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Prepublished online as a Blood First Edition Paper on February 13, 2003; DOI 10.1182/blood-2002-12-3680. This Article Abstract Full Text (PDF) All Versions of this Article: 2002-12-3680v1 101/12/4797    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Dömötör, E. Articles by Zlokovic, B. V. Search for Related Content PubMed PubMed Citation Articles by Dömötör, E. Articles by Zlokovic, B. V. Related Collections Hemostasis, Thrombosis, and Vascular Biology Signal Transduction Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, 15 June 2003, Vol. 101, No. 12, pp. 4797-4801 HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY Activated protein C alters cytosolic calcium flux in human brain endothelium via binding to endothelial protein C receptor and activation of protease activated receptor-1 Eszter Dömötör, Omar Benzakour, John H. Griffin, David Yule, Kenji Fukudome, and Berislav V. Zlokovic From the Frank P. Smith Neurosurgical Research Laboratory, Department of Neurosurgery and Division of Neurovascular Biology, Center of Aging and Development Biology, University of Rochester Medical Center, Rochester, NY; Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA; Department of Physiology and Pharmacology, University of Rochester Medical Center, Rochester, NY; Department of Immunology, Saga Medical School, Saga, Japan; and Socratech Laboratories, Rochester, NY.    Abstract Top Abstract Introduction Materials and methods Results Discussion References   Activated protein C (APC) exerts endothelial protein C receptor (EPCR)–dependent neuroprotective effects in a brain focal ischemia model and direct cellular effects on human umbilical vein endothelial cells (HUVECs) via protease-activated receptor-1 (PAR-1). Because PAR receptors are expressed in brain endothelium and mediate intracellular calcium concentration ([Ca2+]i) signaling, we hypothesized that APC may regulate intracellular [Ca2+] flux in human brain endothelial cells (BECs) via EPCR and PAR-1. Primary cortical BECs derived from human autopsies (early passage) and HUVECs were used for [Ca2+]i imaging fluorometry. Cells were exposed for 1 minute to APC, protein C zymogen, or mutant Ser360Ala-APC, and [Ca2+]i was monitored in the presence or absence of antibodies against PAR-1, PAR-2, PAR-3, or EPCR. APC, but not protein C zymogen or the active site mutant Ser360Ala-APC, induced dose-dependent [Ca2+]i release in human BECs ([Ca2+]i max = 278.3 ± 19.5 nM; EC50 for APC = 0.23 ± 0.02 nM, n = 70 measurements). APC-induced [Ca2+]i signaling was abolished by a cleavage site blocking anti–PAR-1 antibody, whereas anti–PAR-2 and –PAR-3 antibodies were without effect. Antibody RCR252 that ablates APC binding to EPCR blocked APC-mediated [Ca2+]i signaling, whereas anti-EPCR antibody RCR92 that does not block APC binding did not abolish the APC-induced [Ca2+]i response. Experiments using HUVECs confirmed the findings for BECs. Thapsigargin inhibited the APC-induced [Ca2+]i signal, implicating the endoplasmic reticulum as a major source for the APC-induced [Ca2+]i release. These data suggest that APC regulates [Ca2+]i in human brain endothelium and in HUVECs by binding to EPCR and signaling via PAR-1.    Introduction Top Abstract Introduction Materials and methods Results Discussion References   Activated protein C (APC) exerts neuroprotective effects in a brain focal ischemia model that implies a potential therapeutic role for APC in the central nervous system (CNS) vascular and neurodegenerative disorders.1 APC reduces organ damage in animal models of sepsis and of ischemic injury, including stroke, and significantly reduces mortality in patients with severe sepsis.2-4 Prospective epidemiologic data suggest that plasma protein C levels are inversely related to incidence of ischemic stroke.5 APC exerts direct effects on human umbilical vein endothelial cells (HUVECs) via modulation of gene expression that may influence inflammation and apoptosis,6 and it can trigger intracellular signaling via activation of protease activated receptor-1 (PAR-1) and binding to endothelial protein C receptor (EPCR).7 It has been suggested that APC down-regulates nuclear factor B–dependent expression of adhesion molecules in HUVECs.6 PAR-1 and PAR-2 are present on systemic vascular endothelial cells8-10 and on brain microvascular endothelium.11 EPCR is also present on brain endothelial cells12 where it may augment activation of protein C by the thrombin-thrombomodulin complex,13,14 by providing a binding site for protein C on target cell membranes.7,12,15 Recently, it has been shown that both PAR-1 and EPCR are essential for the ability of APC to protect brain endothelium from ischemic injury via down-regulation of p53-dependent proapoptotic pathway.16 Cleavage and activation of PARs by extracellular proteases, however, are also capable of causing intracellular calcium concentration ([Ca2+]i) signaling.17,18 In this study we show that APC induces a potent [Ca2+]i signal in the brain endothelium through PAR-1 and that APC binding to EPCR is a prerequisite for this [Ca2+]i signal.    Materials and methods Top Abstract Introduction Materials and methods Results Discussion References   Materials Purified human plasma–derived APC, protein C zymogen, APC inactivated by heat, and recombinant human mutant Ser360Ala-APC lacking the active serine were prepared as described.19 No residual thrombin activity was detected in any of the APC preparations. All [Ca2+]i experiments were performed in normal HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer containing 135 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 1.2 mM MgCl2, 10 mM glucose, and 20 mM HEPES. RPMI 1640 was purchased from Cellgro (Herndon, VA). For calcium imaging we used the following antibodies: anti-EPCR RCR-292 and RCR-92,14 monoclonal antihuman APC-immunoglobulin G (IgG; C3),20 antihuman PAR-1 (N-19, H-111, and ATAP-2), antihuman PAR-2 (SAM-11), and antihuman PAR-3 (H-103) (Santa Cruz Biotechnology, Santa Cruz, CA). Leech-derived hirudin was obtained from Sigma (St Louis, MO). Isolation and characterization of human brain microvascular endothelial cells (BECs) Human brain capillaries were isolated from brain microvascular fragments from autopsies of neurologically healthy young individuals after trauma, and primary human BECs were characterized and cultured as described previously.21 After fluorescence-activated cell sorter (FACS) analysis using fluorescence-labeled acetylated low-density lipoprotein (Di-ac-LDL, ligand for endothelial scavenger receptor), cells were more than 98% positive for the endothelial markers, factor VIII–related antigen and CD105, and were negative for glial fibrillary acidic protein (GFAP; astrocytes), CD11b (macrophages/microglia), and -actin (smooth muscle cells). Early passage cells were plated on double-coated (polylysine, gelatin) glass coverslips for [Ca2+]i measurements. BEC cultures were kept in RPMI 1640–based medium with HEPES and L-glutamine, containing 20% fetal bovine serum, supplemented with 1% minimal essential medium (MEM) nonessential amino acids, 1% MEM vitamins, 5 U/mL heparin, 30 µg/mL endothelial cell growth supplement, 1 mM sodium pyruvate, and 100 U/mL penicillin/streptomycin. Microarray analysis using U95A Affymetrix (Santa Clara, CA) DNA chips on human BECs confirmed the presence of PAR-1, PAR-2, and PAR-3 mRNA as well as EPCR mRNA (Zlokovic et al, unpublished observation, July 2002). Cell cultures from human umbilical vein endothelium Primary HUVECs were obtained from ScienCell Research Laboratories (San Diego, CA). Cells were cultured with 17% fetal bovine serum-containing RPMI 1640 medium. Early passage cultures were used for experiments. Measurement of [Ca2+]i in single cells Cell cultures on polylysine-gelatin–coated glass coverslips were used on the second to third day after seeding when they reached 70% to 90% confluency. The intracellular calcium level of endothelial cells was imaged using a calcium-sensitive fluorescent dye, Fura-2 AM (Teflabs, Austin, TX). BECs and HUVECs were incubated with 4 µM Fura-2 AM in RPMI 1640 medium for 40 or 20 minutes, respectively. The coverslips were transferred to a perfusion chamber fitted to a stage of an inverted Nikon Diaphot 300 microscope and superfused with normal HEPES buffer for 15 minutes prior to experiments. All reagents were infused via a multitube perfusion system. [Ca2+]i was measured by digital image fluorescence microscopy (objective, Fluor 40/1.3; Nikon) using the Vision 4.0 Software from T.I.L.L. Photonics (Grafelfing, Germany). Excitation wavelengths were 340 and 380 nm, generated by a polychromator illumination system (T.I.L.L. Photonics). Fluorescence emission was monitored at 510 nm. The fluorescent images were collected with a charge-coupled device (CCD) camera (T.I.L.L. Photonics). A fluorescence ratio image (340/380 nm) was acquired in every 2 seconds. The experiments were carried out in HEPES buffer at room temperature (21°C). Fluorescence ratios were converted to free [Ca2+]i using the equation described by Grynkiewicz et al.22 The maximum (Rmax) and the minimum (Rmin) ratio value and the ratio of fluorescence for Ca2+-bound/Ca2+-free dye measured at 380 nm (Sf2/Sb2) were determined using an in vitro calibration method and were corrected for viscosity.23 Experimental results are shown as [Ca2+]i or increase of [Ca2+]i from basal as indicated in the figures. Data analysis and statistics Data analysis was performed with Sigmaplot 2000 (SPSS, Chicago, IL). Calibrated data were pooled and plotted as means ± SEMs of [Ca2+]i. In statistical calculations for comparison of groups Student paired t test was used. For the calculation of agonists potency, dose-response data were fitted to a single sigmoidal curve (Hill equation, 3 parameters).    Results Top Abstract Introduction Materials and methods Results Discussion References   Activated protein C triggers [Ca2+]i signal in human BEC APC administration (50 nM) to BECs for 60 seconds elevated [Ca2+]i from 83.1 nM (± 3.8) to 381.7 ± 26.8 nM (Figure 1Ai,C). Subsequent challenge with APC at 4 minutes after the first APC exposure did not result in any further [Ca2+]i rise, indicating desensitization. Stimulation with APC (10 nM) in the presence of hirudin (40 nM) did not alter the APC-induced [Ca2+]i transients (Figure 1Aii [PDB] ,C). To determine whether the APC-mediated [Ca2+]i signal was dependent on the presence of APC's active site Ser, the active site mutant Ser360Ala-APC was used. Neither recombinant mutant Ser360Ala-APC nor protein C zymogen produced a [Ca2+]i response (Figure 1Aiii-iv,C). Neutralizing anti-APC IgG (C3) antibody abolished APC's effect, and heat-inactivated APC was without effect (Figure 1Av-vi,C). Thus, the change in [Ca2+]i signal in BECs was caused by APC and required APC's active site Ser. The APC-evoked rise in [Ca2+]i was dose dependent (Figure 1B); the threshold APC concentration resulting in a detectable [Ca2+]i signal was between 0.02 and 0.10 nM, whereas the half-maximal response to APC (EC50) was at 0.23 ± 0.02 nM. View larger version (27K): [in this window] [in a new window]   Figure 1.. The effect of APC on [Ca2+]i in human brain endothelial cells. (A) [Ca2+]i was monitored in single cells when challenged with (i) APC (50 nM); (ii) APC (10 nM) and hirudin (40 nM); (iii) mutant Ser360Ala-APC (50 nM); (iv) zymogen protein C (50 nM); (v) APC (50 nM) pretreated for 5 minutes with monoclonal anti-APC IgG (C3) (1:1 molar ratio); and (vi) boiled APC (50 nM). Ten to 20 cells were measured in representative experiments. Positions of bars indicate the stimulation with the noted substances. (B) [Ca2+]i in single cells was measured in the presence of 0.02, 0.1, 0.5, 10, 50, and 100 nM APC administered for 60 seconds. Each set of curves is representative of 3 identical experiments. (C) Bars represent the mean [Ca2+]i ± SEs after exposure to APC (50 nM), APC (10 nM) and hirudin (40 nM), mutant Ser360Ala-APC (50 nM) (mut-APC), zymogen protein C (50 nM) (zym-PC), C3 antibody pretreated APC (50 nM) (C3 APC), and heat-inactivated APC (50 nM). (D) Dose-response relationship for the effect of APC (20 pM to 100 nM) on [Ca2+]i. [Ca2+]i changes from basal levels ± SEs were taken from 3 identical experiments. Data for log [APC] versus the change in [Ca2+]i were fitted to a sigmoidal curve (dashed line).   Effect of PAR blockage on APC-elicited [Ca2+]i signal To study the role of PARs, APC-induced [Ca2+]i responses in BECs were measured in the presence of different antihuman PAR antibodies at 25 µg/mL that were incubated with BECs for 15 minutes at 37°C prior to APC administration. A control antibody (N-19) against an N-terminal epitope of PAR-1 did not alter the APC-induced [Ca2+]i response (Figure 2Ai-ii,B) whereas H-111, a cleavage site-blocking anti–PAR-1 antibody, completely inhibited the APC-induced [Ca2+]i rise (Figure 2Aiii,B). SAM-11, an antibody raised against the PAR-2 cleavage site, did not block or diminish the APC-evoked [Ca2+]i signal (Figure 2Aiv,B). In studies using HUVECs, a different cleavage-blocking anti–PAR-1 antibody (ATAP-2) also abolished the APC-induced [Ca2+], whereas the anti–PAR-3 antibody (H-103) did not inhibit the APC signal (Figure 2C). HUVECs challenged with 150 nM APC responded with an elevation in [Ca2+]i of 161.2 ± 46.2 nM, comparable to BEC (Figure 2C). View larger version (10K): [in this window] [in a new window]   Figure 2.. The effect of pretreatment with antibodies against PAR-1, PAR-2, and EPCR on APC-induced [Ca2+]i signals. (A) [Ca2+]i was monitored in (i) BECs (thin traces) preincubated with antibodies, (ii) N-19, (iii) H-111, (iv) SAM-11, (v) RCR-252, and (vi) RCR-92 (25 µg/mL) at 37°C for 15 minutes. APC (100 nM) was added at the time indicated by arrows. (B) Relative magnitude of changes in [Ca2+]i in BECs in response to APC in which cells were intact or preincubated with antibodies N-19, H-111, SAM-11, RCR-252, or RCR-92. Bars represent normalized mean values of [Ca2+]i ± SEs in response to APC from 3 identical experiments. Normalization was done between paired experiments in which APC-induced changes in signals in antibody-pretreated BECs were compared with APC-induced [Ca2+]i changes performed in subsequent experiments without antibody preincubation. *P < .01. (C) [Ca2+]i increase in HUVECs in response to APC in which cells were intact or preincubated with monoclonal antibodies ATAP-2, H-103, RCR-252, or RCR-92. Bars indicate mean values of [Ca2+]i amplitudes ± SEs in response to APC from 5 identical experiments. *P < .01.   Effect of EPCR blockage on APC-induced [Ca2+]i signal Two antihuman EPCR antibodies, RCR-252 and RCR-92, were used to study the effect of blocking EPCR on APC-evoked [Ca2+]i response. RCR-252 (10 µg/mL), an antibody that blocks APC binding to human EPCR,14 abolished the APC-evoked [Ca2+]i response in both BECs and HUVECs (Figure 2Av,B-C). RCR-92 (10 µg/mL), an antibody raised against an epitope on human EPCR distant from the APC binding site,14 did not block the APC-induced [Ca2+]i signal in either BECs or HUVECs (Figure 2Avi [PDB] ,B-C). Role of intracellular calcium stores for APC-induced [Ca2+]i The amplitude of the APC-induced [Ca2+]i signal was assessed in BECs perfused with media containing either normal extracellular [Ca2+] (1.8 mM) or low extracellular [Ca2+] (50 nM). Perfusing cells with medium containing low Ca2+ for 30 seconds did not alter basal [Ca2+]i. APC treatment of BECs exposed to this condition resulted in a [Ca2+]i signal whose maximum amplitude was within the range of that obtained with BECs perfused with medium containing 1.8 mM Ca2+ (Figure 3A), suggesting that, for the initiation of [Ca2+]i signal in response to APC, normal extracellular Ca2+ concentrations are not required. To confirm the role of intracellular Ca2+ stores in the APC-induced [Ca2+]i signal, thapsigargin (1 µM), a specific blocker of the endoplasmic reticulum Ca2+ adenosine triphosphatase (ATPase),24 was used to deplete endoplasmic reticulum (ER) stores. Addition of thapsigargin for 1 minute gradually elevated [Ca2+]i, which subsequently decreased to basal values in 2 minutes (Figure 3B). Subsequent exposure of cells to APC did not result in a [Ca2+]i signal, confirming an essential role of endoplasmic reticulum [Ca2+]i stores for the APC-evoked [Ca2+]i signals (Figure 3B). View larger version (27K): [in this window] [in a new window]   Figure 3.. The effect of low extracellular [Ca2+]i (50 nM) and thapsigargin on the APC-induced [Ca2+]i signal. (A) BECs in media containing 1.8 mM or 50 nM Ca2+ were subjected to APC (100 nM) perfusions. Traces represent [Ca2+]i of 12 to 15 single cells from a representative experiment. Horizontal line indicates the time of APC addition. (B) Thapsigargin (1 µM; TG) was introduced to BEC culture as indicated by the horizontal line, and APC (100 nM) was administered for 60 seconds at the time indicated by the arrow. Traces show [Ca2+]i in single cells from representative experiments.      Discussion Top Abstract Introduction Materials and methods Results Discussion References   The ability of APC to induce direct cellular responses in endothelial cells6 may involve distinct signal transduction pathways, such as phosphorylation of mitogen-activated protein kinases (MAPKs).7 However, little is known about the wide variety of signaling mechanisms that could mediate APC's direct cellular effects. In this study we demonstrate that APC induces an intracellular [Ca2+]i signal in human BECs and in HUVECs. [Ca2+]i signaling in brain endothelium is of high importance and may lead to changes in cell morphology and blood-brain barrier (BBB) permeability through different mechanisms that include activation of myosin light-chain kinase, and/or conformational and biochemical changes in cytoskeletal proteins comprising the adherens junctions and tight junctions of the BBB.25-33 Brown and Davis noted33 that the exact mechanisms by which calcium flux in brain endothelial cells alters signaling cascades or regulates BBB function are as yet undefined. APC is neuroprotective in vivo1 and appears to protect stressed brain endothelial cells from hypoxic/ischemic damage in vitro and in vivo,18 but the implications of the intracellular [Ca2+]i signal elicited by APC under physiologic and pathophysiologic conditions remains to be elucidated. Because APC is generated from protein C by thrombin, we exposed endothelial cells to APC in the presence of hirudin, a highly specific blocker of thrombin. Hirudin did not abolish or reduce the APC-mediated [Ca2+]i signals, excluding the possibility that the APC-evoked [Ca2+]i was caused by contamination with thrombin.34 The proteolytic activity of APC was required for its effect on [Ca2+]i signaling because replacement of the active site Ser360 by Ala was without effect on [Ca2+]i. APC signaling in BECs and HUVECs was inhibited by PAR-1 cleavage-blocking antibodies (H-111 or ATAP-2), whereas pretreatment with a control anti–PAR-1 antibody (N-19) or with PAR-2 cleavage-blocking antibody (SAM-11) or anti–PAR-3 antibody (H-103) did not prevent the APC-induced [Ca2+]i rise. These results support the hypothesis that APC cleaves and activates PAR-1 that in turn mediates [Ca2+]i signaling in both human BECs and HUVECs, whereas PAR-2 or PAR-3 is not implicated in the APC-mediated [Ca2+]i response. These findings are consistent with the APC-induced effects on PAR-1 signaling in endothelial cells.7,16 Binding of APC to the endothelial receptor, EPCR is essential for the APC-induced MAPK phosphorylation7 and for the activation of APC's antiapoptotic pathway in stressed brain endothelial cells via down-regulation of proapoptotic transcription factor p53. To investigate the necessity of EPCR for APC-induced [Ca2+]i responses, BECs and HUVECs were pretreated with different antibodies against EPCR. An antibody that blocks APC binding to EPCR (RCR-252) blocked the APC-mediated [Ca2+]i signals, implicating binding of APC to EPCR for signaling (Figure 2). Although EPCR expression is relatively low in the brain endothelium,12 the data presented here show that binding of APC to EPCR is required for the APC-mediated [Ca2+]i signal in BECs. The source of intracellular calcium ions responsible for the flux in [Ca2+]i induced by APC was also investigated. When extracellular Ca2+ was lowered to 50 nM, APC induced a [Ca2+]i signal similar in amplitude to that evoked in the presence of 1.8 mM extracellular Ca2+, suggesting that normal extracellular Ca2+ concentrations are not required for the initiation of the APC-mediated [Ca2+]i signal.35 BEC cultures failed to respond to APC when normal endoplasmic reticulum Ca2+ stores had been depleted by thapsigargin, implying that the APC-induced [Ca2+]i flux is mobilized initially from the ER Ca2+ stores (Figure 3B). Because thapsigargin-induced Ca2+ release activates the 44-kDa and 42-kDa MAP kinases in human fibroblasts and epidermal cells,26,36 and, because both MAP kinases are rapidly phosphorylated by APC in HUVECs7 and human BECs,37 it is possible that intracellular [Ca2+] mobilization by APC from the ER results in rapid activation of MAP kinases by a calcium-dependent pathway.26,36 Because of the complexity and multiplicity of the signal transduction mechanisms that potentially contribute to cytoprotection by EPCR-dependent action of APC on PAR-1,6,7,16 future studies on [Ca2+]i regulation by APC in various cells are warranted. In summary, this study shows that human brain microvessel endothelium as well as human umbilical vein endothelium responds to APC with [Ca2+]i signaling, where PAR-1 but not PAR-2 and PAR-3 is involved and where APC binding to EPCR is required for this [Ca2+]i signaling process.    Acknowledgements   We thank Drs. Andrew Gale and José A. Fernández for the gifts of purified APC.    Footnotes   Submitted December 12, 2002; accepted February 5, 2003. Prepublished online as Blood First Edition Paper, February 13, 2003; DOI 10.1182/blood-2002-12-3680. Supported in part by grants HL63290 and HL52246 from the National Institutes of Health. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734. Reprints: Berislav V. Zlokovic, University of Rochester Medical Center, 601 Elmwood Ave, Box 645, Rochester, NY 14642; e-mail: berislav_zlokovic{at}urmc.rochester.edu.    References Top Abstract Introduction Materials and methods Results Discussion References   Shibata M, Kumar SR, Amar A, et al. Anti-inflammatory, antithrombotic, and neuroprotective effects of activated protein C in a murine model of focal ischemic stroke. 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