CD200Fc attenuates inflammatory responses and maintains barrier function by suppressing NF-κB pathway in cigarette smoke extract induced endothelial cells

Biomedicine & Pharmacotherapy 84 (2016) 714–721

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CD200Fc attenuates inflammatory responses and maintains barrier function by suppressing NF-kB pathway in cigarette smoke extract induced endothelial cells Junwei Xua,1, Lu Lub,1, Jing Lua , Jihui Xiaa , Hongjin Lua , Lin Yanga , Wensheng Xiaa,* , Shihai Shena,* a b

Deparment of Vasculocardiology, Taizhou Second People’s Hospital, Taizhou, Jiangsu, PR China Department of Medical Imaging, Jiangsu Traditional Chinese Medical Hospital, Nanjing, Jiangsu, PR China

A R T I C L E I N F O

A B S T R A C T

Article history: Received 24 August 2016 Received in revised form 22 September 2016 Accepted 23 September 2016

Background: Recent evidence suggests that CD200 fusion protein (CD200Fc), a CD200R1 agonist may attenuate inflammatory responses in autoimmune diseases and neuro-degeneration. While, little is known about the function of CD200Fc in cigarette smoke extract (CSE)-induced mouse Cardiac Microvascular Endothelial Cells (mCMECs). The present study was designed to elucidate the effects of CD200Fc on CSE-induced vascular endothelial barrier (VEB) dysfunction and inflammatory responses, which is a highly clinically relevant model of smoking related cardiovascular diseases. Methods: mCMECs were pre-treated with 1, 10 and 100 mg/ml CD200Fc for 24 h respectively, and then treated with 250 mg/ml CSE for different times (24 h or 120 min). The transepithelial electrical resistance (TEER) and transport of fluorescent markers were used to measure VEB function in CSE-induced mCMECs. Western blot and immunofluorescent staining analysis were used to detect the expression of tight junction proteins, such as Zona Occludens-1 (ZO-1) and Claudin-1 in CSE-induced mCMECs. We measured the expression of pro-inflammatory cytokines in CSE-induced mCMECs by using ELISA and RTPCR. In addition, the NF-kB activity in CSE-induced mCMECs were investigated by using nuclear/cytosol fractionation and western blot analysis. Results: In vitro treatment with CSE increased the transport of fluorescent markers and decreased TEER levels in mCMECs, respectively, which were attenuated by CD200Fc (10 and 100 mg/ml) pretreatment. The CSE-induced up-regulation of pro-inflammatory cytokines such as Cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), platelet endothelial cell adhesion molecule-1 (PECAM-1), vascular cell adhesion molecule-1 (ICAM-1), Prostaglandin E2 (PGE2), tumor necrosis factor-a (TNF-a), interleukin-6 (IL-6) and IL-8 in mCMECs was also abrogated by CD200Fc (10 and 100 mg/ml) pretreatment. CD200Fc also inhibited CSE-induced nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) activation in mCMECs, such as inhibition of its DNA binding activity, phosphorylated expression, and translocation to nucleus. Conclusion: Thus, CD200Fc exert anti-inflammatory effect and protect VEB function in CSE-induced mCMECs. The vasoprotective effects of CD200Fc may be specifically beneficial in pathophysiological conditions associated with smoking related cardiovascular diseases. ã 2016 Elsevier Masson SAS. All rights reserved.

Keywords: CD200Fc Inflammatory responses Vascular endothelial barrier Cigarette smoke extract NF-kB pathway Mouse cardiac microvascular endothelial cells

1. Introduction Atherosclerosis is the underlying cause of several cardiovascular diseases (CVDs)-associated complications, including myocardial infarction, heart failure, and stroke [1,2]. A large number of risk

* Corresponding authors. E-mail addresses: [email protected] (W. Xia), [email protected] (S. Shen). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.biopha.2016.09.093 0753-3322/ã 2016 Elsevier Masson SAS. All rights reserved.

factors, physicochemical interactions, cell types, and biological processes were involved in the complexity of CVDs. Cigarette smoking is probably the most complex and the least understood among the risk factors for CVDs [3–5]. To date, apart from a handful of candidate compounds, the relevance of most compounds in cigarette smoke in CVDs initiation, progression, and cardiovascular outcome has not been studied [5,6]. Thus, it seems crucial to investigate the link between proatherogenic cellular and molecular effects of cigarette smoke and initiation of CVDs.

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It is well recognized that cigarette smoking initiates the early stages of CVDs via inducing persistent inflammatory responses and vascular endothelial barrier (VEB) dysfunction, and disrupting vascular homeostasis [7]. VEB dysfunction is an early hallmark of atherosclerosis [3,7]. There is strong evidence suggesting that nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB) is a key cigarette smoking-sensitive event associated with vascular inflammatory responses and dysfunction in atherogenesis [8,9]. Cigarette smoking is known to initiate a series of signaling cascades that result in the activation of NF-kB to induce the release of pro-inflammatory cytokines, causing VEB dysfunction [8,10]. Recent studies have found that inhibition of NF-kB decreases the release of pro-inflammatory cytokines, which is consistent with the improvement of VEB function [7,11,12]. These findings imply that the NF-kB pathway plays an important role in VEB function and it may become an important target in the treatment of CVDs. CD200 is a membrane glycoprotein of the immune-globulin superfamily with immune suppression effect via its receptor CD200R [13,14]. Recent studies suggest that CD200-CD200R signal axis plays a key role in the modulation of inflammatory responses in autoimmune diseases and neuro-degeneration [14,15]. Soluble CD200 fusion protein (CD200Fc) is a CD200 fusion protein consisting of the extracellular domain of CD200 bound to a murine IgG2aFc sequence and modified to eliminate Fc receptor and complement binding regions [16,17]. Experimental studies have demonstrated that CD200Fc attenuates inflammatory diseases [17–19] and reduces LPS-induced microglial activation [20]. However, the anti-inflammatory and vasoprotective effects of CD200Fc on in endothelial cells are not clear. On the basis of the aforementioned studies, we posit that cigarette smoke extract (CSE) activates NF-kB, which elicit the inflammatory responses, and promote endothelial cells dysfunction. We hypothesize that CD200Fc will attenuate these deleterious effects. To test this hypothesis, the vasoprotective effects of CD200Fc treatment on CSE-induced alterations in VEB function and expression of pro-inflammatory cytokines, and NF-kB activation, were investigated. 2. Materials and methods 2.1. Cell culture Primary mouse Cardiac Microvascular Endothelial Cells (mCMECs) were purchased from Cell Biologics (cat # B129-7024, Chicago). The fetal bovine serum (FBS) and endothelial cell medium (ECM) were purchased from Gibco BRL (Grand Island, NY, USA). mCMECs were cultured in ECM supplemented with 10% FBS and maintained at 37
C in 5% CO2 and 95% air. Routine evaluation for FITC-marked CD31 showed that cells were >99% pure (data not shown). Cells at passages 5–8 were used in this study. 2.2. Preparation of CSE The CSE (dissolved in DMSO) was purchased from Murty Pharmaceuticals (Lexington, KY) and was kept at 20
C according to a previously published method [21]. The composition of CSE was the following: 40 mg/ml total particular matter, nicotine content = 6%. mCMECs were randomly divided into five groups: control group, CSE-induced group (An additional 250 mg/ml CSE for 24 h or 120 min was added to the medium), and CD200Fc group (pretreated with 1, 10 and 100 mg/ml CD200Fc for 24 h, and then treated with 250 mg/ml CSE for another 24 h or 120 min). The choice of CSE and CD200Fc concentrations was based on previous studies [18,20,22].

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2.3. Transepithelial electrical resistance (TEER) measurement According to the previous studies [23,24], mCMECs were cultured in Transwell plates until the confluent monolayer achieved a TEER >300 V cm2 (about 15–18 days), demonstrating a tight monolayer. TEER was measured using a voltmeter (MillicellERS; Millipore, USA): TEER (ohms per square centimeter) = (Total resistance-Blank resistance) (ohms)  Area (square centimeter). 2.4. Transport studies mCMECs were seeded on a BD BioCoat Transwell system with 6.5-mm diameter, 0.4-mm polycarbonate pore size inserts (BD Biosciences, Oxford, UK) at a density of 20,000 cells/insert and incubated in 24-well culture plates with a medium change every other day. Confluent mono-layers were washed three times with PBS, left for 30 min at 37
C to equilibrate. PBS containing increasing molecular weight fluorescent dextrans (FDs; 25 mg/ ml) were then added to the apical side: 4 (FD4), 10 (FD10), and 20 kDa (FD20). After 1 h at 37
C, aliquot samples were withdrawn from the basolateral sites. The amounts of different florescence in the samples were determined using a Fluoroskan Ascent FL2.5 fluorometer (Thermo Fisher Scientific, Waltham, MA), with excitation and emission wavelengths of 485 and 520 nm, respectively. The apparent permeability coefficients (Papp) of different fluorescent agents used were measured using the following equation: Papp = (dq/dt)  (A  C
)1, where dq is the amount of fluorescence in the basolateral side (milligrams per milliliter), dt is a function of time per second, A is the surface area of the inserts (0.64 cm2), and C
is the initial concentration of fluorescent applied in apical compartment (milligrams per milliliter). 2.5. Real time polymerase chain reaction (Real time PCR) Total RNA of mCMECs were extracted using Trizol and converted to cDNA by using a cDNA first-strand synthesis system (Fermentas, Canada). The PCR primers were designed based on the NCBI mRNA and genome DNA sequence database. The primers of target genes were as follows: inducible nitric oxide synthase (iNOS) forward primer: 50 -30 -ACA ACA GGA ACC TAC CAG CTC A and reverse primer: 50 -30 -GAT GTT GTA GCG CTG TGT GTC A. Cyclooxygenase-2 (COX-2) forward primer: 50 -30 -CCA GAT GAT ATC TTT GGG GAG C and reverse primer: 50 -30 -CTT GCA TTG ATG GTG GCT G. Platelet endothelial cell adhesion molecule-1 (PECAM-1) forward primer: 50 -30 - CTC TCC CTC CTG TTC CTT GT and reverse primer: 50 30 - CGG TGG ATG AGG TCC AGA TT. Vascular cell adhesion molecule-1 (ICAM-1) forward primer: 50 -30 - GGC CTC AGT CAG TGT GA and reverse primer: 50 -30 - AAC CCC ATT CAG CGT CA. b-Actin forward primer: 50 -30 -GGC GGA CTA TGA CTT AGT TG and reverse primer: 50 30 -AAA CAA CAA TGT GCA ATC AA. DNA fragments of PCR products were designed to amplify within 200 bp length. Results were normalized from b-actin of respective samples. Briefly, each reaction contained 0.8 ml primer (containing 100 pM forward and reverse primers), 2 ml of cDNA (0.1 ml of RNA equivalent), 5 ml of Sofast EvaGreen supermix and 2.2 ml of H2O. Real Time-PCR was performed at 94
C for 45 s, and 55
C for 45 s to denaturation, and at 72
C for 45 s for 50 cycles to extension. All data were analysed by CFX manager software (BioRad, Hercules, CA, USA). 2.6. Cytokine assays The levels of Prostaglandin E2 (PGE2), tumor necrosis factor-a (TNF-a), interleukin-6 (IL-6) and IL-8 were measured using ELISA kits (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions. Briefly, mCMECs were plated in 24-

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well plates. After group-specified experimental conditions, a 100 ml aliquot of culture medium supernatant was collected to determine PGE2, TNF-a, IL-6 and IL-8 concentrations by ELISA kits.

statistically significant. Student’s t-test was used when two groups were compared. One-way ANOVA followed by Tukey multiple comparison was used when three or more groups were compared.

2.7. Western blot analyses

3. Results

mCMECs were lysed using a subcellular protein fractionation kit for cultured cells (Pierce Biotechnology Inc, Rockford, IL, USA) as per manufacturer’s guidelines, such that total protein, nuclear and cytosolic membrane fractions were collected. Sample preparation and the entire process were followed as described in previous report [25]. In brief, denatured samples (20–50 mg protein) was separated with SDS-PAGE and transferred to PVDF membrane (Millipore, Bedford, MA). After blocking with 5% defatted milk in PBS-Tween-20 for 1 h at room temperature, the PDF membrane was incubated with primary antibody against Zona Occludens-1 (ZO-1) (anti-rabbit, 1:400), Claudin-1 (anti-mouse, 1:500), b-actin (anti-mouse, 1:800), phosphorylated (p)- IkB (anti-rabbit, 1:300), total (t)- IkB (anti-rabbit, 1:500), p-P65 (anti-rabbit, 1:500) and tP65 (anti-rabbit, 1:800) at 4
C overnight. After washed with PBSTween-20 every 15 min for three times, the membrane was incubated with goat-anti-rabbit or goat-anti-mouse second antibody conjugated horseradish peroxidase (1:10,000; Abgent) for 2 h and then scanned with the Odyssey infrared imaging system (LICOR Bioscience).

3.1. CD200Fc attenuates TEER changes in CSE-induced mCMECs

2.8. Immunofluorescent staining After group-specified experimental conditions, mCMECs were fixed with 4% paraformaldehyde and then blocked in 1% bovine serum albumin and 0.2% Triton X-100 (both from Sigma-Aldrich, St. Louis, MO, USA) and then incubated with primary antibody ZO-1 (anti-rabbit, 1:100, Cell Signaling, Danvers, MA, USA) or Claudin-1 (anti-mouse, 1:200, Cell Signaling, Danvers, MA, USA) at 4
C overnight, and then incubated with fluorescein isothiocyanate (FITC)-conjugated anti-rabbit or anti-mouse IgG. mCMECs were incubated with DAPI for 10 min. Images were observed under confocal microscope (Carl Zeiss, Oberkochen, Germany). 2.9. NF-kB assay Nuclear extracts from treated mCMECs were prepared using the Nuclear Extract Kit (Active Motif, Carlsbad, CA). NF-kB activity was measured by a NF-kB p65 assay kit (Active Motif) according to the manufacturer’s protocol according to a previously published method [26]. 2.10. Statistical analysis Statistical analyses were performed with GraphPad Prism (v6.0) (GraphPad Software Inc.). In all cases, P < 0.05 was considered

According to the previous studies [23,24], TEER was used to measure paracellular permeability and integrity of cell monolayers in cell culture experiments, which indicate VEB function. In our study, we first tested various concentrations (1, 10 or 100 mg/ ml) of CD200Fc at 6 h (Fig. 1A), 12 h (Fig. 1B) or 24 h (Fig. 1C) and found that 10 and 100 mg/ml produced the strongest effects on TEER. Compared with control group, TEER was reduced in CSEinduced mCMECs. Exposure to CD200Fc for various concentrations (10 mg/ml and 100 mg/ml) significantly increased TEER in CSEinduced mCMECs, with the most substantial increase observed at 12 h (Fig. 1B) and 24 h (Fig. 1C). 3.2. CD200Fc attenuates transport of fluorescent markers changes in CSE-induced mCMECs To further explore the role of CD200Fc in preventing CSEinduced VEB dysfunctions, we measured transport of fluorescent markers changes in CSE-induced mCMECs. In Fig. 2, the results showed the effects of various CD200Fc concentrations (1, 10 and 100 mg/ml) on the increasing sizes of fluorescent markers FD4 (Fig. 2A), FD10 (Fig. 2B) and FD20 (Fig. 2C) in CSE-induced mCMECs. 1 mg/ml wogonin did not alter the transport of any of the fluorescent markers compared with CSE-induced group. Pretreatment of 10 and 100 mg/ml CD200Fc for 24 h significantly decreased transport of fluorescent markers: FD4 (Fig. 2A), FD10 (Fig. 2B) and FD20 (Fig. 2C) compared with CSE-induced group. 3.3. CD200Fc increases expression of ZO-1 and claudin-1 in CSEinduced mCMECs The tight junction is formed by a protein complex that is widely used as a marker of paracellular permeability and integrity [23]. The integrity of the tight junction in mCMECs was examined by Western blot and immunofluorescent staining results through analyses of expression of the integral membrane proteins, ZO-1 and Claudin-1. In our study, Western blot results demonstrated that treatment with 10 and 100 mg/ml CD200Fc for 24 h attenuated the down-regulation of ZO-1 and Claudin-1 protein levels (Fig. 3A– C) in CSE-induced mCMECs. In addition, our immunofluorescent staining results (Fig. 3D) were consistent with the Western blot results. Above all, the results in Figs. 1–3 showed the obviously vasoprotective effects of CD200Fc on the VEB function in CSEinduced mCMECs.

Fig. 1. Effect of CD200Fc on TEER level changes in CSE-induced mCMECs. mCMECs were pre-treated with 1, 10 and 100 mg/ml CD200Fc for 6 h (A), 12 h (B) or 24 h (C), and then treated with 250 mg/ml CSE for another 24 h. Data were shown as mean  SEM (n = 3, #P < 0.001, compared to control group; ***P < 0.001, **P < 0.01, *P < 0.05 compared to CSE-induced group).

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Fig. 2. Effect of CD200Fc on transport of fluorescent markers changes in CSE-induced mCMECs. mCMECs were pre-treated with 1, 10 and 100 mg/ml CD200Fc for 24 h, and then treated with 250 mg/ml CSE for another 24 h. (A–C). Effects of CD200Fc on the paracellular transport (permeability coefficients) of fluorescein isothiocyanate-dextrans of 4 (FD4), 10 (FD10), and 20 kDa (FD20). Data were shown as mean  SEM (n = 3, #P < 0.001, compared to control group; ***P < 0.001, **P < 0.01 compared to CSE-induced group).

Fig. 3. Effect of CD200Fc on LPS-induced intestinal barrier function changes in CSE-induced mCMECs. mCMECs were pre-treated with 1, 10 and 100 mg/ml CD200Fc for 24 h, and then treated with 250 mg/ml CSE for another 24 h. (A). mRNA levels of ZO-1 and Claudin-1 were evaluated by western blot analysis. Densitometric analysis of effects of CD200Fc on expression of ZO-1 (B) and Claudin-1 (C). (D). Immunofluorescent staining showing the effects of CD200Fc on the expressions of ZO-1 and Claudin-1 exposed to CSE. The phenotype of nuclei was also investigated via DAPI staining. Scale Bar = 25 mm. Data were shown as mean  SEM (n = 3, #P < 0.001, compared to control group; ***P < 0.001, **P < 0.01 compared to CSE-induced group).

3.4. CD200Fc modulates expressions of pro-inflammatory cytokines in CSE-induced mCMECs Inflammatory responses is known to induce destruction of paracellular permeability and integrity as well as cause VEB dysfunctions [11,27]. In order to confirm that pro-inflammatory cytokines release could be inhibited by CD200Fc in CSE-induced mCMECs, we tested the iNOS, COX-2, PECAM-1, ICAM-1, PGE2, TNFa, IL-8 and IL-6 levels. The Real Time PCR results showed that the mRNA expressions of COX-2, iNOS, PECAM-1 and ICAM-1 were increased in CSE-induced mCMECs. Conversely, these markers were decreased after 10 and 100 mg/ml CD200Fc pretreatment

(Fig. 4A–D). Moreover, our ELISA results indicated that the levels of PGE2, TNF-a, IL-6 and IL-8 were significantly increased in CSEinduced mCMECs compared to control group, which were reduced after 10 and 100 mg/ml CD200Fc pretreatment (Fig. 5A–D). This finding suggested that CD200Fc could inhibit the activation of the inflammatory cascade in CSE-induced mCMECs. 3.5. CD200Fc reduces activation of NF-kB pathway in CSE-induced mCMECs NF-kB is activated by a wide variety of agents including oxidative stresses such as cigarette smoke [28,29]. As shown in

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Fig. 4. Effect of CD200Fc on release of inflammatory mediators in CSE-induced mCMECs. mCMECs were pre-treated with 1, 10 and 100 mg/ml CD200Fc for 24 h, and then treated with 250 mg/ml CSE for another 24 h. mRNA levels of iNOS (A), COX-2 (B), PECAM-1 (C) and ICAM-1 (D) were evaluated by Real Time PCR. Data were shown as mean  SEM (n = 3, #P < 0.001, compared to control group; ***P < 0.001, **P < 0.01, *P < 0.05 compared to CSE-induced group).

Fig. 6, CSE significantly enhanced the DNA binding activity of nuclear NF-kB p65 in mCMECs. The increase in NF-kB activity was significantly decreased by pretreating mCMECs with 10 and 100 mg/ml CD200Fc. Treatment of mCMECs with CSE leads to the phosphorylation of IkB. These phosphorylation events target IkB for ubiquitindependent degradation, resulting in the release and nuclear translocation of P65 [28]. As shown in Fig. 7A–C, p-IkB was increased and total t-IkB was decreased 120 min after CSE treatment, indicating that IkB was phosphorylated and degraded 120 min after CSE treatment. Pretreatment of mCMECs with 10 and 100 mg/ml CD200Fc decreased phosphorylation and degradation of IkB in CSE-induced mCMECs. NF-kB P65 is inactivated in the cytosol by binding to IkB, and becomes phosphorylation and translocation to the nucleus and then induces inflammatory responses [28,29]. As shown in Fig. 7A, D and E, p-P65 was increased 120 min in CSE-induced mCMECs. Pretreatment of mCMECs with 10 and 100 mg/ml CD200Fc decreased p-P65 expression in response to CSE. In addition, western blot data also showed a marked increased translocation of P65 from cytoplasm to nucleus in CSE-induced mCMECs. However, CSE-induced P65 level in the nuclear fractions was reduced by 10 and 100 mg/ml CD200Fc pretreatment (Fig. 8A–C), indicating that the subsequent NF-kB inactivation induced by CD200Fc in CSEinduced mCMECs. 4. Discussion CVDs, such as myocardial infarction, heart failure and stroke involving a change in vessel microenvironment are

characteristically accompanied by VEB dysfunction, thereby emphasizing the necessity of maintaining a proper barrier function and integrity to conserve the vessel microenvironment [2,3]. As mentioned earlier, CSE have been considered to increase the chances of developing and propelling the above-mentioned vascular pathological conditions [3,6]. Recent findings demonstrated that CSE exposure induced VEB dysfunction and strong inflammatory responses [2,3,30]. This included a down-regulation and redistribution of tight junction protein expression and an increase in release of pro-inflammatory cytokines [27,30]. Results of this study demonstrated a progressive down-regulation and disruption of ZO-1 and Claudin-1 expression following 24 h exposure to CSE, and a strong increase in expressions of proinflammatory cytokines (iNOS, COX-2, PECAM-1, ICAM-1, PGE2, TNF-a, IL-8 and IL-6) was observed at 24 h under CSE conditions. Additionally, our paracellular permeability and integrity results in mCMECs showed a strong reduction in TEER level and a markedly increased transport of fluorescent markers by exposure to CSE. Thus, exposure to CSE induced VEB dysfunction and inflammatory responses, which could be a highly clinically relevant model of smoking related cardiovascular diseases. CD200 is a member of the immunoglobulin super-family of glycoproteins [31], and is expressed in a wide variety of cells, such as neurons [14], microglial cells [18] and renal proximal tubular epithelial cells [19]. Interaction of CD200 with its receptor family (CD200R1-R4) has an immunoregulatory role and attenuates various types of inflammatory diseases [14,31,32]. CD200Fc is a CD200 fusion protein consisting of the extracellular domain of mouse CD200 linked to a murine IgG2a Fc sequence and modified to eliminate Fc receptor and complement binding regions.

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Fig. 5. Effect of CD200Fc on release of PGE2, TNF-a, IL-8, and IL-6 in CSE-induced mCMECs. mCMECs were pre-treated with 1, 10 and 100 mg/ml CD200Fc for 24 h, and then treated with 250 mg/ml CSE for another 24 h. Levels of PGE2 (A), TNF-a (B), IL-8 (C) and IL-6 (D) in mCMECs were determined by ELISA analysis. Data were shown as mean  SEM (n = 3, #P < 0.001, compared to control group; ***P < 0.001, **P < 0.01, *P < 0.05 compared to CSE-induced group).

Fig. 6. Effect of CD200Fc on DNA binding activity of nuclear NF-kB p65 in CSEinduced mCMECs. mCMECs were pre-treated with 1, 10 and 100 mg/ml CD200Fc for 24 h, and then treated with 250 mg/ml CSE for another 120 min. Nuclear extracts were prepared by using a nuclear extract kit. NF-kB activity was measured using an ELISA kit. Data were shown as mean  SEM (n = 3, #P < 0.001, compared to control group; *** P < 0.001 compared to CSE-induced group).

Recently, it has been shown that CD200Fc could activate CD200R1 expression and inhibits the release of the pro-inflammatory cytokines in activated microglial cells [20] and human renal proximal tubular epithelial cells [19]. Intrathecal administration of CD200Fc induces very rapid suppression of neuro-inflammatory reactions associated with glial activation and neuropathic pain

development [18]. Based on this information, we postulate that CD200Fc may play a key role in the inhibition of inflammatory responses, which may be associated with CSE-induced VEB dysfunction in mCMECs. In this present study, pretreatment with CD200Fc could reduce the levels of proinflammatory cytokines in CSE-induced mCMECs. This notion was in accordance with previous reports demonstrating that CD200Fc was able to ameliorate inflammatory responses in activated microglial cells [20] and human renal proximal tubular epithelial cells [19]. Moreover, pretreatment with CD200Fc alleviated CSE-induced reduction in TEER level and a marked increase of transport of fluorescent markers in mCMECs. The NF-kB pathway plays an important role in cigarette smoking-induced vascular inflammatory responses [8,9,12,33]. In quiescent cells, the NF-kB P65 complex is present in the cytoplasm is an inactive form, bound to its inhibitory protein known as IkB [8]. In response to a diversity of stimuli such as CSE, IkB is phosphorylated by the activation of the IkB kinase (IKK) complex [8]. The IKK complex phosphorylates IkB, leading to its ubiquitylation and subsequent degradation, which allows NF-kB P65 protein translocation into nucleus, which induce the release of pro-inflammatory cytokines and cause endothelial dysfunction [8,33]. In our present study, we found CD200Fc significantly inhibited NF-kB activity by blocking NF-kB translocation to nucleus, inhibiting the DNA binding NF-kB P65 activity and IkB phosphorylation. Therefore, CD200Fc might exert anti-inflammatory effect via inhibition of NF-kB pathway which might be a reason for the protection of VEB function in CSE-induced mCMECs. In conclusion, we propose that CD200Fc may exert a vasoprotective effect through the attenuation of inflammatory

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Fig. 7. Effect of CD200Fc on expression of p-Ik-B, t-Ik-B, p-NF-kB and t-Ik-B in CSE-induced mCMECs. mCMECs were pre-treated with 1, 10 and 100 mg/ml CD200Fc for 24 h, and then treated with 250 mg/ml CSE for another 120 min. (A). Protein levels of p-Ik-B, t-Ik-B, p-NF-kB and t-Ik-B were evaluated by western blot analysis. b-actin was used to ensure equal loading. (B–E). Densitometric analysis of effects of CD200Fc on expression of p-Ik-B (B), t-Ik-B (C), p-P65 (D) and t-P65 (E). Data were shown as mean  SEM (n = 3, #P < 0.001, compared to control group; ***P < 0.001, **P < 0.01, *P < 0.05 compared to CSE-induced group). p: phosphorylated; t: total.

Fig. 8. Effect of CD200Fc on P65 translocation into nuclei in CSE-induced mCMECs. mCMECs were pre-treated with 10 and 100 mg/ml CD200Fc for 24 h, and then treated with 250 mg/ml CSE for another 120 min. (A). The levels of P65 in both nuclear and cytosolic fractions were measured by western blot assay. Expression of HDAC1 and b-actin (an internal control) was used for monitoring P65 translocation into nuclei. (B). The bar chart showed the ratio of P65 to HDAC1 in nuclear fractions at each group. (C). The bar chart showed the ratio of P65 to b-actin in cytosolic fractions at each group. Data were shown as mean  SEM (n = 3, #P < 0.001, compared to control group; ***P < 0.001, **P < 0.01 compared to CSE-induced group). N: Nucleus; C: Cytoplasm.

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