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Research Article

The angiopoietin1–Akt pathway regulates barrier function of the cultured spinal cord microvascular endothelial cells through Eps8 Xinchun Liun, Xiaoshu Zhou, Wei Yuan Department of Orthopedics, The First Hospital of China Medical University, 155 Nanjing Street, Heping District, Shenyang 110001, China

article information

abstract

Article Chronology:

In mammalian central nervous system (CNS), the integrity of the blood–spinal cord barrier (BSCB),

Received 11 April 2014

formed by tight junctions (TJs) between adjacent microvascular endothelial cells near the basement

Received in revised form

membrane of capillaries and the accessory structures, is important for relatively independent activities

5 August 2014

of the cellular constituents inside the spinal cord. The barrier function of the BSCB are tightly regulated

Accepted 13 August 2014

and coordinated by a variety of physiological or pathological factors, similar with but not quite the

Available online 20 August 2014 Keywords: Ang1 Akt SCMEC Blood–spinal cord barrier Eps8

same as its counterpart, the blood–brain barrier (BBB). Herein, angiopoietin 1 (Ang1), an identified ligand of the endothelium-specific tyrosine kinase receptor Tie-2, was verified to regulate barrier functions, including permeability, junction protein interactions and F-actin organization, in cultured spinal cord microvascular endothelial cells (SCMEC) of rat through the activity of Akt. Besides, these roles of Ang1 in the BSCB in vitro were found to be accompanied with an increasing expression of epidermal growth factor receptor pathway substrate 8 (Eps8), an F-actin bundling protein. Furthermore, the silencing of Eps8 by lentiviral shRNA resulted in an antagonistic effect vs. Ang1 on the endothelial barrier function of SCMEC. In summary, the Ang1–Akt pathway serves as a regulator in the barrier function modulation of SCMEC via the actin-binding protein Eps8. & 2014 Elsevier Inc. All rights reserved.

Introduction In vertebrates, the blood–spinal cord barrier (BSCB) provides an independent microenvironment for the spinal cord parenchyma. As a morphological extension of the blood–brain barrier (BBB), BSCB shares similar building blocks, including capillary endothelium, basal

lamina, pericytes, and astrocyte end-foot. Increasing evidences have shown that BSCB is involved in the occurrence and development of several diseases or pathological conditions related to the neurological system, such as traumatic spinal cord injury [1,2], radiation injury, spinal cord ischemia [3,4], amyotrophic lateral sclerosis [5–7], inflammation [8], etc. In the central neural system (CNS), neural signaling

Abbreviations: Ang1, angiopoietin1; SCMEC, spinal cord microvascular endothelial cell; BSCB, blood–spinal cord barrier; SC, spinal cord; SCM, spinal cord microvessels; Eps8, epidermal growth factor receptor pathway substrate 8; ABPs, actin binding proteins; CNS, central nervous system; TJ, tight junction; AJ, adherence junction; WM, wortmannin; TEER, transendothelial electrical resistance; Na-F, sodium fluorescein; EBA, Evans blue albumin; shRNA, short hairpin RNA; co-IP, co-immunoprecipitation n

Corresponding author. E-mail address: [email protected] (X. Liu).

http://dx.doi.org/10.1016/j.yexcr.2014.08.019 0014-4827/& 2014 Elsevier Inc. All rights reserved.

Antibodies used in this report were found to cross-react with the corresponding proteins in the rat as indicated by the manufacturers. IB, immunoblot; IF, immunofluorescence; IP, immunoprecipitation; and HRP, horseradish peroxidase. a

– – – – – HRP HRP HRP Alexa Fluor 488

Mouse Rabbit Goat Rabbit Rabbit Rabbit Rabbit Rabbit Goat Bovine Bovine Bovine Donkey

BD Transduction Santa Cruz Biotechnology Santa Cruz Biotechnology Santa Cruz Biotechnology Zymed/Invitrogen Zymed/Invitrogen Abcam Zymed/Invitrogen Santa Cruz Biotechnology Santa Cruz Biotechnology Santa Cruz Biotechnology Santa Cruz Biotechnology Invitrogen

– –

1:1000 1:250 1:250 1:250 1:250 1:250 1:1000 1:250 1:250 1:3000 1:3000 1:3000

1:100

1:40 1:40 1:50 1:50 1:50 1:50

IP IF IB

Working dilution Conjugation Vender Host

610,143 sc-9026 sc-1618 sc-33437 71-1500 61-7300 ab27775 71-2700 sc-1616 sc-2370 sc-2350 sc-2371 A-31572

Sprague–Dawley rats were obtained from the Department of Laboratory Animals, China Medical University (CMU). Animals

Eps8 Tie-2 Akt1 p-Akt1 Occludin ZO-1 VE-cadherin β-Catenin Actin Rabbit IgG Goat IgG Mouse IgG Rabbit IgG

Animals and reagents

Catalog no.

Materials and methods

Antibody

requires a highly controlled microenvironment. To achieve this, the paracellular diffusion of molecules across capillary endothelium are restricted by tight junctions (TJs) between adjacent endothelial cells [9], which are assembled partially depending on the adherence junction (AJ) framework [10]. These cell–cell junction apparatus, together with the associated cytoskeletal networks [11], ultimately determine the integrity and barrier function of the microvascular endothelial monolayer in the BBB or the BSCB. Of the three major cytoskeletal networks (F-actin, microtubules and intermediate filaments) in eukaryotic cells, F-actin cytoskeleton, which comes in diversity of structures depending on proteins that bind to it, is the most abundant and affects the endothelial barrier function in multiple ways [12,13]. Epidermal growth factor receptor pathway substrate 8 (Eps8), a known actin binding protein of 97 kDa, is one of the verified actin bundling regulator by activating and synergizing with IRSp53 [14,15]. On the other hand, Eps8 also facilitates the formation of a branched actin network through its actin barbed-end capping activity and Rac GTPase activation [16,17]. Recently, a study in the blood–testis barrier (BTB) revealed an important role of Eps8 in maintaining germ cell adhesion and sertoli cell epithelial barrier integrity [18]. By using cultured sertoli cell epithelium that mimicks the BTB in vivo, the knockdown of Eps8 was found to lead to F-actin disorganization and mislocalization of TJ proteins [18]. Nevertheless, the role of Eps8 in endothelial barrier function including the blood–CNS barrier, which also comprises multiple actin-based cell junctions, still remains unveiled. Angiopoietin1 (Ang1), the endothelium-specific ligand known to be crucial for vascular stabilization, angiogenesis, and branching morphogenesis [19], is one of the strongest endogenous promoters of BBB stabilization and necessary to activate the Tie-2 receptor tyrosine kinase with Ang2 acts as its competing inhibitor [20–22]. It can lead to autophosphorylation of Tie-2 and thus cause a chemotatic effect on endothelial cells [21]. In developed vasculature, Ang1 is constitutively secreted by pericytes and smooth muscle at a basal level. In BBB, Ang1 was found to maintain blood vessel quiescence by limiting permeability and controlling barrier integrity [23,24]. A recent study also reported a stabilization effect of Ang1 on brain endothelial junctions by up-regulating ZO-2 expression [25]. In microvascular endothelial cells (MVEC), Ang1 was shown to exert its role via phosphatidyl inositol-3 kinase (PI3K)/Akt pathway. For instance, an anti-apoptotic effect through the Akt/survivin was verified in Ang1treated bovine MVEC [26]. In human umbilical vein endothelial cells (HUVECs), it was reported that Ang1 could induce activation of β-catenin through PI3K/Akt, which subsequently enhances Notch signal-induced Dll4 expression and leads to vascular stabilization [27]. In the present study, we confirmed the enhancing effects of the Ang1–Akt pathway on barrier integrity in the spinal cord microvascular endothelial cell (SCMEC) of rat, which was isolated and applied as a model to mimick the BSCB in vitro. More importantly, the actin bundling protein Eps8, was uncovered to participate in F-actin re-organization and junction protein interactions during the enhancement of SCMEC barrier function induced by Ang1.

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Table 1 – Antibodies used for different experiments in this reporta.

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were raised at 22 1C and a light:dark cycle of 12 h:12 h with free access to water and standard chow ad libitum. Animal handling protocols and treatment procedures were approved by the CMU Animal Care and Use Committee. Reagents used in this report, unless otherwise specified, were from Sigma Aldrich. All the antibodies used in different experiments herein are listed in Table 1.

Isolation of glial cells and preparation of feeder layer for permeability assay To get a high yield of glial cells to provide the feeder layer for microvascular endothelial cells, brains from 2 day postpartum Sprague–Dawley rats were used. Briefly, whole brains were separated and placed in cold DMEM/F-12 (Dulbecco's Modified Eagle Medium-Nutrient Mixture F-12, Gibco) after decapitation. Thereafter, brain cortices were isolated and meninges were removed by rolling the tissue on a filter paper under aseptic conditions. Cell suspension was collected after homogenizing and was then passed through a 70 mm cell strainer (BD Falcon). The cells were resuspended in DMEM/F12 medium containing 10% FBS and glutamine, and plated in culture flasks. Cultures were incubated at 37 1C with 95% air and 5% CO2 (v/v) in a humidified atmosphere and the medium was replaced every 5–7 days. After culturing for 15 days, the cells were passaged with 0.125% trypsin–0.02% EDTA before frozen gradually and cryopreserved in culture medium containing 10% DMSO in liquid nitrogen. When needed for microvascular endothelial cell culturing, the cryopreserved glia were thawed quickly in 37 1C water bath to revive and cultured in the condition as described above until 95% confluence. Then the glia were passaged with 0.125% trypsin and plated into 24-well plates before addition of the upper inserts of the twochamber culture wells with microvascular endothelial cells.

Isolation of spinal cord microvessels and spinal cord microvascular endothelial cell (SCMEC) Microvessels were isolated from the spinal cord according to the method described elsewhere previously [28,29] with modifications. Adult Sprague–Dawley rats (275–300 g body weight) were euthanized with CO2 humanely, followed by separation of the spinal column with ophthalmological instruments. The separated spinal column was then immersed in pre-cold D-Hanks balanced salt solutions (Hyclone) and the spinal cord was discharged by injecting cold D-Hanks solution into the spinal column. After clearance of meninges and big vessels, the dissected tissues were washed with fresh solution for three times and cut into 1 mm3 pieces with ophthalmic scissors in cold DMEM/F-12. The smashed tissues were then added into a glass homogenizer and homogenated on ice several times manually. Resulting homogenates were digested in pre-warmed DMEM/F-12 containing collagenase II (1 mg/ml) in a shaker for 30 min at 37 1C and then centrifuged at 200g for 5 min at 4 1C. The pellets were resuspended in 22% bovine serum albumin, followed by another centrifugation at 2000g for 10 min at 4 1C. Then the upper myelin layer was aspirated and the pellet containing the vascular fraction was washed three times with DMEM/F-12. Macrovascular and red blood cells were further eliminated by passing the suspension through sterile stainless steel filter of 295 mm and 40 mm cell strainer (BD Falcon) respectively. Thereafter, the spinal cord

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microvessel (SCM) components left on the cell strainer were collected by rinsing the underside of the strainer with DMEM/F12 medium and centrifuged at 1000g for 5 min. The acquired SCMs were then digested with 0.1% collagenase/ dipase (Roche) at 37 1C for approximately 30 min with occasional agitation and observation under inverted microscope, until endothelial cells began to protrude from the microvessel fragment which appeared like “beads on a string”. The digestion was terminated by a centrifugation at 1000g for 5 min. Then the digested microvessel fragments were washed and resuspended in SCMEC culture medium [DMEM/F12, supplemented with 10% fetal bovine serum, 10% horse serum, 2 mM glutamine, 1 mg/ml heparin, 1 ng/ml basic fibroblast growth factor (PeproTech), 10 ng/ ml vascular endothelial growth factor (PeproTech), 50 mg/ml gentamicin, 3 mM puromycin] before plated onto collagen type IV and fibronectin (both 5 mg/cm2, BD Biosciences)-coated culture plates at a density of 1
104/cm2. Confluenced monolayer was formed 6 days after isolation. The cultured SCMEC were identified by immunofluorescence using anti-factor VIII (von Willebrand factor) antibody (Abcam) and SCMEC purity was counted under fluorescence microscope to be  95%. Then the expanded SCMEC were digested with 0.125% trypsin–0.02% EDTA and subcultured into different vessels at different ratios depending on subsequent experiments: (i) culture dishes at 1:1 for lysate preparation; (ii) microscopic coverslips placed in culture dishes at 1:5 for immunofluorescence analysis; or (iii) upper inserts of the two-chamber culture wells (Millipore, 1.0 mm pore size) at 1:1 for transendothelial electrical resistance (TEER) measurement and permeability evaluation, which was then placed in the 24-well dishes (lower chamber) pre-seeded with glial cells in advance. Cultures were incubated at 37 1C with 95% air and 5% CO2 (v/v) in a humidified atmosphere for an additional week (days 0–7) with the medium replaced every 2–3 days before further analysis. Each experiment including both control and treatment groups was repeated at least three times, using SCMEC cultured from different batches of rats excluding pilot experiments.

Treatment of Ang1 and wortmannin To assess the effects of Ang1 on the cultured SCMEC permeability barrier, on day 7 the cultured SCMEC in some experiments were incubated with Ang1 (100 ng/ml, PeproTech), with or without wortmannin (the known inhibitor of PI3K/Akt pathway, 100 nM, Gene Operation) for 12 h for TEER measurement, permeability evaluation, immunocytochemistry or F-actin staining. For immunoblot or Akt activity assay, the treated SCMEC were terminated at different time points. The concentrations of Ang1 and wortmannin used herein were selected based on earlier reports [30–33] and pilot experiments of this study.

Transendothelial electrical resistance (TEER) measurement Barrier function of the SCMEC co-cultured with glial cells was assessed by quantifying TEER across the SCMEC endothelium cultured on the upper inserts of the two-chamber culture wells with an EVOM2 resistance meter. The resistance (R) of an intact cell barrier to the current passage between the two electrodes placed respectively at the upper and the lower chamber of the system was recorded at each designated time point in different treatment groups. TEER (Ω cm2) was calculated by the following

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formula: (Runknown  Rblank)
effective surface area. For each time point, triplicate samples were set and each experiment was repeated at least three times using different batches of cells.

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selected LV-shNC or LV-shEps8 infected cells were subjected to Ang1 treatment as described above for further experiments.

Lysate preparation and immunoblot analysis Transendothelial permeability evaluation To evaluate the permeability of SCMEC monolayer, sodium fluorescein (Na-F, MW 376 Da) and Evans blue albumin (EBA, MW 67 kDa) were used as markers of different molecule size. To be specific, 0.5 ml assay buffer (136 mM NaCl, 0.9 mM CaCl2, 2.7 mM KCl, 0.5 mM MgCl2, 10 mM NaH2PO4, 1.5 mM KH2PO4, 25 mM glucose, and 10 mM HEPES, pH 7.4) was added to the lower chamber of the two-chamber culture wells, then the culture medium in the upper inserts was replaced by 0.5 ml assay buffer containing 10 mg/ml Na-F and 165 mg/ml EBA. At 0, 4, 8, or 12 h, 50 ml samples were removed from the basal chamber which was immediately replenished with equal amount of assay buffer. The retrieved samples were analyzed at 535 nm for Na-F emission and at 595 nm for EBA absorbance (RF-5301PC, Shimadzu). The permeability efficient (Pe, cm/min) for both markers was calculated by the following formula: Pe¼ Vlower/(S
Cupper)
(Clower/T), in which Vlower is the volume of the lower chamber (0.5 cm3), S is the surface area of SCMEC monolayer (0.6 cm2), Cupper is the initial marker concentration in upper inserts, Clower is the marker concentration in lower chamber at sampling time T (min).

Lentivirus-mediated short hairpin RNA (shRNA) silencing of Eps8 expression in cultured SCMEC To knock down Eps8 expression, three pairs of oligonucleotides targeting at rat Eps8 gene (NC_005103.3, the targeting site showed the best silencing effect herein was as follows: 5'-TTCGTGCCAAATAACATTCTGGATA-3'), each composed by two reverse complementary sequences that were connected by a 9-bp loop (TTCAAGAGA), were designed and synthesized by Genechem Co. Ltd. In addition, a non-targeting control shRNA sequence (5'-TTCTTCTCCGAACGTGTCACGTCCA-3') that showed no homology to rat genes aligned by GenBank BLAST was also synthesized as above. After annealing, these oligonucleotides pairs were cloned into the linearized pGreenPuro Lentivector driven by EF1 promotor (System Biosciences) between BamHI and EcoRI. Then these recombinant vectors were transformed into DH5α competent cells followed by purification with an endotoxin-free plasmid purification kit (Qiagen). Polymerase chain reaction and sequencing analysis were used to confirm successful ligations. For lentivirus particle packaging, 293T cells (Shanghai Institute of Biochemistry and Cell Biology) were simultaneously transfected with the shRNA lentiviral vector and pPACKH1 Packaging Plasmid Mix (System Biosciences) using Lipofectamine™ 2000 (Invitrogen) according to the manufacturer's protocols. Viral supernatants were harvested and subjected to a 0.45 mm filter after 48 h, and the titers were determined with serial dilutions of the concentrated lentivirus. The resulting lentivirus containing the recombinant non-targeting shRNA or Eps8-specific shRNA vector was referred to as LV-shNC and LV-shEps8, respectively. For SCMEC infection, the cells were added with the lentivirus at a multiplicity of infection of 2 in the presence of 4 mg/ml polybrene on day 2 and cultured for 12 h before replaced with fresh SCMEC culture medium containing 3 mM puromycin. On day 7, some of the

Lysate of spinal cord (SC), spinal cord microvessels (SCM), and SCMEC after different treatments at designated time points were prepared in Nonidet P-40 lysis buffer [50 mM Tris, 150 mM NaCl, 2 mM EGTA, 1% Nonidet P-40 (v/v), 10% glycerol (v/v), 2 mM PMSF, 1 mM sodium orthovanadate, 2 mM N-ethylmaleimide, 1 mg/ml leupeptin, and 1 mg/ml aprotinin, pH 7.4]. Protein concentrations were quantified by using the DC protein assay kit (Bio-Rad Laboratories). Approximately 35 mg protein from each sample was applied per lane and resolved by SDS-PAGE under reducing conditions for immunoblot with specific antibodies (Table 1). Then the enhanced chemiluminescence was applied using kit from Pierce Chemical Co. Further densitometric analysis was made using Scion Image software (version 4.0.3). All samples within an experiment group were simultaneously analyzed by immunoblot to avoid inter-experimental variations.

Co-immunoprecipitation (co-IP) Interactions between junction proteins were detected by co-IP experiments in this study. To avoid nonspecific IgG-interaction, a pre-cleaning step was applied by adding 2 mg normal rabbit IgG to 500 mg protein lysate from SCMEC and incubated for 1 h. Then 10 ml protein A/G agarose (GenDEPOT) was added to the samples for another 1 h incubation, followed by a concentration at 4 1C. Subsequently, the supernatant was collected and incubated with 2 mg normal rabbit IgG, which served as negative control, anti-ZO-1 or anti-VE-cadherin antibody overnight at room temperature. The immunocomplexes were precipitate by adding 20 ml protein A/G agarose beads and gently washed in lysis buffer. After extracted by SDS-PAGE sample buffer at 100 1C for 5 min, the immunocomplexes were at last resolved by SDS-PAGE and subjected to immunoblot analysis using anti-occludin or anti-β-catenin antibody.

Akt activity assay The activity of Akt was determined by the phosphorylation state of H2B. In brief, SCMEC lysates were pre-incubated with protein G-agarose at 4 1C for 30 min before immunoprecipitated with anti-Akt antibody for 2 h. Then the immunoprecipitates were washed sequentially with lysis buffer, MilliQ water, and kinase buffer [50 mM HEPES, 10 mM MnCl2, 10 mM MgCl2, 1 mM dithiothreitol, 1 mM protein kinase A inhibitor peptide (Seleckchem), pH 7.2], followed by incubation in kinase buffer supplemented with 20 mCi/ml [γ-32P]ATP (Beijing FuRui Biotechnology) and 0.2 mg/ml histone H2B (Enzo Life Sciences) as substrate at 30 1C for 10 min. Afterwards, the reaction aliquots were spotted on Whatman p81 paper and the reaction was stopped using 5% H3PO4 solution. After thorough washing, the radioactivity on the filter paper was counted with a BECKMAN scintillation counter. A parallel experiment was performed to further confirm the phosphorylation state of H2B by incubating the immunoprecipitates with kinase buffer described above containing 50 mCi/ml [γ-32P] ATP at 37 1C for 30 min. Then SDS sample buffer was used to stop the reaction before resolving the samples by SDS-PAGE and the phosphorylation state of H2B was detected by autoradiograghy.

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Immunofluorescence analysis and F-actin staining SCMEC plated on coverslips pre-coated with collagen type IV and fibronectin were fixed in 4% paraformaldehyde for 4 min. Cells were then permeabilized in 0.1% Triton X-100 in PBS for another 4 min before blocking with 5% BSA for 30 min. Primary antibodies diluted in 1% BSA at specific concentrations (Table 1) were added and incubated overnight at room temperature. Secondary antibodies (Table 1) conjugated with fluorescein isothiocyanate (FITC)-488 and diluted in 1% BSA were then applied and incubated at room temperature for 1 h. For F-actin staining, SCMEC were incubated with rhodamine phalloidin (Invitrogen) at the same

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step with the secondary antibodies. Cells were then mounted with 4, 6-iamidino-2-phenylindole (DAPI)-contained Prolong Gold Antifade reagent (Invitrogen). Images were visualized with a fluorescence microscope (BX60, Olympus) and captured using a digital camera (SpotRT, Diagnostic Instruments). It is noted that micrographs reported herein are representative images of at least three independent experiments using different batches of SCMEC.

Statistical analysis GB-STAT statistical analysis software (Dynamic Microsystems) was used to analyze data from the TEER experiment, permeability

Fig. 1 – Characterization of isolated SCMEC and expression of Eps8 during the assembly of the endothelial monolayer. (A) Duallabeled immunofluorescence showing the expression of factor VIII (green) in cell cytosol around nucleus and junction protein β-catenin (red) at cell–cell interface in cultured SCMEC on day 7 after passaging at a lower ratio of 1:5. To get a better visualization of SCMEC, the “polygon-like” cells were selected to be shown herein. Nuclei were stained with DAPI (blue). Scale bar, 25 lm, applying to all micrographs in A. (B) Line chart represents SCMEC barrier integrity by quatifying TEER across the cell endothelium cultured on collagen type IV and fibronectin-coated inserts daily after isolation. (C) Immunoblot analysis of Eps8, Tie-2 receptor and junction proteins, i.e. occludin, ZO-1, VE-cadherin and β-catenin in lysates of rat spinal cord (SC), spinal cord microvascular (SCM) and SCMEC cultured in vitro with actin served as a loading control. (D) Histograms correspond to the results of C describing the relative protein levels in SC, SCM, and SCMEC on different culture day after each data point was normalized against actin. Protein level at day 1 in SCMEC was arbitrarily set at 1 against which statistical analysis was performed. Each bar is a mean7SD of n¼3 batches of samples. *Po0.05, **Po0.01.

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evaluation, and immunoblot analysis. All data presented in this report are results from at least 3 independent experiments. One-way ANOVA coupled with two-tailed Dunnett's test was applied to determine statistical significance between groups. Values represent mean7SD.

Results

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Ang1 enhances kinase activity of Akt in SCMEC barrier Since it has been reported that Ang1 has a strong endothelial survival effect through the Akt/phosphatidyl inositol-3 kinase (PI3K) pathway [26], we explored its role in the in vitro BSCB model herein. SCMEC cultured on day 7 after passaging were treated with either vehicle solution or Ang1, or Ang1 in combination with wortmannin (WM), a known inhibitor of PI3K/Akt pathway, and terminated at specified time points. The results

Cultured SCMEC permeability barrier assembly was accompanied with an increment of Eps8 expression Microvascular endothelial cells were isolated from rat spinal cord in this study. In this in vitro model, the branched microvessels began to adhere 2–3 h after isolation. After 2 days of culturing, the endothelial cells were observed to protrude from the germinal center. The confluence monolayer was formed 6 days after isolation, with the SCMEC represented typically “short spindlelike” or “polygon-like” appearance, which was coincident with the previous reports on CNS-originated microvascular endothelial cells [28,29,34–37]. To get a better visualization of the interested proteins, the primary SCMEC were passaged and seeded onto the collagen and fibronectin-coated coverslips at a relatively lower ratio of 1:5 in immunofluorescence analysis. It should be noted that at this ratio, the proportion of the “polygon-like” cells (Z65%) is larger than that of the “spindle-like” cells. Though the two different shaped cells display similar attributes in subsequent morphological assays, the polygonal SCMECs were selected to be shown to get a clearly demonstration of the junction proteins or F-actin distribution. After the first subculturing, a purity of (95.871.84)% was identified by using a specific antibody to factor VIII (von Willebrand Factor), which exhibited a distribution in SCMEC cytosol around the nucleus (Fig. 1A). In order to establish a functional SCMEC permeability barrier that mimics the BSCB in vivo, we also subcultured the confluenced endothelial cells on the upper inserts of the two chamber culture wells at a ratio of 1:1, which was then placed in the lower chambers that pre-seeded with glial cells as the feeder layer. From day 1 to day 7 after passaging, an increasing TEER value across the endothelium was detected, indicating the assembly of a functional SCMEC permeability barrier (Fig. 1B). Due to the importance of actin cytoskeleton organization in endothelial barrier establishment, the protein levels of several actin-binding proteins (ABPs), including espin, vinculin, fimbrin, Eps8 and Cdc42, were examined by immunoblot using SCMEC lysates on different culture days. The result showed that the expression level of Eps8 was significantly induced during the formation of the endothelial monolayer (Fig. 1C and D). This expression trend of Eps8 coincided with the increasing barrier function of SCMEC in vitro as indicated by TEER assay (Fig. 1B), implying that Eps8 might participate in the assembly of SCMEC barrier. Since the effects of angiopoietin1 on SCMEC were investigated in the following study, the steady-state level of Tie-2, the receptor of angiopoietin1 that can induce the growth and maturation of blood vessels in vivo [21], was also detected by immunoblot (Fig. 1C and D). Moreover, expression levels of tight junction (TJ)-associated proteins (occludin and ZO-1) and adherence junction (AJ)-associated proteins (VE-cadherin and β-catenin) exhibited no significant difference among SCMEC on different culture days (Fig. 1C and D).

Fig. 2 – Effects of Ang1 on phosphorylation state and activity of Akt in SCMEC cultured in vitro. (A) Immunoblot analysis of p-Akt1 and total Akt1 using lysate of SCMEC (35 lg) treated with vehicle, Ang1 (100 ng/ml), or a mixture of Ang1 (100 ng/ml) and WM (100 nM) and terminated at specified time points on culture day 7, with actin serving as a loading control. (B) Histogram corresponds to the result of (A) and depicts the ratio of p-Akt1/total Akt1 at each time point in the three groups. (C and D) Akt activity analysis using SCMEC lysates after Ang1 or Ang1þWM treatment. For each time point, cells were lysed and Akt kinase activity was detected by scintillation counting (C) or autoradiography (D) with H2B as the substrate. Each bar is a mean7SD of n¼ 3 batches of samples. Comparisons were made between vehicle and Ang1 group, or between Ang1þWM group and Ang1 group at the same time point, and significant differences were labeled by star signs (*) or pound signs (#), respectively. * or #, Po0.05. ** or ##, Po0.01.

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showed that the phosphorylation state of Akt (p-Akt, Ser473) detected by immunoblot increased after Ang1 treatment for 4 h as compared with the vehicle group. However, this effect of Ang1 was significantly suppressed in the presence of WM (Fig. 2A and B). Consistently, the kinase activity assay using H2B as substrate also revealed an enhanced Akt activity in the Ang1 group in a WM-sensitive manner in the lysates of cultured SCMEC (Fig. 2C and D).

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The enhancing effect of Ang1–Akt pathway on barrier integrity and Eps8 expression in SCMEC endothelial monolayer In order to reveal the possible effects of Ang1–Akt pathway on barrier function of SCMEC endothelial monolayer, we next quantified TEER across the SCMEC endothelium and evaluated its permeability to Na-F or EBA as described in the section Materials and

Fig. 3 – The functional roles of Ang1 in the integrity of cultured SCMEC endothelium barrier. (A) The establishment of a functional SCMEC barrier was assessed by quantifying TEER across the cell endothelium. On culture day 7 when the BSCB in vitro was fully established with a stable TEER, cells were treated in the same way as in Fig. 2 and TEER was quantified at specified time points. (B and C) The permeability of each group was assessed by transendothelial permeability of transport markers Na-F (B) and EBA (C) across the monolayer. Permeability values are calculated in the way described in the section Material and methods and indicated as a percent of the vehicle group at time 0. (D) Representative immunoblots showing the steady-state levels of Eps8 and junction proteins using SCMEC lysates terminated at each time points after different treatments, with actin serving as a loading control. (E) Histograms summarizing immunoblotting results shown in D and normalized against actin. Protein levels of the vehicle group at time 0 were arbitrarily set at 1. All data are presented as the mean7SD of at least n¼ 3 replicates. Comparisons were made between vehicle and Ang1 group, or between Ang1þWM group and Ang1 group at the same time point, and significant differences were labeled by star signs (*) or pound signs (#), respectively. * or #, Po0.05. ** or ##, Po0.01.

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methods. After Ang1 treatment, the SCMEC barrier was strengthened as indicated by a significant increase in TEER across the cell endothelium (Fig. 3A) and a decreased permeability to both Na-F and EBA (Fig. 3B and C) as compared with the control group. In addition, an up-regulation of the protein level of Eps8, but not the junction proteins such as occludin, ZO-1, VE-cadherin, and β-catenin, was also observed in Ang1-treated group (Fig. 3D and E). Furthermore, the enhancing effects of Ang1 on SCMEC barrier, including the increased TEER and the decreased permeability, were at least partially antagonized by the addition of wortmannin in the treatment solution (Fig. 3A–C). Besides, the stimulating effect of Ang1 on Eps8 expression also disappeared in the presence of the PI3K/Akt inhibitor (Fig. 3 D and E). These results implied the participation of Ang1–Akt in SCMEC barrier functions maintaining, which might involve the function of the actin-bundling protein, Eps8.

Angiopoietin1 regulates distribution of F-actin and junction protein interactions at the SCMEC interface via Akt activity Due to the change of Eps8 level after Ang1 treatment, F-actin distribution in SCMEC was visualized using rhodamineconjugated phalloidin. It was observed that Ang1 treatment alone

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led to a more concentrated alignment of F-actin at the cell–cell interface (Fig. 4A, b vs. a), while the addition of wortmannin weakened this effect (Fig. 4A, c vs. b). This suggested the role of F-actin organization in Ang1 induced barrier function increment. To further clarify the mechanisms by which Ang1 strengthens the barrier integrity of SCMEC, junction proteins that contribute to endothelium integrity were observed by immunofluorescence in Ang1-treated cells. The result showed that TJ-associate protein ZO-1 (Fig. 4A, h vs. g) and AJ-associate protein VE-cadherin (Fig. 4A, k vs. j) displayed intensified signals at the cell junction sites after Ang1 treatment but remained unchanged when wortmannin was applied additionally (Fig. 4A, i vs. g, and l vs. j). The intensified distribution of ZO-1 and VE-cadherin at cell junctions in response to Ang1 was further supported by co-IP experiments to detect junction protein interactions in the BSCB in vitro. As indicated by Fig. 4, after Ang1 treatment for 12 h, the interactions between occludin and ZO-1, and between VE-cadherin and β-catenin were both up-regulated to over 200% and over 150%, respectively. Similar with the above effects, these enhancements in junction protein interactions were greatly attenuated in the Ang1þWM group (Fig. 4 B and C). These findings indicated that Ang1–Akt pathway could regulate barrier function of SCMEC by modulating F-actin distribution, junction protein recruitment and interactions.

Fig. 4 – Ang1–Akt pathway regulates F-actin organization, junction proteins distribution and interactions in SCMEC. (A) F-actin (red) staining using rhodamine-conjugated phalloidin and immunofluorescence analysis of junction proteins (green), i.e. occludin, ZO-1, VEcadherin, and β-catenin, in SCMEC cultured on coverslips after treatment of vehicle, Ang1, or Ang1þWM for 12 h on day 7. Cell nuclei were visualized with DAPI (blue). Yellow arrowheads indicate more intense fluorescence signals as compared with the vehicle control group. To get a better visualization, the “polygon-like” SCMEC were shown herein. Scale bar, 25 lm, applying to all micrographs in A. (B) Co-IP results assess changes in protein–protein interactions between occludin and ZO-1, and between VE-cadherin and β-catenin using 500 lg protein from SCMEC lysates after treatment described above. Normal rabbit IgG was used to substitute for the precipitating antibodies in the negative control group. Equal IgG or antibody loading among groups was confirmed by the blot of IgGH and IgGL chains in the bottom blot. (C) Histogram summarizing the result presented in B in which the relative protein–protein interactions in vehicle group was set at 1 arbitrarily. Each bar represents a mean7SD of at least n¼ 3 independent experiments. Comparisons were made between vehicle and Ang1 group, or between Ang1þWM group and Ang1 group at the same time point, and significant differences were labeled by star signs (*) or pound signs (#), respectively. ** or ##, Po0.01.

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Knockdown of Eps8 by lentivirus-mediated shRNA has no effects on junction protein expression and Akt activity in SCMEC Based on findings reported in Figs. 1, 3, and 4, it appeared that the increasing expression of Eps8 in SCMEC following Ang1 treatment was related to the effects of Ang1 on SCMEC, including F-actin reorganization and junction protein interactions. Therefore, we investigated the role of Eps8 in this process using lentivirus-mediated shRNA targeting at Eps8 in cultured SCMEC as stated in the section

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Materials and methods. After infection with LV-shNC or LV-shEps8 for 12 h on culture day 2 and Ang1 treatment for 12 h on day 7, the lysates of SCMEC were detected by immunoblot and [32P] incorporation tests. The result showed that LV-shEps8 infection in this experiment led to 75% or 90% Eps8 knockdown in Ang1-untreated or Ang1-treated groups, respectively, as compared with the nontargeting group (Fig. 5A and B, LV-shEps8 vs. LV-shNC, or LVshEps8þAng1 vs. LV-shNCþAng1). That is to say, Ang1 could not induce the increment of Eps8 expression in SCMEC after LV-shEps8 infection. In addition, no changes in the expression of junction

Fig. 5 – Effects of Eps8 knockdown by lentiviral shRNA on junction protein expression and Akt activity in Ang1-treated and Ang1untreated SCMEC. (A) SCMEC were cultured alone for 2 days after passaging and were infected with specific LV-shEps8 or nontargeting LV-shNC for 12 h. Cultures were terminated on day 7 after treatment of Ang1 (100 ng/ml) for 12 h to obtain lysates for immunoblot analysis, which detected no effect on junction proteins (i.e. occludin, ZO-1, VE-cadherin, and β-catenin) expression and the ratio of p-Akt1/total Akt1 when Eps8 was silenced significantly either in Ang1-treated or Ang1-untreated groups. (B and C) Histograms based on immunoblot results shown in A after normalizing the data against actin (B) or calculating the ratio of p-Akt1/ Akt1 (C) in each group. (D and E) Akt activity analysis after Eps8 silencing in both Ang1-treated and Ang1-untreated SCMEC by scintillation counting (D) and autoradiography (E). Each bar is a mean7SD of at least n¼3 independent experiments. *Po0.05, ** Po0.01, NS, no significant difference between groups.

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proteins, including occludin, ZO-1, VE-cadherin, and β-catenin, were found within different groups (Fig. 5A and B). Although Ang1 could not change the steady-state level of Eps8 in LV-shEps8 infected SCMEC, the activity of Akt displayed an up-regulation after Ang1 treatment in both LV-shEps8 and LV-shNC infected groups (Fig. 5A, C–E, LV-shEps8 vs. LV-shEps8þAng1, or LV-shNC vs. LV-shNCþAng1).

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Another point should be notified was that knockdown of Eps8 did not affect Akt activity in either Ang1-treated or Ang1-untreated groups (Fig. 5A, C–E, LV-shEps8 vs. LV-shNC, or LV-shEps8þAng1 vs. LVshNCþAng1). These facts further prompt the assumption that Eps8 functions as a regulator of BSCB permeability in vitro downstream of Ang1–Akt pathway.

Fig. 6 – Eps8 knockdown antagonizes the effects of Ang1 on SCMEC endothelium. (A and B) The permeability of each group was assessed by transendothelial permeability of transport markers Na-F (A) and EBA (B) across monolayers on day 7 after Ang1 was added to LV-shNC or LV-shEps8 infected SCMEC at time 0. Permeability values are indicated as a percent of the LV-shNC group at time 0. (C) The integrity of SCMEC barrier was assessed by quantifying TEER across the cell endothelium from time 0 after Ang1 treatment in either LV-shNC or LV-shEps8 infected SCMEC. (D) F-actin (red, a–d) and junction proteins (green), i.e. ZO-1 (e–h), VE-cadherin (i–l), were stained by rhodamine-conjugated phalloidin and immunofluorescence, respectively, in SCMEC cultured on coverslips after treatment of Ang1 for 12 h in either LV-shNC or LV-shEps8 infected SCMEC. Yellow arrowheads indicate intensed fluorescence signals as compared with the non-targeting control group. Scale bar, 25 lm, applying to all micrographs in D. (E) Co-IP results assess changes in protein–protein interactions between occludin and ZO-1, and between VE-cadherin and β-catenin using 500 lg protein from SCMEC lysates after Ang1 treatment for 12 h on day 7 in both LV-shNC and LV-shEps8 groups. Equal antibody loading among groups was confirmed by the blots of IgGH and IgGL chains at the bottom. (F) Histogam summarizing the result presented in E in which the relative protein–protein interactions in LV-shNC infected group was set at 1 arbitrarily. Each bar represents a mean7SD of at least n ¼3 independent experiments. Comparisons were made vs. LV-shNC group at the same time point, *Po0.05, **Po0.01. NS, no significant difference between specified groups.

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Eps8 knockdown antagonizes the effects of Ang1 on SCMEC integrity To confirm the role of Eps8 in Ang1-induced SCMEC barrier function improvement, we investigated the permeability and integrity alteration after Eps8 knockdown in Ang1-treated or Ang1-untreated SCMEC. On day 7 after subculturing, the Ang1untreated SCMEC endothelium that had been infected with LV-shEps8 showed a discounted barrier function vs. the LV-shNC group, as indicated by the increased permeability to Na-F and EBA (Fig. 6A and B) and the decreased TEER value (Fig. 6C). This demonstrated the importance of Eps8 in SCMEC barrier function maintaining in vitro. Consistent with the results listed above, in the LV-shNC groups, a significant decrease in barrier permeability paralleled with an increment in TEER was detected in response to Ang1 treatment (Fig. 6A–C, LV-shNCþAng1 vs. LV-shNC). However, in Eps8-knockdown groups, no change in SCMEC barrier function was observed after Ang1 treatment (Fig. 6A–C, LV-shEps8 vs. LV-shEps8þAng1). These findings indicated Ang1 participates in the regulation of SCMEC barrier, at least partially, via its positive effect on Eps8 expression. Since Ang1 was shown to influence F-actin and junction proteins distribution as well as interactions in SCMEC (Fig. 4), we next explored the relationship between these effects and Eps8 expression. The results showed that in the LV-shEps8 groups, the F-actin signal remained unchanged after Ang1 treatment (Fig. 6D, c and d), which was quite different from the case in the LV-shNC groups (Fig. 6D, a and b). This implied the effect of Ang1 on F-actin re-organization in SCMEC was through Eps8, the F-actin bundling and barbed-end capping protein. Interestingly, after Eps8 knockdown, the distribution of ZO-1 (Fig. 6D, g and h) and VE-cadherin (Fig. 6D, k and l) and their respective interactions with occludin and β-catenin (Fig. 6E and F) also displayed no response to Ang1 treatment. These discrepant effects of Ang1 based on Eps8 expression level collectively confirmed the importance of Eps8 during SCMEC barrier function regulation by Ang1– Akt pathway.

Discussion In microcirculation of the spinal cord, endothelial cells composed capillaries form an anatomically unique structure, the BSCB, which strictly regulates microenvironment of the spinal cord. In the studies of blood–CNS barriers, in vitro models that could mimic the BBB or the BSCB have been established and developed during the past decades [28,29,38,39]. In this report, microvascular endothelial cells from rat spinal cord (SCMEC) were isolated and co-cultured with mixed glial cells from postnatal rats, which has long been demonstrated to induce barrier function at the blood–CNS barriers [40]. By referring to the previously reported methods with minor modifications, the purity of cultured SCMEC was optimized to 95% when detected with the factor VIIIspecific antibody [28,29]. As expected, a reproducibly increasing barrier integrity was observed after the primary SCMEC were passaged and co-cultured with glial cells in two chamber culture wells, with the TEER value reaching a maximum of 45–50 Ω cm2 on days 6–7. Ang1 has its unique role in vascular maintenance. The Aktmediated anti-apoptotic effects were reported to participate in

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Ang1 stabilization of vascular structures [23,26]. In coronary artery endothelium, the activation of PI3K/Akt by Ang1 was confirmed to induce NO synthesis via endothelial nitric oxide synthase (eNOS) and further regulate vascular reactivity to endotoxin shock [41]. At this point, we detected the activity of Akt by [32P]-incorporation test using lysates from SCMEC cultured in the presence of Ang1. As the result shown in Fig. 2, Akt1 activity increased significantly vs. the vehicle group after 4-htreatment of Ang1. Nevertheless, this effect of Ang1 was found to disappear in the presence of wortmannin, the known inhibitor of PI3K/Akt pathway. These findings indicate the regulation of PI3K/Akt by Ang1 might be a general phenomenon in endothelial cells. The strengthening effects of Ang1 on endothelial monolayer integrity were also confirmed in this in vitro BSCB model by TEER assay and permeability evaluation. Interestingly, the enhancing effects of Ang1 on SCMEC barrier were also partly reversed by wortmannin, which further confirmed the role of Akt in Ang1induced SCMEC barrier maintaining. In endothelial barriers, the TJ complexes between adjacent cells are composed of three types of proteins (claudins, occludin, and junctional adhesion molecules) which bind to PDZ domains of zonula occludens (ZO-1,-2, and -3) via their cytoplasmic tails. Adherence junctions (AJs) formed by homophilic interactions between cadherins and nectins, whose cytoplasmic tails associate with a series of intracellular catenins or afadins, are located immediately underneath TJs [40,42,43]. The integrity of these junction apparatus determines permeability and barrier function of the endothelium. Herein, the expressions of occludin, ZO-1, VE-cadherin and β-catenin in the SCMEC lysates were detected to decide whether it was by modifying the protein level of these proteins that Ang1–Akt pathway exerted its role on SCMEC barrier. Nevertheless, the result showed no changes in the steady-state level of these proteins after treatment of Ang1. Thus, we further investigated the distribution of these proteins and the organization of F-actin cytoskeleton by fluorescence analysis. It was shown that after Ang1 treatment, the signals of F-actin, ZO-1 and VE-cadherin became more concentrated and intensified at the cell–cell interface. Moreover, the Ang1 treatment also led to an up-regulation in junction protein interactions, which, together with the localization changes, could be antagonized at least partially by wortmannin. These observations strongly suggested that Ang1 could affect the barrier function of SCMEC through the activity of PI3K/Akt pathway and this process might involve the modulation of actin dynamics and protein interactions at the cell junction sites. Intact and functional organization of actin microfilaments is crucial for the maintenance of endothelial and epithelial barriers including the blood–CNS barriers by keeping cell–cell adhesion and proper permeability [44,45]. The dynamics of actin comprises a series of biochemical events such as bundling, branching, nucleation, cross-linking, polymerization, depolymerization, severing and capping, which involve an array of actinbinding and regulatory proteins. In the present study, we found the assembly of the BSCB in vitro was accompanied with an increasing expression of Eps8, an important actin dynamic regulator which controls actin-based motility by capping the barbed ends and regulates actin organization by activating Rac GTPase [14,16]. Moreover, the expression level of Eps8 was revealed to rise in response to Ang1, which was also partly reversed by the additional treatment of wortmannin. Therefore,

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it can be speculated that the Ang1–Akt pathway might regulate SCMEC endothelial barrier function via Eps8. Actually, in vascular endothelial cells, Akt activation has been reported to potentially influence cell motility through modulating actin [46,47]. Although individual studies reported that actin could be a direct substrate of Akt [48], more investigations discovered that Akt participated in actin cytoskeleton dynamics by affecting the status of actin-binding proteins, such as girdin [49], filamin A [50], the sodium–hydrogen exchanger isoform 1 (NHE1) [51], tuberous sclerosis complex 2 (TSC2) [52], palladin [53], etc. Eps8 was originally identified as an intracellular substrate for the EGFR, in which tyrosyl phosphorylation was increased in response to EGF [54]. Through its SH3 domain, Eps8 binds to Abi and further forms a ternary complex with Sos1, representing Rac-specific GEF (guanine exchange factors) activity. The C-terminal “effector region” of Eps8 determines its localization within the cell and the activation of Rac GTPase, which leads to actin cytoskeletal remodeling [17]. To verify the speculation that the Ang1-induced Akt activity elevation could regulate SCMEC barrier function via the actin-bundling capability of Eps8, we proceeded on to knockdown Eps8 by lentivirus-mediated shRNA in the in vitro BSCB. After Eps8 expression was silenced by 75–90%, the Ang1-induced barrier enhancement disappeared, including the reduction in Na-F or EBA permeability and the increment in TEER value. This might be explained by the failure of Ang1 in causing the redistribution of F-actin and junction proteins at the cell–cell interface in the Eps8-knockdown SCMEC. Furthermore, the up-regulation in interactions between junction proteins in Ang1-treated SCMEC was also not observed after Eps8 silencing. Thus, it can be concluded that Eps8 participated in the barrier function strengthening caused by Ang1 in SCMEC. This coincides with the observation in the blood– testis barrier in which the knockdown of Eps8 also led to a discounted barrier function in the seminiferous epithelium [18]. Although the data we got at present could not describe the precise mechanisms by which Eps8 regulates F-actin and junction protein distribution in response to Ang1, it will be helpful to investigate the existence and GEF activity of the Eps8–Abi–Sos1 complex in this in vitro model in the near future due to the importance of Rac activity in junction protein complex stabilization [55]. In summary, we here report that Ang1 acts on cultured SCMEC via its effect on Akt activity and further on Eps8, the actin-bundling protein, which enhances the F-actin organization at cell junction sites and causes the strengthening of the BSCB model in vitro. Notably, after Ang1 treatment in this study, the increment of Akt activity in Eps8-silenced group resembled that in the non-targeting control group. That is to say, Eps8 knockdown could antagonize the barrier function enhancement caused by Ang1 without influencing Ang1induced Akt activity elevation, which meant that Eps8 functions downstream of Akt in the regulation of SCMEC endothelial barrier. However, in the studies investigating the role of Eps8 in squamous carcinogenesis, Eps8 was found to activate Akt by stimulating PI3K. In the Eps8-overexpression HN4 tumer cells, the MMP-9 activity was also elevated in a PI3K/Akt manner [56]. In this context, much work is needed in future studies to elucidate the exact interaction relationship between Eps8 and Akt in different cell types.

Conflict of interest statement The authors declare that they have no conflicts of interest, financial or otherwise.

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Disclosure statement The author has nothing to disclose.

Acknowledgments This work was supported by Scientific Research Fund of the First Affiliated Hospital of China Medical University (FSFH1206) to Xinchun Liu.

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