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Cudratricusxanthone A inhibits endothelial protein C receptor shedding in vitro and in vivo Cudratricusxanthone A inhibits endothelial protein C receptor shedding in vitro and in vivo
Ku, Sae-Kwang; Han, Min-Su; Jeong, Gil-Saeng; Bae, Jong-Sup
2014-01-02 00:00:00
MOLECULAR & CELLULAR BIOLOGY Animal Cells and Systems, 2014 Vol. 18, No. 1, 9–16, http://dx.doi.org/10.1080/19768354.2014.886619 a b c d* Sae-Kwang Ku , Min-Su Han , Gil-Saeng Jeong and Jong-Sup Bae Department of Anatomy and Histology, College of Korean Medicine, Daegu Haany University, Gyeongsan 712-715, Republic of Korea; Laboratory for Arthritis and Bone Biology, Fatima Research Institute, Daegu Fatima Hospital, Daegu 701-724, Republic of Korea; c d College of Pharmacy, Keimyung University, Daegu 704-701, Republic of Korea; CMRI, Research Institute of Pharmaceutical Sciences, College of Pharmacy, Kyungpook National University, Daegu 702-701, Republic of Korea (Received 2 November 2013; received in revised form 21 December 2013; accepted 20 January 2014) Increasing evidence has demonstrated that beyond its role in activation of protein C, endothelial cell protein C receptor (EPCR) is involved in vascular inflammation. EPCR activity is markedly changed by ectodomain cleavage and released as the soluble EPCR. EPCR can be shed from the cell surface, which is mediated by tumor necrosis factor-α converting enzyme (TACE). Cudratricusxanthone A (CTXA), a natural bioactive compound extracted from the roots of Cudrania tricuspidata Bureau, is known to possess hepatoprotective, antiproliferative, and anti-inflammatory activities. However, little is known about the effects of CTXA on EPCR shedding. Data from this study showed that CTXA induced potent inhibition of phorbol-12-myristate 13-acetate (PMA), tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and cecal ligation and puncture (CLP)– induced EPCR. CTXA also inhibited expression and activity of TACE induced by PMA in endothelial cells. In addition, treatment with CTXA resulted in reduced PMA-stimulated phosphorylation of p38, extracellular regulated kinases (ERK) 1/2, and c-Jun N-terminal kinase (JNK). These results demonstrate the potential of CTXA as an anti-sEPCR shedding reagent against PMA and CLP-mediated EPCR shedding. Keywords: cudratricusxanthone A; EPCR shedding; vascular inflammation Introduction sEPCR circulates in plasma, retaining its ability to bind both PC and APC, but does not enhance protein C The protein C (PC) anticoagulant pathway plays an activation (Kurosawa et al. 1997). It inhibits anticoagulant essential role in regulation of fibrin formation via proteo- activity of APC by formation of a complex, which does lytic degradation of the procoagulant cofactors such as not bind to phospholipid membranes (Liaw et al. 2000). factor Va and VIIIa by activated protein C (APC) (Fulcher sEPCR can be detected in plasma, resulting from shedding et al. 1984). PC circulates in plasma as a zymogen, which of membrane EPCR, with a plasma concentration of ap- is activated on the surface of endothelial cells by the proximately 100 ng/mL; high levels of sEPCR in systemic thrombin-thrombomodulin complex (Mosnier et al. 2007). inflammatory diseases have been reported (Kurosawa et al. Another endothelial cell–specific protein involved in the 1998). In vitro studies have reported a dramatic increase in PC anticoagulant pathway is the endothelial cell protein C EPCR shedding from the endothelium by a wide variety receptor (EPCR). EPCR is a type I transmembrane of inflammatory mediators (IL-1β,H O , and phorbol protein, which is highly expressed on the endothelium of 2 2 myristate acetate) and thrombin, and EPCR shedding is large vessels, while it is present at trace levels in most potentiated by the microtubule disrupting agent, nocoda- capillary beds (Mosnier et al. 2007). It binds PC on the zole (Xu et al. 2000). In addition, phosphorylation of endothelial cell surface, where it enhances the rate of PC p38MAPK, ERK1/2, and JNK was increased by stimula- activation (Mosnier et al. 2007), possibly by decreasing the K of PC for activation by the thrombin-thrombomo- tion with PMA (Leng et al. 2004; Menschikowski et al. 2009; Han et al. 2010), and activation of TACE occurs dulin complex (Mosnier et al. 2007). EPCR is found upon activation of ERK or p38 (Huovila et al. 2005; primarily on the endothelium of arteries and veins and Murphy 2008). exhibits high affinity toward PC and APC. This effective location of protein C may ensure efficacious activation of The common names of Cudrania tricuspidata Bureau protein C on the surface of large vessels. (Moraceae), a rich source of xanthones and flavonoids, A soluble form of EPCR (sEPCR) is generated in vitro have been investigated phytochemically and biologically through proteolytic cleavage by metalloprotease activity (Zou et al. 2004). C. tricuspidata is a species of deciduous inducible by thrombin and other inflammatory mediators trees that are widely cultivated in Korea, China, and (Xu et al. 2000); this process is known as shedding. Japan, some of which have anti-cancer, hepatoprotective, *Corresponding author. Email: baejs@knu.ac.kr © 2014 Korean Society for Integrative Biology 10 S.-K. Ku et al. anti-inflammatory, and anti-oxidant activities (Kim et al. the aqueous MeOH layer was subsequently extracted with 2007). This species has been used as a traditional Chinese CHCl . Finally, the 60% aqueous MeOH mixture was medicine for treatment of lumbago, hemoptysis, and evaporated in vacuo and partitioned between n-BuOH and contusion (Jeong et al. 2009). In addition, the root bark H O. A portion (5.3 g) of the CHCl soluble fraction (23.7 2 3 of C. tricuspidata has been used for treatment of g) was chromatographed using a silica gel column with gonorrhea, rheumatism, jaundice, and dysmenorrhea CHCl /MeOH/H O (9:1:0.1 → 4:1:0.1 → 6:4:1) to afford 3 2 (Hwang et al. 2007). During our continued search for six fractions (Fr. A-F). Fr. C (2.02 g) was subjected to natural products capable of modulating EPCR shedding, silica gel column chromatography (eluent: CHCl /MeOH, in this study, we found that the methanol extract of 40:1 → 10:1) to afford five fractions (Fr. C1-C5). Fr. C3 C. tricuspidata exhibited potent anti-EPCR shedding (190 mg) was purified by Sephadex LH-20 column activity in both cell and animal models. Activity-guided chromatography with CHCl /MeOH (15:1) to give cudra- purification of the extract of C. tricuspidata resulted in tricusxanthone A (34.6 mg). The structure of cudratricus- isolation of one active principle, which was subsequently xanthone A (Figure 1, CTXA) was identified by identified as cudratricusxanthone A (Figure 1A, CTXA). comparison of spectral data with those reported in the Cudratricusxanthone A exhibited various biological prop- literature (Tian et al. 2005) and dissolved in TBS (50 mM erties, including hepatoprotective, antiproliferative, and tris-buffered physiological saline solution pH 7.4) for anti-inflammatory activities (Tian et al. 2005; Kim et al. further experiments. 2007; Jeong et al. 2009). However, to the best of our knowledge, the effects of CTXA on EPCR shedding have Cell culture not yet been studied. Noting that sEPCR serves as a marker of vascular barrier integrity in vascular inflammat- Primary human umbilical vein endothelial cells (HUVECs) ory disease and that sEPCR is involved in the pathophy- were obtained from Cambrex Bio Science (Charles City, siology of sepsis (Kurosawa et al. 1998; Borgel et al. IA, USA) and maintained as previously described (Bae & 2007), we hypothesized that CTXA might possess anti- Rezaie 2011). HUVECs of passage numbers 3 or 4 were sEPCR shedding activity. Therefore, in the current study, used in the experiments. we investigated the effect of CTXA against PMA-induced EPCR shedding in human endothelial cells and in a cecal Animals and husbandry ligation and puncture (CLP) model of septicemia in mice. Male C57BL/6 mice (6–7 weeks old, weighting 18–20 g) were purchased from Orient Bio Co. (Sungnam, Kyung- Materials and methods KiDo, Korea) and used after a 12-day acclimatization period. Animals were housed five per polycarbonate cage Reagents under controlled conditions (20°C–25°C/RH 40–45%) sEPCR and TNF-α were purchased from Abnova (Tai- under a 12:12 h light/dark cycle and supplied a normal wan). Phorbol-12-myristate 13-acetate (PMA) and IL-1β rodent pellet diet and water ad libitum. All animals were were purchased from Sigma (St. Louis, MO, USA). treated in accordance with the Guidelines for the Care and Use of Laboratory Animals issued by Kyungpook National University (IRB No. KNU 2012–13). Plant material, extraction, isolation, and high- performance liquid chromatography (HPLC) Dried and pulverized root bark of C. tricuspidata (2 kg) Enzyme-linked immunosorbent assay (ELISA) for was extracted twice with hot MeOH (2 × 20 L) for 2 h. membrane EPCR expression The dried MeOH extract (86 g) was partitioned between Modified whole-cell ELISA was performed as previously equal volumes of n-hexane and 60% aqueous MeOH, and described for determination of expression levels of EPCR on HUVECs (Kim et al. 2011). Briefly, confluent mono- layers of HUVECs were treated with or without CTXA for 6 h, followed by treatment with PMA, tumor necrosis factor (TNF)-α, or interleukin (IL)-1β for 1 h. Media were then removed and cells were washed with PBS and fixed with 50 µL of 1% paraformaldehyde for 15 minutes at room temperature. After washing, 100 µL of EPCR antibodies (Abnova) was added, and, 1 h (37°C, 5% CO ) later, cells were washed three times, followed by treatment with 100 µL of 1:2000 peroxidase-conjugated Figure 1. Structure of cudratricusxanthone A. anti-rabbit IgG antibodies (Sigma) for 1 h. Cells were then Animal Cells and Systems 11 washed three times and developed using o-phenylenedia- TACE activity assay mine substrate (Sigma). Colorimetric analysis was per- For the TACE activity assay, a commercially available formed by measurement of absorbance at 490 nm. All TACE activity kit (Innozyme TACE activity assay kit, measurements were performed in triplicate wells. EMD Millipore, Billerica, MA, USA) was used as described previously (Miller et al. 2013). Competitive enzyme-linked immunosorbent assay (Competitive ELISA) for sEPCR and TACE Cecal ligation and puncture (CLP) Ninety-six-well plastic flat microtiter plates (Corning, NY, For induction of sepsis, male mice were anesthetized with USA) were coated with sEPCR or TACE protein in 20 3% isoflurane (Forane®, Choongwae Pharma. Corp., mM carbonate–bicarbonate buffer (pH 9.6) containing Seoul, Korea). The CLP-induced sepsis model was 0.02% sodium azide overnight at 4°C. Lyophilized culture prepared as previously described (Wang et al. 2004). In media were prepared for sEPCR and total cell lysates were brief, a 2-cm midline incision was made in order to allow prepared for TACE using lysis buffer containing (mM): exposure of the cecum and adjoining intestine. The cecum Tris–HCl (20) pH 7.5, EGTA (0.5), EDTA (2), dithio- was then tightly ligated using a 3.0-silk suture at 5.0 mm threitol (2), p-methylsulfonyl fluoride (0.5), and 10 µg/mL from the cecal tip, punctured once with a 22-gauge needle, leupeptin. Plates were then rinsed three times in PBS- gently squeezed in order to extrude a small amount of 0.05% Tween 20 (PBS–T) and kept at 4°C. Prepared feces and returned to the peritoneal cavity. The laparo- samples from cell culture media and mouse plasma for tomy site was then stitched with 4.0-silk. In sham sEPCR or from cell lysates for TACE were pre-incubated controls, the cecum was exposed but not ligated or with anti-EPCR antibodies (1:500, Abnova) or anti-TACE punctured and then returned to the abdominal cavity. antibodies (1:500, Santa Cruz) in 96-well plastic round microtiter plates for 90 min at 37°C, transferred to pre- This protocol was approved in advance by the Animal coated plates, and incubated for 30 min at room temper- Care Committee at Kyungpook National University (IRB ature. Plates were then rinsed three times with PBS-T, No. KNU 2012–13). incubated for 90 min at room temperature with perox- idase-conjugated anti-rabbit or anti-goat IgG antibodies (1:2000, Amersham Pharmacia Biotech), rinsed three Immunohistochemistry times in PBS-T, and incubated for 60 min at room For analysis of the expression pattern of EPCR, aortas temperature in the dark with 200 µl of substrate solution from CLP-induced septic (Day 4) and sham-operated mice (100 µg/mL o-phenylenediamine containing 0.003% were removed and fixed in 4% formaldehyde solution H O ). The reaction was then stopped by addition of 50 2 2 (Junsei, Japan) in PBS for 20 h at 4°C. Following fixation, µl of 8N H SO , and absorbance was read at 490 nm. 2 4 the aortas were dehydrated through an ethanol series, embedded in paraffin, and cut into 3-µm sections. Deparaffinized sections were quenched in 3% H O in 2 2 Western blotting methanol, washed in PBS, placed in boiled 1 mM Tris Confluent monolayers of HUVECs were treated with or solution (pH 9.0), supplemented with 0.5 mM EGTA without CTXA for 6 h, followed by treatment with PMA solution in order to reveal the antigens, and blocked in for 1 h. Total cell extracts were prepared by lysing the PBS, supplemented with 1% bovine serum albumin, 0.2% cells, and protein concentration was determined using gelatin, and 0.05% saponin for 1 h at RT. Sections were Bradford assay. Equal amounts of protein were separated incubated with anti-EPCR antibody (Abcam) diluted by SDS-PAGE (10%) and electroblotted overnight onto 1:500 in PBS and supplemented with 0.1% BSA and immobilon membranes (Millipore, Billerica, MA, USA). 0.3% Triton X 100 for 16 h at 4°C in a humidified The membranes were blocked for 1 h with 5% low-fat chamber. After washing in PBS, supplemented with 0.1% milk-powder TBS (50 mM Tris-HCl, pH 7.5, 150 mM BSA, 0.2% gelatin, and 0.05% saponin, the sections were NaCl) containing 0.05% Tween 20, followed by incuba- incubated with peroxidase-conjugated anti-rabbit IgG tion with anti-phospho-p38 or total p38 (1:100, Abnova), antibody (DAKO, Glostrup, Denmark) for 1 h at RT and anti-phospho-ERK1/2, total ERK1/2, anti-phospho-JNK then developed using the Liquid DAB+ Substrate-Chro- or total JNK (1:1000, Cell Signaling Tech.), TACE anti- mogen System (DAKO). Counterstaining was performed EPCR antibodies (1:500), or anti-TACE antibodies (Santa with 0.5% methyl green in ddH2O. Non-immune rabbit Cruz, Dallas, TX, USA; diluted 1:500 in PBS-T) at room IgG (DAKO, at the same concentration instead of EPCR temperature for 1.5 h, followed by incubation with antibody) and anti-CD31 antibody (1:200, Abcam) were horseradish peroxidase-conjugated secondary antibody used as negative and positive control for immunohisto- and ECL-detection according to the manufacturer’s instructions. chemistry, respectively. 12 S.-K. Ku et al. ELISA for total and phospho p-38MAPK, ERK1/2, Effects of CTXA on PMA-stimulated expression and and JNK activity of TACE HUVECs were cultured in 96-well microplates for quant- A previous study reported that PMA-stimulated EPCR itative determination of p38MAPK, ERK1/2, and JNK shedding is mediated by TNF-α converting enzyme/ ADAM17 (TACE) (Qu et al. 2007). In order to determine phosphorylation. On the day that experiments were per- whether CTXA could inhibit stimulation of TACE expres- formed, culture medium was replaced with serum-free sion and activity, endothelial cells were pretreated with growth medium. Cells were then treated with or without increasing concentrations of CTXA for 6 h, followed by CTXA for 6 h, followed by treatment with PMA (1 µM) stimulation with 1 µM PMA for 1 h. Data showed that for 1 h. Activation of p38MAPK, ERK 1/2, and JNK CTXA inhibited expression (Figure 3A and B) and was quantified in nuclear lysates using ELISA kits for activity of TACE (Figure 3C) induced by PMA in total/phosphorylated p38MAPK (Invitrogen, for total endothelial cells. p38MAPK or Cell Signaling Technology, for phosphory- lated-p38MAPK), total/phospho ERK1/2, and JNK (R&D Systems, Minneapolis, MN, USA) according to the Effect of CTXA on CLP-induced EPCR shedding manufacturer’s instructions. In experiments to confirm the inhibitory effects of CTXA on EPCR shedding in vivo, a CLP mouse model was used because it closely resembles human sepsis (Yang et al. Statistical analysis 2004; Buras et al. 2005). At 24 h after the operation, Results are expressed as mean ± standard error of the animals manifested signs of sepsis, including shivering, bristled hair, and weakness. To examine the trend in mean (SEM) of at least three independent experiments. mortality after CLP, groups of septic and sham-operated Statistical significance was determined using two-way mice were monitored for survival for 120 h (5 days). ANOVA of variance (SPSS, version 14.0, SPSS Science, CLP resulted in a survival rate of 20% 96 h after CLP Chicago, IL, USA) and p values of <0.05 (p < 0.05) were (Figure 4A). In CLP-induced septic mice, immunohisto- considered significant. chemical analysis showed decreased expression of mem- brane EPCR compared to normal control (Figure 4B). Administration of CTXA at a single dose (7.93 µg, 12 h Results and discussion after CLP) did not prevent CLP-induced EPCR shedding Effect of CTXA on PMA, TNF-α,orIL-1β-induced (Figure 4C); therefore, it was administered twice (28.4 µg EPCR shedding per mouse, once 12 h, then 50 h after CLP), resulting in a Previous studies have reported on stimulation of EPCR decrease in EPCR shedding (Figure 4D). Assuming an shedding from HUVECs by PMA (Qu et al. 2006, 2007). average body weight of 20 g and an average blood volume of 2 mL, the amounts of CTXA produced a concentration In agreement with previous results, results of our study of approximately 10 µM in peripheral blood. This marked showed that PMA from 1 µM fully stimulated benefit achieved by administration of CTXA suggested EPCR shedding from HUVECs (Figure 2A) and mem- that inhibition of EPCR shedding provides a therapeutic brane EPCR on HUVECs showed a dose-dependent strategy for management of severe vascular diseases. decrease by PMA (Figure 2B). EPCR shedding by TNF-α or IL-1β also showed an increase (Figure 2C and D), in agreement with a previous study (Menschi- Effects of CTXA on PMA-stimulated phosphorylation of kowski et al. 2009). p38MAPK, ERK1/2, and JNK To investigate the effect of CTXA on PMA-mediated Previous studies have reported involvement of p38MAPK, EPCR shedding, endothelial cells were pretreated with ERK1/2, and JNK in cytokine-induced EPCR shedding, increasing concentrations of CTXA for 6 h, followed by and increased phosphorylation of p38MAPK, ERK1/2, and stimulation with 1 µM PMA for 1 h. As shown in Figure JNK was known to occur by stimulation with PMA (Leng 2A and B, treatment with CTXA resulted in inhibition of et al. 2004; Menschikowski et al. 2009; Han et al. 2010). EPCR shedding induced by PMA in endothelial cells, Therefore, in order to determine the molecular mechanisms with an optimal effect at 5–10 µM. As shown in Figure of suppression of PMA-induced EPCR shedding by 2A, treatment with CTXA alone (10 uM) did not have CTXA, the effects of CTXA on PMA-stimulated phos- an effect on shedding of EPCR. Therefore, CTXA alone phorylation of p38MAPK, ERK1/2, and JNK were tested. (10 uM) did not affect the expression of membrane bound As shown in Figure 5, treatment with CTXA resulted EPCR (Figure 2B). CTXA also induced suppression of in reduction of PMA-stimulated phosphorylation of TNF-α or IL-1β-mediated EPCR shedding in HUVECs p38MAPK (Figure 5A), ERK1/2 (Figure 5B), and JNK (Figure 2C and D). (Figure 5C). These results were confirmed by western Animal Cells and Systems 13 Figure 2. Effect of CTXA on PMA, TNF-α, and IL-1β-induced EPCR shedding. The effects of various concentrations of CTXA on PMA (1 µM, 1 h)-induced EPCR shedding were monitored by measurement of sEPCR (A) or membrane EPCR on HUVECs (B). (C and D) The same as A and B, except that HUVECs were incubated with TNF-α (25 ng/mL for 1 h, white bar) or IL-1β (25 ng/mL for 1 h, black bar). Results indicate the mean ± SEM of three separate experiments. *p < 0.05 or **p < 0.01 vs. PMA alone (A, B) or TNF-α/IL-1β alone (C, D). Figure 3. Effect of CTXA on PMA-stimulated expression and activity of TACE. The effects of various concentrations of CTXA on PMA (1 µM, 1 h)-induced expression (A, B) or activity (C) of TACE were monitored by measurement using TACE ELISA (A), western blotting (B), or TACE activity assay Kit (C). All results indicate the mean ± SEM of three separate experiments. *p < 0.05 or **p < 0.01 vs. PMA alone. 14 S.-K. Ku et al. Figure 4. Effect of CTXA on CLP-induced EPCR shedding. (A) Male C57BL/6 mice underwent CLP and animal survival was monitored every 6 h after CLP for 126 h. (B) Immunohistochemical staining of blood vessel for membrane EPCR from CLP-operated mice (4 days after CLP), as indicated in the text. Staining results from sham-operated mice are compared. Results are representative of three to six stainings from two independent experiments per condition. (C–D) Serum was obtained from sham-operated (white bar) or CLP-induced septic mice (gray bar) on the indicated day after CLP surgery (n = 5) on different days for different mice. Or, CTXA was administered once (B, black bar, 7.93 µg per mouse via IV, once 12 h after CLP) or twice (C, black bar, 7.93 µg per mouse via IV, once 12 h, then 50 h after CLP) on different days for different mice. EPCR shedding was then monitored by measurement of sEPCR. All results indicate the mean ± SEM of three separate experiments. **p < 0.01 vs. CLP alone. blotting (Figure 5D). A panel of pharmacological inhibitors expression of TACE by CTXA, we investigated involve- of MAP kinases was used to confirm involvement of ment of MAPK signaling pathways in PMA-stimulated MAPK in EPCR shedding. As shown in Figure 5E, distinct condition. MAPKs comprise a family of highly conserved attenuation of sEPCR release in HUVECs was observed serine/threonine protein kinases, which are believed to after treatments with PD-98059 as a pharmacological have key roles in mediation of inflammation (Thalhamer inhibitor of ERK 1/2 activity, SB-203580 as an inhibitor et al. 2008). Three major classes of MAPKs are repre- of p38MAPK, and SP-600125 as an inhibitor of JNK. sented by ERK 1/2 and the two stress-activated protein Increased phosphorylation of p38MAPK, ERK1/2, kinase families, JNK and p38 MAPK. As shown in and JNK is known to occur by stimulation with PMA Figures 2 and 5, PMA stimulated expression of TACE (Leng et al. 2004; Menschikowski et al. 2009; Han et al. and phosphorylation of p38 MAPK, ERK1/2, JNK, and 2010), and activation of TACE occurs upon activation of CTXA inhibited these responses by PMA. Therefore, ERK or p38 MAPK (Huovila et al. 2005; Murphy 2008); CTXA inhibited shedding of EPCR by inhibition of therefore, in order to define the processes responsible for PMA-stimulated expression of TACE and activation of inhibition of PMA-stimulated shedding of EPCR and MAPKs. Animal Cells and Systems 15 Figure 5. Effect of CTXA on PMA-induced phosphorylation of p38MAPK, ERK1/2, and JNK. PMA (1 µM, 1 h)-mediated phosphorylation of phospho-p38MAPK (A, white bar) or total p38MAPK (A, black bar), phospho-ERK1/2 (B, white bar) or total ERK1/2 (B, black bar), and phospho-JNK (C, white bar) or total JNK (C, black bar) were analyzed after treatment of cells with the indicated concentrations of CTXA. Results are expressed as fold increase over control values. (D) Western blotting for phosphorylated or total p38, ERK1/2, or JNK. (E) Cells were pre-incubated with 50 µM PD-98059, 10 µM SB-203580, or 20 µM SP-600125 as indicated for 30 min and thereafter exposed to PMA at a final concentration of 1 µM for an additional 1 h. EPCR shedding was monitored by measurement of sEPCR ELISA. All results indicate the mean ± SEM of three separate experiments. *p < 0.05 or **p < 0.01 vs. PMA alone. Jeong et al. (2009) previously reported that the active 1/2, and JNK. A large number of cell membrane bound compound, CTXA, an important component of the roots proteins were shed by TACE, and, in this study, EPCR of C. tricuspidata Bureau, exhibited anti-inflammatory shedding was mediated by TACE, which is inhibited by responses in lipopolysaccharide (LPS)-treated cells. They CTXA. Although the use of CTXA for therapeutic demonstrated that CTXA inhibited expression of cycloox- purposes could have non-specific effects, the data pre- ygenase-2 (COX-2) and inducible nitric oxide (NO) sented in this study provide novel information on the role synthase (iNOS), thereby reducing production of COX- of CTXA in EPCR shedding. Therefore, our findings 2–derived prostaglandin E2 (PGE2) and iNOS-derived indicate the potential of CTXA as a candidate for NO in LPS-stimulated macrophages (Jeong et al. 2009). treatment of severe vascular inflammatory diseases, such Similarly, CTXA suppressed production of TNF-α and IL- as sepsis and septic shock. 1β and inhibited the induced phosphorylation and degradation of IκB-α as well as the LPS-induced increase Acknowledgement in p65 in the nuclear fraction of macrophages. This study was supported by the National Research Foundation Noting involvement of EPCR shedding in the patho- of Korea (NRF) funded by the Korean government [MSIP] physiological pathway in vascular inflammatory diseases, [Grant No. 2013-067053]. the hypothesis that CTXA could be used as a candidate therapeutic for treatment of vascular inflammatory dis- References eases has been strengthened by the finding of a previous Bae JS, Rezaie AR. 2011. 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