School of Medicine, Indianapolis, IN, USA Department of Pharmacology, Indiana University School of Medicine, Indianapolis, IN, USA Department of Neurological Surgery, Indiana University School of Medicine, Indianapolis, IN, USA d Division of Clinical Neuroscience, Edinburgh University, Edinburgh, UK
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Justin 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,⁎
Arachidonic 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 mechanisms remain unknown. Challenge of primary human brain endothelial cells (HBECs) with AA activated a stress response resulting in caspase-3 activation, poly(ADP-ribose) polymerase cleavage, and disruption of monolayer integrity. AA also induced loss of mitochondrial membrane potential and cytochrome c release consistent with activation of intrinsic apoptosis. HBEC stimulation with AA resulted in sustained p38-MAPK activation and subsequent phosphorylation of mitogen-activated protein kinase activated protein-2 (MAPKAP-2) kinase and heat shock protein-27 (Hsp27). Conversely, other unsaturated and saturated fatty acids had no effect. Pharmacological and RNA interference-mediated p38α or p38β suppression abrogated AA signaling to caspase-3 and Hsp27, suggesting involvement of both p38 isoforms in AA-induced HBEC apoptosis. Hsp27 silencing also blocked caspase-3 activation. AA stimulated intracellular calcium release, which was attenuated by inositol 1,4,5-trisphosphate (IP3) receptor antagonists. Blockade of intracellular calcium release decreased caspase-3 activation, but had no effect on AA-induced p38-MAPK activation. However, inhibition of p38-MAPK or blockade of intracellular calcium mobilization abrogated AA-induced cytochrome c release. AA-induced caspase-3 activation was abrogated by pharmacological inhibition of lipooxygenases. These findings support a previously unrecognized signaling cooperation between p38-MAPK/MAPKAP-2/Hsp27 and intracellular calcium release in AA-induced HBEC apoptosis and suggest its relevance to neurological disorders associated with vascular inflammation. © 2014 Published by Elsevier Inc.
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anatomical and functional recovery following insults to the brain. Evidence from experimental and clinical studies indicates that injuries to the 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 may offer novel therapeutic strategies to prevent loss of BBB integrity, limit neuronal damage, and enhance brain recovery. Development of such therapies, however, awaits a better understanding of the signaling events that contribute to the destabilization of brain endothelial cell monolayer integrity and death. Dysregulated neuroinflammation constitutes a common feature of several neurological disorders (Glass et al., 2010). Arachidonic acid (AA), a biologically active polyunsaturated fatty acid, plays an important role 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 and exerts structural, signaling, and homeostatic functions (Brash, 2001;
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Arachidonic acid induces brain endothelial cell apoptosis via p38-MAPK and intracellular calcium signaling
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Introduction
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The ability of brain endothelial cells to survive environmental stressors is a prerequisite for brain development and homeostasis. A functional endothelium is essential to ensure the proper metabolic exchange between blood and the brain parenchyma, preserve the integrity of the blood brain barrier (BBB), and support neuroaxonal growth (Jin et al., 2002; Guo et al., 2008; Weiss et al., 2009). Apoptosis constitutes a mode of death in the cerebral endothelium and contributes to disruption of endothelial monolayer integrity and subsequent breakdown of the BBB, vasogenic edema, and neuronal damage (Rizzo and Leaver, 2010). Moreover, apoptotic endothelial cells are impaired in their ability to sustain neurogenesis or initiate neovascularization, thus delaying
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⁎ Corresponding author at: Signal Transduction Laboratory, Methodist Research Institute, Indiana University Health, 1800 North Capitol Avenue, Noyes Bldg, Room E504E, Indianapolis, IN 46202, USA. Fax: +1 317 962 9369. E-mail address:
[email protected] (M.T. Rizzo).
http://dx.doi.org/10.1016/j.mvr.2014.04.011 0026-2862/© 2014 Published by Elsevier Inc.
Please cite this article as: Evans, J., et al., Arachidonic acid induces brain endothelial cell apoptosis via p38-MAPK and intracellular calcium signaling, Microvasc. Res. (2014), http://dx.doi.org/10.1016/j.mvr.2014.04.011
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Hanks' balanced salt solution (HBSS), phosphate buffered saline (PBS), endothelial cell tissue culture medium (EBM-2) and supplements (EGM-2) were from Lonza (Walkersville, MD). Primary HBECs, attachment 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-specific p38-MAPK antibodies (diphosphorylated p38-MAPK; clone p38-TY) were from Sigma (Saint Louis, MO). Tris-glycine gels, See Blue Plus-2 protein standard, and Lipofectamine RNAiMAX were from Invitrogen (Carlsbad, CA). Caspase-3 inhibitor II, SB203580, SB202474, and acetoxymethylester of Fura-2 (Fura-2 AM) were from EMB Bioscience (La Jolla, CA). Aminoethyldiphenylborinate (2-APB), SC-560, and Xestospongin C were from Cayman (Ann Arbor, MI). RWJ67657 was from Tocris (Ellisville, MO). Caspase-3/CPP32 colorimetric assay and MitoCapture assay kits were from Biovision Inc. (Mountain View, CA).
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SignalSilence siRNA p38α MAPK RNAII, SignalSilence siRNA p38β MAPK RNAII, Signal Silence Hsp27 RNAII, SignalSilence siRNA control, caspase-3, phospho-Hsp27, phospho-MAPKAP-2 (clone 27B7), cytochrome c (clone 136F3), poly(ADP-ribose) polymerase (PARP), and GAPDH antibodies were from Cell Signaling Technology (Danvers, MA). Hsp27 and p38-MAPK antibodies and horseradish peroxidaselinked secondary antibodies were from Santa Cruz (Santa Cruz, CA).
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Rapoport, 2008). During neuroinflammation, levels of unesterified AA rapidly 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 signaling events that are ultimately involved in the initiation, maintenance, and exacerbation of vascular inflammation and endothelial cell damage (De Caterina et al., 2000). Early studies suggest that AA and its metabolites are major contributing factors to the breakdown of the BBB (Chan and Fishman, 1978; Ohnishi et al., 1992). The pathophysiological relevance of AA and its bioactive metabolites in cerebral endothelium injury is further supported by the elevated levels of AA detected in patients with traumatic brain injury, hemorrhagic or ischemic stroke, and neurodegenerative disorders (Bazan et al., 2002; Pilitsis et al., 2002; Ward et al., 2011). Importantly, inhibition of AA metabolism provides neuroprotection in animal models of focal cerebral ischemia and neurodegeneration (Scali et al., 2003; Gopez et al., 2005). Despite the current evidence for a role of AA in neuroinflammation and cerebral vascular injury, the underlying signaling mechanisms are not understood. Previous studies support the role of p38-MAPK in vascular inflammation and endothelial cell apoptosis (Jung et al., 2008; Yang et al., 2010; Wolfson et al., 2011). Interestingly, Nito et al. (2008), using a model of cerebral ischemia and reperfusion, demonstrated p38-MAPK involvement upstream to activation of cytosolic PLA2 (cPLA2) and subsequent disruption of the BBB. Although these findings suggest the involvement of AA downstream to cPLA2 activation, a direct role of the fatty acid in apoptosis of brain endothelial cells has not yet been established. Fluctuations in intracellular calcium levels regulate cell death in response to a variety of stimuli, including AA (Penzo et al., 2004; Fang et al., 2008; Giorgi et al., 2008). Moreover, calcium release from the IP3-sensitive intracellular stores can be rapidly taken up by the mitochondria, causing cytochrome c release and apoptosis (Giorgi et al., 2008). Brain endothelial cells are especially susceptible to mitochondrial apoptotic insults because of their higher number of mitochondria compared to peripheral endothelial cells (Oldendorf et al., 1977). While the contribution of AA to mitochondrial dysfunction and apoptosis has been shown in several cellular system (Hillered and Chan, 1988; Takeuchi et al., 1991; Scorrano et al., 2001; Penzo et al., 2002; Penzo et al., 2004), the extent to which AA activates mitochondrial-dependent apoptosis signaling in brain endothelial cells has not yet been investigated. The present study was undertaken to test the hypothesis that AA induces apoptosis of brain endothelial cells and elucidate the underlying signaling mechanisms. We report that AA induces HBEC mitochondrial apoptosis via a signaling pathway involving p38-MAPK, its downstream targets, MAPKAP-2 and Hsp27, and intracellular calcium mobilization. Conversion of AA via the lipooxygenase pathway is required for its proapoptotic effects on HBECs.
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Fig. 1. AA stimulates intrinsic apoptosis in HBECs. (A). Time course of HBEC detachment in response to AA (50 μM). Data are mean ± SD (n = 3). ⁎⁎p b 0.01 and ⁎⁎⁎p b 0.001 vs control at each time interval. (B). Representative contrast microscopy images (×20). AA (50 μM; 2 h). (C). Fluorescence microscopy images of Hoechst-stained HBECs. Apoptotic nuclei are shown by arrows (×20). (D). Stimulation of HBECs with AA (50 μM) for the indicated 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 indicated concentrations for 2 h. Western blotting of caspase-3, cleaved caspase-3 fragments and β-actin. Representative blot and quantitative densitometry (n = 3). Data are mean ± SD (n = 3). ⁎⁎⁎p b 0.001 vs 0. (F). Stimulation with AA (50 μM) for the indicated times. Western blotting of full-length PARP (116 kDa), cleaved PARP (89 kDa) and β-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-treated HBECs were stained with MitoCapture and visualized by fluorescence microscopy (×40). Red specks in control cells indicate health mitochondria. Apoptotic cells (diffuse cytosolic green-orange fluorescence and intact plasma membrane) together with necrotic cell debris are present in AA-treated cells. (H). Western blotting of cytochrome c and β-actin. Stimulation with AA (50 μM) for the indicated time intervals. Representative blot and quantitative densitometry (n = 3). Data are mean ± SD. ⁎⁎⁎p b 0.001 vs control at 120 min. (I). HBECs were 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. Extracts were incubated with the labeled caspase-3 substrate, DEVD-pNA. Cleaved pNA was quantified by spectrophotometry. Data are mean ± SD (n = 3).⁎⁎⁎p b 0.001 vs control.
Please cite this article as: Evans, J., et al., Arachidonic acid induces brain endothelial cell apoptosis via p38-MAPK and intracellular calcium signaling, Microvasc. Res. (2014), http://dx.doi.org/10.1016/j.mvr.2014.04.011
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MAPKAP-2 antibodies were from Novus Biologicals (Littleton, CO). Hyperfilms were from Phenix Research Products (Candler, NC).
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Primary HBECs were cultured in EBM-2 tissue culture medium containing EGM-2 supplements, 10% Bac-Off and 2% fetal bovine serum at 37 °C in a humidified atmosphere of 95% air and 5% CO2 (Rush et al., 2007). Confluent cells exhibited a cobblestone appearance. Experiments were performed using cells at passages 3–12. Cells did not show changes in morphology over this passage range.
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HBECs were seeded onto attachment factor-coated 6-well plates in complete EBM-2 medium at 5 × 105/well. The following day, cells were stimulated with AA or vehicle in serum-free and growth factorfree EBM-2 medium, unless otherwise indicated. AA was dissolved in ethanol 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.
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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 addition of 1 ml lysing solution (PBS containing 1% sodium dodecyl sulfate) and kept overnight at − 80 °C. Following incubation for 30 min at 37 °C, the staining intensity was measured using a Bio Tek Uquant microplate reader at 620 nm. The absorbance of lysing solution from a tissue culture well containing no cells and stained exactly as described above, was used as background reading and subtracted from the absorbance of each sample.
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HBECs were plated in 24-well plates (5 × 104/well) in EBM-2 medium and incubated at 37 °C and 5% CO2. The following day, cells were stimulated with AA in complete EBM-2 medium for 48 h at 37 °C and 5% CO2. 3H-thymidine (2.5 μCi) was added to each well during the last 8 h of incubation. 3H-thymidine incorporation was assayed as previously described (Payner et al., 2006).
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Please cite this article as: Evans, J., et al., Arachidonic acid induces brain endothelial cell apoptosis via p38-MAPK and intracellular calcium signaling, Microvasc. Res. (2014), http://dx.doi.org/10.1016/j.mvr.2014.04.011
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HBECs (~ 40–50% confluent) were transfected under serum-free conditions with p38α (100 nM), p38β (100 nM), Hsp27 (200 nM) or control siRNA sequences using Lipofectamine RNAiMAX. Six hours later, EBM-2 medium containing growth factors and serum was added, and incubation was continued for 48 h. Cells were stimulated and subjected to Western blotting as indicated.
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Western blotting
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To detect apoptotic nuclei, HBECs grown on attachment factorcoated glass coverslips, were stimulated as indicated, fixed with 4% paraformaldehyde for 15 min at room temperature, and stained with Hoechst 33342 (10 μg/ml) for 30 min at room temperature. HBECs were washed in PBS, air dried, and visualized by a Leica DM RB fluorescence microscope. Mitochondrial apoptosis was detected with the
HBECs were plated in 6-well plates the day prior to stimulation. Following stimulation, detached and attached cells were collected together by centrifugation at 4 °C on ice-cold PBS containing 5 mM NaF and 1 mM Na3VO4. Proteins were extracted in lysis buffer (50 mM Tris–HCl pH 8.0, 150 mM NaCl, 5 mM EDTA, 10 mM NaF, 10 mM disodium pyrophosphate, 1 mM Na3VO4, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 10 μg/ml
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Caspase-3 activity was measured by quantification of p-nitroanilide (pNA) released from the labeled caspase-3 substrate DEVD-pNA (AspGlu-Val-Asp-pNA) following the manufacturer's instructions. Briefly, HBECs were plated in 6-well plates (5 × 105/well) the day prior to the experiments and stimulated as indicated. Attached and suspended HBECs were pooled, washed in PBS, lysed and subjected to centrifugation (10,000×g; 1 min). Lysates (25–50 μg) were incubated in a buffer containing DEVD-pNA (200 μM) and 10 mM DTT (37 °C; 2 h). pNA release was quantified by spectrophotometry at 405 nm. The absorbance of lysis buffer containing identical amounts of the DEVD-pNA was subtracted from the absorbance of each sample.
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Please cite this article as: Evans, J., et al., Arachidonic acid induces brain endothelial cell apoptosis via p38-MAPK and intracellular calcium signaling, Microvasc. Res. (2014), http://dx.doi.org/10.1016/j.mvr.2014.04.011
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Changes in intracellular calcium concentrations were measured using Fura-2 AM (Rizzo et al., 1999). HBECs were grown overnight on attachment factor-coated glass coverslips (22 × 9 mm). The following day, cells were loaded for 30 min in phenol-free HBSS containing 1 mM CaCl2 and 5 μM Fura-2 AM at 37 °C and 5% CO2. Coverslips were washed with HBSS and equilibrated for 10 min to allow de-esterification of Fura-2 AM. Calcium signals were detected via continuous recording at 37 °C in a PerkinElmer Luminescence Spectrometer LS50B at alternating excitation wavelengths of 340/380 nm. Emission fluorescence intensity was collected at 510 nm. Data are expressed as 340/380 Fura-2 fluorescence ratio.
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Data are means ± SD. Differences between groups were evaluated by ANOVA and Tukey post hoc (GraphPad Software). Differences between two groups were analyzed using the two-tailed t test. p b 0.05 was considered statistically significant.
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AA induces HBEC mitochondrial apoptosis
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To determine whether AA stimulates HBEC apoptosis, cells were seeded onto 6-well plates in complete EBM-2 medium at 5 × 105/well. HBECs formed a confluent monolayer 24 h after plating and were challenged with 50 μM AA in serum- and growth factor-free EBM-2 medium. HBECs were monitored microscopically for several time intervals. Treatment with AA significantly decreased HBEC attachment to the culture 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 180 min, respectively) compared with control (Fig. 1A). Representative photographs of HBECs following 2 h exposure to vehicle or AA are shown (Fig. 1B). AA-treated HBECs also displayed morphological features of apoptosis, including condensed and fragmented nuclei (Fig. 1C). These morphologic changes were accompanied by marked caspase-3 activation. As shown in Fig. 1D, AA stimulated cleavage of the inactive caspase-3 precursor into the active caspase-3 fragments in a time-dependent manner. Levels of active caspase-7 also increased with identical kinetics following treatment with 50 μM AA (not shown). AA stimulated caspase-3 activation in a dose-dependent manner. Caspase-3 activation was detected following stimulation with 35 μM and 50 μM AA, while 10 μM or 25 μM AA had no 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 experiments were 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, was examined. AA stimulated PARP cleavage into the 89 kDa active fragment with kinetics identical to those of caspase-3 (Fig. 1F). To determine whether AA induced caspase-3 activation via the intrinsic mitochondrial pathway of apoptosis, we assessed loss of
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mitochondrial membrane potential, an intracellular event that leads to the release of cytochrome c from mitochondria into the cytosol and subsequent caspase-9 and caspase-3 activation (Galluzzi et al., 2012). We used MitoCapture, a cationic dye that, in healthy cells, accumulates in mitochondria emitting red punctuated fluorescence, whereas, in cells in which the mitochondrial membrane potential is lost, the dye remains in the cytosol and emits green fluorescence. HBECs showed a diffuse cytosolic 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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aprotinin, 10 μg/ml leupeptin, 1 mM phenylmethylsulphonylfluoride), resolved by gel electrophoresis and transferred to nitrocellulose membranes. Immunoblotting was performed using the following primary antibodies: phospho- p38-MAPK (1:2000); p38-MAPK (1:500); phosphoHsp27 (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 appropriate horseradish-peroxidase-linked secondary antibodies, immunoreactive bands were visualized by enhanced chemiluminescence (ECL). Densitometry analyses of immunoreactive bands were performed using the UN-Scan-IT Software Program (Silk Scientific, Orem, UT).
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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, DEVDpNA. 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-DEVDFMK control vs vehicle control. (B). 3H-thymidine incorporation following 48 h stimulation 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 treated with AA (50 μM; 48 h) without or with apocynin. Data are mean ± SD of triplicate determinations of a representative experiment. ⁎⁎⁎p b 0.001 vs control.
Please cite this article as: Evans, J., et al., Arachidonic acid induces brain endothelial cell apoptosis via p38-MAPK and intracellular calcium signaling, Microvasc. Res. (2014), http://dx.doi.org/10.1016/j.mvr.2014.04.011
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To establish the functional consequences of AA-induced apoptosis, we measured HBEC proliferation in the presence or absence of ZDEVD-FMK, a cell-permeable irreversible inhibitor of caspase-3. Pretreatment with Z-DEVD-FMK blocked basal and AA-induced caspase-3 activity (Fig. 2A). Moreover, AA-induced inhibition of HBEC proliferation was partially rescued by Z-DEVD-FMK (57.8% ± 8.5 compared to cells treated with AA only). In contrast Z-DEVD-FMK failed to block
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AA-induced inhibition of HBEC proliferation is partially rescued by inhibition of caspase-3
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To test the hypothesis that p38-MAPK contributes to AA-induced HBEC apoptosis, cells were exposed to 50 μM AA or vehicle, and activation of p38-MAPK was assessed by immunoblotting using an antibody that recognizes activated p38-MAPK (phosphorylated at Tyr182 and Thr180 residues within its activation loop) (Cuadrado and Nebreda, 2010). As shown in Fig. 3A, p38-MAPK phosphorylation significantly increased in a time-dependent manner reaching maximal levels at 2 h stimulation with AA and declined thereafter. AA stimulated p38-MAPK phosphorylation at concentrations of 35–50 μM, whereas lower concentrations (10–25 μM) had no effect (Fig. 3B). However, similar to caspase3 activation, 25 μM AA stimulated p38-MAPK phosphorylation in subconfluent HBECs, whereas 5–10 μM AA had no effect (Fig. S1B). Myristic acid, stearic acid, palmitic acid, oleic acid or linoleic acid, each 50 μM concentrations, failed to stimulate p38-MAPK phosphorylation (Fig. 3C). An immunoreactive band migrating at approximately 50 kDa was 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 activate MAPKAP-2 and Hsp27, two downstream targets of p38-MAPK (Cuadrado and Nebreda, 2010).
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AA stimulates p38-MAPK, MAPKAP-2, and Hsp27 phosphorylation
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the antiproliferative effect of C2-ceramide (Fig. 2B). The antioxidant 307 apocynin also failed to prevent AA-induced inhibition of HBEC prolifer- 308 ation (Fig. 2C). 309
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To confirm activation of intrinsic apoptosis, we measured cytochrome c release following treatment with 50 μM AA for various time intervals (0.5, 1, 2 h). Cytochrome c release significantly increased following 2 h stimulation with AA (Fig. 1H). Taken together, these findings implicate the intrinsic pathway of apoptosis in AAinduced HBEC death. Next, the effect of AA on caspase-3 activity was compared to that of other fatty acids with various chain length and degree of unsaturation. AA (50 μM; 2 h) stimulated caspase-3 activity, as shown by increased pNa release from the labeled caspase-3 selective substrate (pNaDEVD), compared to control (Fig. 1I). In contrast, equimolar concentrations 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.
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Fig. 3. AA stimulated p38-MAPK, MAPKAP-2 and Hsp27 phosphorylation. (A, B). AA-induced time- and dose-dependent p38-MAPK activation detected by immunoblotting with an antibody that recognizes p38-MAPK phosphorylated at Tyr-182/Thr-180 (phospho-p38) followed by stripping and reprobing with an antibody against total p38-MAPK (p38). 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 point; ⁎⁎⁎p b 0.001 vs 0. (C). HBECs were 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. p38-MAPK activation was detected as described in A,B. Shown is a representative blot and quantitative densitometry (n = 6). Data are mean ± SD. ⁎⁎⁎p b 0.001 vs control. (D). Pretreatment of HBECs with SB203580 or SB202474 prior to stimulation with AA (50 μM; 2 h). Immunoblotting with antibodies against p-MAPKAP-2 or MAPKAP-2. Representative blot and quantitative densitometry (n = 4). Data are mean ± SD. ⁎⁎⁎p b 0.001 AA vs control; and ⁎⁎⁎p b 0.001 AA + SB203580 vs AA. (E). HBECs were treated as in D. Immunoblotting with antibodies against p-Hsp27 or Hsp27. Representative blot and quantitative densitometry (n = 3). Data are mean ± SD. ⁎⁎⁎p b 0.001 vs control; and ⁎⁎⁎p b 0.001 AA + SB203580 vs AA.
Please cite this article as: Evans, J., et al., Arachidonic acid induces brain endothelial cell apoptosis via p38-MAPK and intracellular calcium signaling, Microvasc. Res. (2014), http://dx.doi.org/10.1016/j.mvr.2014.04.011
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J. Evans et al. / Microvascular Research xxx (2014) xxx–xxx
Fig. 3 (continued).
Please cite this article as: Evans, J., et al., Arachidonic acid induces brain endothelial cell apoptosis via p38-MAPK and intracellular calcium signaling, Microvasc. Res. (2014), http://dx.doi.org/10.1016/j.mvr.2014.04.011
Pharmacological inhibition of p38-MAPK and Hsp27 knockdown abrogate 337 AA-induced apoptosis 338 To establish the contribution of p38-MAPK to AA-induced apoptosis, HBECs were pretreated with SB203580 or SB202474 prior to measurement of AA-induced caspase-3 activation. SB203580 abrogated AAinduced cleavage of caspase-3 and release of the labeled caspase-3
O
3000
C
O
74 24
74
20
24
35 20
B +S
SB
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A
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74 24 20
24
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20 A
+S
SB A
A
A
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SB
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20
35
74
80
80 35
A
ol tr
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0
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20 B
∗∗∗
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+S
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A 2.5
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on
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35
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80
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ol tr on C 3.0
∗∗∗
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74
E
0.04
2.5
C
∗∗∗
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N
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J6 76 57
57 A
A
+R
R
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W
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76
A
U ol
A
∗∗∗
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0
A
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E
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0.5
80
80
35
tr on C 3.0
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D
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C
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∗∗
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A
Caspase-3
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AA+SB202474
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SB202474
SB203580
AA
Control
A
on tr
336
C
334 335
A620 nm
332 333
AA significantly increased MAPKAP-2 phosphorylation at the p38-MAPKdependent site, Thr334 (Fig. 3D). Hsp27 phosphorylation at Ser82 also increased after AA stimulation (Fig. 3E). Pretreatment with SB203580, a p38-MAPK inhibitor, attenuated AA-induced MAPKAP-2 and Hsp27 phosphorylation (70.2% ± 11.3 and 76.3% ± 18.3, respectively). In contrast, SB202474, the inactive SB203580 analogue, had no effect (Figs. 3D,E).
Caspase-3 activity (pNA at 405 nm)
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J. Evans et al. / Microvascular Research xxx (2014) xxx–xxx
ol
8
Fig. 4. p38-MAPK and Hsp27 are required for AA-induced apoptosis. (A). Pretreatment of HBECs with 25 μM SB203580 or SB202474 prior to stimulation with AA (50 μM; 2 h). Western blotting of cleaved caspase-3 and β-actin. Representative blot and quantitative densitometry. Data are mean ± SD (n = 3). ⁎⁎⁎p b 0.001 AA vs control; and ⁎⁎p b 0.01 AA + SB203580 vs AA. (B). Caspase 3-activity monitored by the release of pNA. Data are mean ± SD (n = 3). ⁎⁎⁎p b 0.001 AA vs control; ⁎⁎⁎p b 0.001 AA + SB203580 vs AA. (C,D). Pretreatment of HBECs with SB203580, SB202474, or RWJ67657 (10 μM) prior to stimulation with AA (50 μM; 2 h). HBEC detachment was measured at 620 nm. Data are mean ± SD (n = 3). ⁎⁎⁎p b 0.001 AA vs control; ⁎⁎⁎p b 0.001 AA + SB203580 vs AA; ⁎⁎⁎p b 0.001 AA + RWJ67657 vs AA. (E). HBECs were transfected as indicated and stimulated with AA (50 μM; 2 h) or vehicle. Western blotting of caspase-3, Hsp27, or GAPDH. Representative blots and quantitative densitometry (n = 3). Data are mean ± SD. Upper panel: ⁎⁎⁎p b 0.001 control (siRNA Hsp27) vs control (siRNA control). Lower panel: ⁎⁎⁎p b 0.001 AA (siRNA control) vs control (siRNA control); and ⁎⁎⁎p b 0.001 AA (siRNA Hsp27) vs AA (siRNA control). (F). Cell detachment in HBECs transfected with siRNA Hsp27 or siRNA control. Data are mean ± SD (n = 4). ⁎⁎⁎p b 0.001 AA (siRNA control) vs control (siRNA control); and ⁎⁎⁎p b 0.001 AA (siRNA Hsp27) vs AA (siRNA control).
Please cite this article as: Evans, J., et al., Arachidonic acid induces brain endothelial cell apoptosis via p38-MAPK and intracellular calcium signaling, Microvasc. Res. (2014), http://dx.doi.org/10.1016/j.mvr.2014.04.011
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J. Evans et al. / Microvascular Research xxx (2014) xxx–xxx
E siRNA
Control
decreased following transfection with Hsp27 targeting siRNA sequences compared to control siRNA-transfected cells (76.4% ± 6.6 and 78.6% ± 3.0 control and AA-treated cells, respectively) (Fig. 4E). Moreover, Hsp27 knockdown markedly attenuated AA-induced caspase-3 activation (Fig. 4E) and cell detachment (Fig. 4F). These findings support the involvement of the p38-MAPK/MAPKAP-2/Hsp27 cascade in AA-induced HBEC apoptosis.
Hsp27
AA Hsp27
Because SB203580 and RWJ67657 selectively inhibit p38α and p38β isoforms of p38-MAPK (Lee et al., 1994; Wadsworth et al., 1999), the results shown above suggested that both or either p38α or p38β mediated AA-dependent apoptotic signaling. To distinguish between these possibilities, HBECs were transfected with siRNA sequences targeting p38α or p38β. Non-targeting siRNA sequences were used as controls. The degree of knockdown was evaluated 48 h later by immunoblotting using selective p38α or p38β antibodies. Caspase-3 activation and Hsp27 phosphorylation were monitored as readouts of p38α and p38β silencing after AA stimulation (50 μM; 2 h). As shown in Figs. 5A,B, p38α or p38β protein expression significantly decreased following transfection with p38α or p38β targeting siRNA compared to 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 markedly decreased AA-induced caspase-3 activation and Hsp27 phosphorylation. Importantly, the siRNA sequences used here selectively silenced p38α or p38β protein expression (Fig. 5C). Thus, p38α and p38β are both involved in transmitting AA-induced apoptotic signaling in HBECs.
O
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R O
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Hsp27/GAPDH ratio
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349 350 351 352 353 354 355
Requirement of p38α and p38β for AA-induced caspase-3 activation and 356 Hsp27 phosphorylation 357
Cleaved caspase-3
U
9
∗∗∗
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∗∗∗
0 siRNA Control
siRNA Hsp27
Fig. 4 (continued).
358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377
AA-induced intracellular calcium mobilization does not mediate p38-MAPK 378 phosphorylation but is involved in caspase-3 activation 379 To understand early signaling events involved in activation of p38MAPK by AA, we focused on calcium dynamics because intracellular calcium mobilization is required for AA-induced activation of the c-jun amino-terminal kinase (JNK) (Rizzo et al., 1999). Moreover, increases in intracellular calcium are implicated in regulation of apoptotic events in response to a variety of agonists, including AA (Penzo et al., 2004; Giorgi et al., 2008). Thus, we asked whether AA stimulated intracellular calcium release in HBECs. AA (50 μM) added to Fura 2-AM-labeled HBECs induced a rapid release of intracellular calcium (Fig. 6A). This release resulted from depletion of intracellular calcium stores and was followed by calcium influx upon addition of calcium in the extracellular medium (Fig. 6A). 2-APB, a membrane-permeable IP3 receptor antagonist (Maruyama et al., 1997), attenuated AA-dependent depletion of intracellular calcium stores and subsequent calcium entry (Fig. 6A). However, 2-APB did not attenuate AA-induced p38-MAPK activation, although abrogated AA-induced caspase-3 activation (Figs. 6B,C). Similarly, the IP3 receptor-selective antagonist, Xestospongin C (Gafni et al., 1997), failed to block p38-MAPK activation but significantly attenuated AA-induced caspase-3 activation (Figs. 6D,E). Thus, AA-induced p38MAPK activation occurs independently of intracellular calcium release. However, intracellular calcium release contributes to AA-induced caspase-3 activation.
380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401
AA-dependent p38-MAPK activation and intracellular calcium mobilization 402 converge to stimulate mitochondrial apoptosis 403 343 344 345 346 347 348
substrate, whereas SB202474 had no effect (Figs. 4A,B). SB203580 or RWJ67657, another selective p38-MAPK inhibitor, significantly decreased AA-induced HBEC detachment (78.9% ± 1.9 and 67.2% ± 22 respectively), whereas SB202474 did not (Figs. 4C,D). Next, we determined the consequence of Hsp27 knockdown on AA-induced caspase-3 activation. Hsp27 protein expression significantly
The findings that AA-induced caspase-3 activation was abrogated by inhibiting either intracellular calcium release or p38-MAPK activation suggest that both signaling events contribute to AA-dependent mitochondrial apoptosis. To test this possibility, we measured cytochrome c release in HBECs pretreated with 2-APB, SB203580, or RWJ67657.
Please cite this article as: Evans, J., et al., Arachidonic acid induces brain endothelial cell apoptosis via p38-MAPK and intracellular calcium signaling, Microvasc. Res. (2014), http://dx.doi.org/10.1016/j.mvr.2014.04.011
404 405 406 407 408
414 415
To determine whether AA itself or its conversion into eicosanoids was required for HBEC apoptosis, HBECs were stimulated for 2 h with AA (50 μM) or ETYA (50 μM), a non-metabolizable AA analogue that mimics 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-3 activity compared to control cells, while ETYA had no effect (Fig. S2A). Similarly, ETYA failed to disrupt HBEC monolayer integrity or activate p38-MAPK (not shown). To gain insights into the requirement of eicosanoids for AA-induced apoptosis, HBECs were preincubated with SC-560 (1–10 μM), a selective cyclooxygenase-1 (COX-1) inhibitor, prior to measuring AA-dependent caspase-3 activity. SC-560 did not prevent AA-induced caspase-3 activation (Fig. S2B). In contrast, pretreatment with NDGA, an antioxidant and pan-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 staurosporineinduced caspase-3 activation (not shown). Similarly, the antioxidant
The present study provides mechanistic insights into the previously reported effects of AA on breakdown of the BBB and, as summarized in Fig. 8, emphasizes a previously unrecognized signaling cooperation between p38-MAPK and intracellular calcium mobilization in mediating AA-induced brain endothelial cell apoptosis. While previous studies have highlighted the critical role of AA in activation of p38-MAPK in several cell types (Hii et al., 1998; Rizzo et al., 2002), to our knowledge this is the first study to demonstrate activation of p38-MAPK by AA in brain endothelial cells and its contribution to apoptosis and disruption of brain endothelial monolayer integrity. Our findings also support the involvement of MAPKAP-2 and Hsp27, two downstream p38-MAPK substrates, which have been implicated in p38-MAPK-induced apoptosis and disruption of monolayer integrity in pulmonary endothelial cells and human umbilical vein endothelial cells (Yang et al., 2010; Wolfson et al., 2011). Consistent with this possibility, pharmacological inhibition or siRNA-mediated knockdown of p38-MAPK attenuated AA-induced MAPKAK-2 and Hsp27 phosphorylation. The involvement of p38-MAPK/MAPKAP-2/Hsp27
435
D
A
E
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T
p38
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Cleaved caspase-3
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p-Hsp27
-actin
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AA
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0 siRNA Control Control
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∗∗
siRNA p38 Control AA
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p-Hsp27/ -actin (% of control)
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434
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R
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O
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N
420 421
Cleaved caspase-3/ -actin ratio (% of control)
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U
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Discussion
F
AA metabolism is required for AA-induced apoptosis of HBECs
O
413
apocynin did not prevent AA-induced apoptosis (not shown). These re- 431 sults suggest that lipooxygenase metabolites mediate AA-induced HBEC 432 apoptosis. 433
R O
411 412
AA-induced cytochrome c release was abrogated by 2-APB, SB203580, or RWJ67657 (Figs. 7A,B,C,D). Thus, intracellular calcium mobilization and p38-MAPK activation converge to stimulate AA-dependent apoptosis via the release of cytochrome c.
p-38 / -actin ratio (% of siRNA control)
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J. Evans et al. / Microvascular Research xxx (2014) xxx–xxx
P
10
∗∗
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∗
500 250
0
0 siRNA Control
siRNA p38
siRNA Control
siRNA p38
Fig. 5. p38α and p38β mediate AA-induced caspase-3 activation and phosphorylation of Hsp27. (A). HBECs were transfected as indicated and subjected to immunoblotting with antibodies against p38α, caspase-3, p-Hsp27, or β-actin. Representative blots and quantitative densitometry (n = 3). Data are mean ± SD. Upper right panel: ⁎p b 0.05 control (siRNA p38α) vs control (siRNA control); and ⁎p b 0.05 AA (siRNA p38α) vs AA (siRNA control). Lower left panel: ⁎⁎p b 0.01 AA (siRNA control) vs control (siRNA control); and ⁎⁎p b 0.01 AA (siRNA p38α) vs AA (siRNA control). Lower right panel: ⁎⁎p b 0.01 AA (siRNA control) vs control (siRNA control); and ⁎p b 0.05 AA (siRNAp38α) vs AA (siRNA control). (B). HBECs were transfected as indicated and subjected to immunoblotting with antibodies against p38β, caspase-3, p-Hsp27, or β-actin. Representative blots and quantitative densitometry (n = 3). Data are mean ± SD. Upper right panel: ⁎p b 0.05 control (siRNAp38β) vs control (siRNA control); ⁎p b 0.05 AA (siRNA p38β) vs AA (siRNA control). Lower left panel: ⁎⁎p b 0.01 AA (siRNA control) vs control (siRNA control); ⁎p b 0.05 AA (siRNA p38β) vs AA (siRNA control). Lower right panel: ⁎⁎p b 0.01 AA (siRNA control) vs control (siRNA control); and ⁎p b 0.05 AA (siRNA p38β) vs AA (siRNA control). (C). Membranes were stripped and reprobed with antibodies against p38α (upper), p38β (lower) or β-actin.
Please cite this article as: Evans, J., et al., Arachidonic acid induces brain endothelial cell apoptosis via p38-MAPK and intracellular calcium signaling, Microvasc. Res. (2014), http://dx.doi.org/10.1016/j.mvr.2014.04.011
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p38
Fig. 5 (continued).
in AA-induced apoptosis is further supported by abrogation of AAinduced caspase-3 activation and cell detachment following Hsp27 silencing. These findings support the role of Hsp27 as a crucial downstream component of the p38-MAPK apoptotic signaling cascade activated by AA. It should be emphasized that while Hsp27 phosphorylation contributes to p38-MAPK-dependent apoptosis and antiproliferative signaling in endothelial cells (Trott et al., 2009), in other cell types Hsp27 phosphorylation results in cytoprotection (Garrido et al., 2006; Stetler et al., 2012). The reasons for these divergent cellular outcomes are presently unclear, but Hsp27 interaction with distinct effectors, substrates, or binding partners among different cell types, could explain the opposing responses on cell survival. The p38-MAPK family includes four isoforms, p38α, p38β, p38γ, p38δ, which differ in tissue distribution profiles, substrate specificity and cellular outcomes (Cuadrado and Nebreda, 2010). p38α and p38β are the main p38-MAPK isoforms expressed in the brain (Jiang et al., 1996). Their contribution to brain endothelial cell apoptosis is not known. Our findings indicate that both isoforms contribute to AAinduced brain endothelial cell apoptosis. Consistent with this possibility, pharmacologic inhibitors of both p38α and p38β abrogated AA-induced phosphorylation of MAPKAP-2 and Hsp27, and caspase-3 activation. Moreover, silencing of either p38 isoform blocked AA-induced caspase-
U
459 460
AA
C
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p38
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-actin
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P
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p38
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-actin
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Cleaved caspase-3/ -actin ratio (% of control)
siRNA Control
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20
-actin
3 activation. The marked reduction of caspase-3 activation, following silencing of either p38α or p38β, further suggests a redundant and noncompensatory role of both isoforms in mediating AA-induced brain endothelial cell apoptosis. Our findings are also consistent with AA-induced mitochondrial apoptosis as shown by loss of mitochondrial membrane potential and release of cytochrome c. Inhibition of p38-MAPK abrogated AAinduced cytochrome c release, suggesting the involvement of p38MAPK in regulation of the intrinsic apoptotic pathway (Owens et al., 2009). However, we observed that AA induced caspase-3 and PARP cleavage as early as 1 h, whereas cytochrome c release was detected at 2 h. These findings indicate that AA-induced mitochondrial apoptosis accounts only in part for caspase-3 activation and suggest the coparticipation of the extrinsic apoptotic pathway. Perturbation of intracellular calcium levels has been reported to mediate AA-dependent apoptotic signals (Penzo et al., 2004; Fang et al., 2008). Moreover, we previously demonstrated that AA-induced calcium release from the endoplasmic reticulum was a critical component of the signaling pathway leading to JNK activation in bone marrow stromal cells (Rizzo et al., 1999). These observations led us to postulate the participation of intracellular calcium in the upstream signaling events leading to AA-induced p38-MAPK activation and subsequent apoptosis.
Please cite this article as: Evans, J., et al., Arachidonic acid induces brain endothelial cell apoptosis via p38-MAPK and intracellular calcium signaling, Microvasc. Res. (2014), http://dx.doi.org/10.1016/j.mvr.2014.04.011
475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496
12
2.51
A
while intracellular calcium mobilization is an important upstream sig- 500 nal for AA-induced mitochondrial apoptosis, is not required for p38- 501 MAPK activation. 502
AA 2-APB+AA
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∗
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on g AA +X e
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sp to Xe s
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Cleaved caspase-3/ -actin ratio (% of control)
Caspase-3 activity (pNA 405 nm)
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ol
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200
600
es
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po
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Phospho-p38/p38 ratio (% of control)
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p38
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Phospho-p38/p38 ratio (% of control)
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499
Pharmacological inhibition of IP3R-dependent intracellular calcium attenuated AA-dependent calcium signals, cytochrome c release and caspase-3 activation, but failed to prevent p38-MAPK activation. Thus,
U
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J. Evans et al. / Microvascular Research xxx (2014) xxx–xxx
Please cite this article as: Evans, J., et al., Arachidonic acid induces brain endothelial cell apoptosis via p38-MAPK and intracellular calcium signaling, Microvasc. Res. (2014), http://dx.doi.org/10.1016/j.mvr.2014.04.011
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J. Evans et al. / Microvascular Research xxx (2014) xxx–xxx
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AA
on
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507 508 509
In 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 reaching micromolar concentrations in response to experimental and pathological conditions (Rehncrona et al., 1979; Yoshida et al., 1980; Siesjo et al., 1982; Pilitsis et al., 2002; Artwohl et al., 2003; Farias et al., 2011). Importantly levels of AA in the CSF of patients with ischemic or hemorrhagic stroke
U
505 506
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R
Fig. 7. Requirement of p38-MAPK and intracellular calcium for AA-induced cytochrome c release. (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 and quantitative 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 SB203580 prior 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 AA vs control; and ⁎⁎p b 0.01 AA + SB203580 vs AA. (C). HBECs were preincubated with RWJ67657 prior 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 0.01 AA vs control; and ⁎p b 0.01 AA + RWJ67657 vs AA.
503 504
563
D
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E
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T
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+S
SB
B2
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03
35
58
80
AA
ol tr on C AA+RWJ67657
RWJ67657
AA
Control Cyt c
600
Conflicts of interest
O
400
0
C
510 511
F
∗∗∗
500
correlated with worse clinical outcome (Pilitsis et al., 2003). Thus, the concentrations of AA, negatively affecting HBEC survival in the present study, is within the range of those detected in vivo during brain injuries and vascular inflammation. Intriguingly, 25 μM AA stimulated caspase-3 and p38-MAPK activation in subconfluent HBECs but had no effect on confluent HBECs. These findings underscore the complex interactions between AA concentrations and endothelial monolayer density in regulating AA-dependent apoptotic responses. Because subconfluent endothelial cells release higher amounts of AA compared to confluent endothelial cells (Whatley et al., 1994), it is possible that the endogenous release of AA from subconfluent HBECs contributes to the proapoptotic effects of 25 μM AA added exogenously. Our studies also indicate that the proapoptotic effects of 50 μM AA are unlikely a result of a nonspecific lipid or detergent effect because the other fatty acids tested failed to stimulate p38-MAPK and caspase-3 activation when used at concentrations of 50 μM. Cultured brain endothelial cells largely produce PGE2 and monohydroxyeicosatetraenoic acids (HETEs) when exposed to high concentrations of AA (Moore et al., 1988). Moreover, lipooxygenase metabolites are implicated in mitochondrial apoptosis (Nazarewicz et al., 2007). In here we observed that ETYA failed to stimulate caspase-3 activity, while NDGA, at concentrations previously reported to suppress lipooxygenase activity and HETE production in cultured brain endothelial cells (Moore et al., 1988), blocked AA-induced brain endothelial cell apoptosis. The antioxidant properties of NDGA are not likely responsible for its rescue effects because the antioxidant apocynin did not prevent AA-induced inhibition of brain endothelial proliferation and disruption of monolayer integrity. On the other hand, SC-560, at concentrations reported to suppress prostaglandin production (Smith et al., 1988), had no effect on AA-induced caspase-3 activation. Collectively, these findings support the requirement of AA metabolic transformation via the lipooxygenase pathway for its proapoptotic effects on brain endothelial cells. A comprehensive assessment of the involved lipooxygenase metabolites and their contributions to each of the two signaling components of the apoptotic pathway activated by AA requires further study. In summary, the present study provides novel insights into the mechanisms of human brain endothelial cell injury following exposure to exogenous AA, a lipid mediator known to critically contribute to neuroinflammation and BBB destabilization. Our findings are consistent with a cooperative role of p38-MAPK/MAPKAP-2/Hsp27 activation and intracellular calcium mobilization in activating a mitochondrial apoptotic pathway responsible for AA-induced endothelial cell injury and death. Although additional studies are required to characterize further the upstream events leading to AAinduced p38-MAPK activation and intracellular calcium mobilization, as well as the involvement of additional members of the MAPK family, the signaling network elucidated in here may have important mechanistic implications in neurological disorders initiated, maintained, or exacerbated by inflammation of the cerebral endothelium. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.mvr.2014.04.011.
R O
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Cyt c/ -actin ratio (% of control)
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AA
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AA
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on
tr o
l
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Fig. 6. Intracellular calcium is not involved in AA-induced p38-MAPK phosphorylation but mediates AA-induced caspase-3 activation. (A). Fura-2 AM-labeled HBECs were pretreated with 2-APB or vehicle prior to AA (50 μM) stimulation (solid arrow) in the absence of extracellular Ca2+. Ca2+ added to the extracellular medium (dashed arrow). Data are expressed as 340/380 Fura-2 fluorescence ratio. Representative recording (n = 3). (B). Preincubation with 2-APB or vehicle and stimulation with AA. Immunoblotting of phospho-p38-MAPK and total p38MAPK. Representative blot and quantitative densitometry (n = 3). Data are mean ± SD (n = 3). ⁎⁎p b 0.01 AA vs control. (C). HBECs were stimulated as in B. Caspase-3 activity was monitored by the release of pNA. Data are mean ± SD (n = 3). ⁎⁎⁎p b 0.001 AA vs control; and ⁎⁎⁎p b 0.001 AA + 2-APB AA vs AA. (D). Preincubation with Xestospongin C or vehicle prior to stimulation with 50 μM AA. Immunoblotting of phospho-p38-MAPK and total p38-MAPK. Representative blots and quantitative densitometry (n = 3). Data are mean ± SD. ⁎⁎⁎p b 0.001 AA vs control. (E). Cells were treated as in D. Immunoblotting of caspase-3 and β-actin. Representative blots and quantitative densitometry (n = 4). Data are mean ± SD. ⁎⁎⁎p b 0.001 AA vs control; ⁎p b 0.05 AA + Xestospongin vs AA.
Please cite this article as: Evans, J., et al., Arachidonic acid induces brain endothelial cell apoptosis via p38-MAPK and intracellular calcium signaling, Microvasc. Res. (2014), http://dx.doi.org/10.1016/j.mvr.2014.04.011
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Arachidonic Acid
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Fig. 8. Proposed signaling pathway mediating AA-induced HBEC apoptosis. Stimulation of HBECs with AA triggers signaling events characterized by intracellular calcium mobilization and phosphorylation of p38-MAPK, MAPKAP-2 and Hsp27. Intracellular calcium and activated p38-MAPK/MAPKAP-2/Hsp27-dependent signaling converge into the mitochondria to induce caspase-3-dependent apoptosis and cell detachment. Conversion of AA by lipooxygenases (LOXs) is required for AA-dependent apoptotic signaling. The solid and dashed arrows indicate direct and indirect experimental evidence in support of the proposed targets/pathways, respectively. Pharmacological or siRNA-mediated manipulation of the proposed targets/pathways is indicated. Arrowheads: ►activation: │ inhibition.
Acknowledgments
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We thank Dr. Dino Rotondo for the critical suggestions, Dr. Rafat Siddiqui for the use of the spectrometer, Elaine Bammerline for the illustrations, Marilyn Michael Yurk for the editorial assistance, Jamie Heitzenrater and Andrea Doell for the technical assistance. This work was supported by Institutional Funds to M.T.R.
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