A novel anti-inflammatory mechanism of high density lipoprotein through up-regulating annexin A1 in vascular endothelial cells

    A novel anti-inflammatory mechanism of high density lipoprotein through Up-regulating annexin A1 in vascular endothelial cells Bing Pan, Jinge Kong, Jingru Jin, Jian Kong, Yubin He, Shuying Dong, Liang Ji, Donghui Liu, Dan He, Liming Kong, David K. Jin, Belinda Willard, Subramaniam Pennathur, Lemin Zheng PII: DOI: Reference:

S1388-1981(16)30074-9 doi: 10.1016/j.bbalip.2016.03.022 BBAMCB 57942

To appear in:

BBA - Molecular and Cell Biology of Lipids

Received date: Revised date: Accepted date:

19 October 2015 1 March 2016 18 March 2016

Please cite this article as: Bing Pan, Jinge Kong, Jingru Jin, Jian Kong, Yubin He, Shuying Dong, Liang Ji, Donghui Liu, Dan He, Liming Kong, David K. Jin, Belinda Willard, Subramaniam Pennathur, Lemin Zheng, A novel anti-inflammatory mechanism of high density lipoprotein through Up-regulating annexin A1 in vascular endothelial cells, BBA - Molecular and Cell Biology of Lipids (2016), doi: 10.1016/j.bbalip.2016.03.022

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ACCEPTED MANUSCRIPT A Novel Anti-inflammatory Mechanism of High Density Lipoprotein Through Up-regulating Annexin A1 in Vascular Endothelial Cells

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Bing Pan1#, Jinge Kong2#, Jingru Jin3, 5, Jian Kong4, Yubin He5, Shuying Dong4, Liang Ji1,

Pennathur9, *Lemin Zheng1

The Institute of Cardiovascular Sciences and Institute of Systems Biomedicine, School of

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# B. P. and J. K. contributed equally to this work.

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Donghui Liu1, Dan He1, Liming Kong6, David K. Jin7, Belinda Willard8, Subramaniam

Basic Medical Sciences, and Key Laboratory of Molecular Cardiovascular Sciences of

Neuroscience Research Institute, Key Laboratory for Neuroscience (Ministry of Education),

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Ministry of Education, Peking University Health Science Center, Beijing 100191, China.

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Key Laboratory for Neuroscience (National Health and Family Planning Commission), Department of Neurobiology, School of Basic Medical Sciences, Health Science Center,

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Peking University, Beijing 100191, China. 3

Southern Medical University, Guangzhou 510515, China.

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Department of Hepatobiliary Surgery, West Campus, Beijing Chao-yang Hospital Affiliated

to Capital Medical University, No. 5 Jingyuan Road, Shijingshan District, Beijing, China. 5

The Military General Hospital of Beijing, Beijing 100700, China.

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Department of Pediatrics, Beijing Ren-he Hospital, Beijing 102600, China.

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Ansary Stem Cell Institute, Weill Cornell Medical College of Cornell University, New York,

NY 10027, USA. 8

Proteomics Laboratory, Cleveland Clinic, Cleveland, OH 44195, USA. 1 / 42

ACCEPTED MANUSCRIPT 9

Department of Medicine, University of Michigan, Ann Arbor, MI 48109, USA.

* Corresponding author contact information

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Lemin Zheng, Ph.D.

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Institute of Cardiovascular Sciences, Peking University Health Science Center Address: 38 Xueyuan Road, Haidian District, Beijing 100191, China. Phone: 086-010-82805452, fax: 086-010-82802769

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E-mail: [email protected]

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ACCEPTED MANUSCRIPT Abstract

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High density lipoprotein (HDL) as well as annexin A1 has been reported to be associated with

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cardiovascular protection. However, the correlation between HDL and annexin A1was still unknown. In this study, HDL increased endothelial annexin A1 and prevented decrease of annexin A1 in TNF-α-activated endothelial cells in vitro and in vivo, and above effects were

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attenuated after knockdown of annexin A1. Annexin A1 modulation affected HDL-mediated

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inhibition of monocyte adhesion to TNF-α-activated endothelium (45.2±13.7% decrease for Annexin A1 RNA interference; 78.7±16.3% decrease for anti-Annexin A1 antibody blocking;

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11.2±6.9% increase for Ad-ANXA1 transfection). Additionally, HDL up-regulated annexin

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A1 through scavenger receptor class B type I, involving ERK, p38MAPK, Akt and PKC

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signaling pathways, and respective inhibitors of these pathways attenuated HDL-induced annexin A1 expression as well as impaired HDL-mediated inhibition of monocyte-endothelial

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cells adhesion. Apolipoprotein AI also increased annexin A1 and activated similar signaling pathways. Endothelial annexin A1 from apolipoprotein AI knockout mice was decreased in comparison to that from wild type mice. Finally, HDL-induced annexin A1 inhibited cell surface VCAM-1, ICAM-1 and E-selectin, and secretion of MCP-1, IL-8, VCAM-1 and E-selectin, thereby inhibiting monocyte adhesion.

Keywords: High density lipoprotein, annexin A1, endothelial cell, monocyte adhesion

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ACCEPTED MANUSCRIPT Abbreviations HDL: high-density lipoprotein;

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CVD: cardiovascular disease;

ROS: reactive oxygen species; TNF-α: tumor necrosis factor α;

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HUVECs: human umbilical vein endothelial cells;

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ANXA1: annexin A1; ANXA5: annexin A5;

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SR-BI: scavenger receptor class B type I; apo: apolipoprotein

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NO: nitric oxide;

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TGF-β: transforming growth factor β; PKC: protein kinase C;

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MAPK: mitogen-activated protein kinase; ERK: extracellular regulated protein kinases; EMPs: endothelium microparticles;

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ACCEPTED MANUSCRIPT Introduction

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Cardiovascular disease (CVD), which accounted for almost 30% of all deaths according to the

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2013 Global Burden of Disease Study, is reported to be one of the leading causes of death worldwide. [1] Many cardiovascular risk factors can overwhelm defense mechanisms of the vascular endothelium and trigger endothelial dysfunction, ultimately leading to CVD.[2]

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Endothelial dysfunction, which plays a significant role in the initiation and progression of

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CVD, is commonly associated with an imbalance of many factors including Nitric Oxide (NO) bioavailability, reactive oxygen species (ROS) and Thromboxane A2, exhibiting

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pro-inflammatory, pro-oxidant, proliferative, pro-coagulation and pro-vascular adhesion

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therapy of CVD.

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features.[3] Therefore, to prevent endothelial dysfunction will be of great importance for

Annexin A1 (ANXA1), the first identified member of the annexin superfamily, is

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characterized by harboring the Ca2+ and phospholipid-binding sites with high biological and structural homology to other annexin superfamily members. It is a key modulator with potent inhibition of phospholipase A2 activity in the early inflammatory responses.[4, 5] Recent studies have revealed that endothelial expression of ANXA1 was impaired during endothelial dysfunction and vascular inflammation.[6, 7] ANXA1 expression in atherosclerotic plaques was higher in patients without clinical symptoms compared to those with cerebrovascular symptoms.[8] Moreover, ANXA1 (or ANXA1 receptor) and ApoE double-mutant enhanced atherosclerotic lesion formation, arterial myeloid cell adhesion, and recruitment.[9] ANXA1 peptide Ac2-26 can reduce atherosclerotic lesion sizes and lesion macrophage 5 / 42

ACCEPTED MANUSCRIPT accumulation.[10, 11] These observations indicated that ANXA1 might be a novel protective factor or target for endothelium integrity, atherosclerotic plaques and CVD. Furthermore,

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further exploring the role and protective mechanism of ANXA1 in endothelium may provide

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clues in preventing progression of atherosclerosis as well as CVD, since endothelial dysfunction is a crucial pathological process involved.

It is well-known that HDL is identified as an athero-protective factor involving its endothelial

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protection, including vaso-relaxation, anti-oxidant, anti-inflammatory and anti-thrombotic

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effects.[12] The anti-inflammatory mechanism of HDL is related to inhibition of many factors including E-selectin, cell adhesion molecules [13, 14] and several cytokines [15]. Interestingly,

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our primary work demonstrated up-regulation of ANXA1 by HDL, implicating a relationship

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between anti-inflammation of HDL and ANXA1 in endothelium. Therefore, our present work

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sets forth the role and mechanism of HDL-induced ANXA1 in endothelium.

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ACCEPTED MANUSCRIPT Materials and Methods

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Ethics Statement

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Human umbilical vein endothelial cells (HUVECs) were isolated by collagenase digestion of umbilical veins from fresh cords which were from donors with written informed consent.[16] This study protocol was approved by the ethics committee of the Military General Hospital of

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Beijing. All animal experimental procedures were approved by the Ethics Committee of

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Animal Research, Peking University Health Science Center. The investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the US National

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Institutes of Health.

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Cell Culture and Materials

Monocytic THP-1 cells were provided by Cell Resource Center, Chinese Academy of Medical

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Sciences, Peking Union Medical College. THP-1 cells were cultured in RPMI 1640 medium (R1640; GIBCO, UK), supplemented with 10% fetal bovine serum (FBS) in a humidified incubator at 37 °C with an atmosphere of 5% CO2. HUVECs were cultured in Endothelial Cell Medium (ECM; Science, USA) supplemented with 5% fetal bovine serum, 1% endothelial cell growth supplement and 1% penicillin/streptomycin solution. All cells were grown on 0.1% (w/v) gelatin-coated culture ware and then cultured in a humidified incubator at 37 °C with an atmosphere of 5% CO2. HUVECs were used at passages 2-5. Mouse aortas endothelial cells (MAECs) were isolated as described previously.[17] Briefly, 7 / 42

ACCEPTED MANUSCRIPT wild type C57BL/6 (WT) and SR-BI knockout (SR-BI -/-) mice, provided by Prof. George Liu from Peking University, were used at the age of 8-12 weeks. Genotyping was done using

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genomic DNA extracted from tails by PCR (see supplemental figure I). Mouse aortas were

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excised under anaesthesia with sodium pentobarbital (50 mg/kg, i.p.). The adequacy of anaesthesia was monitored through pinching the hind paw, and the sufficiently sedated mice were euthanized through cervical dislocation. 2 mm sections of mouse aortas were placed on

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matrigel pre-coated plates and cultured in ECM at 37°C for 7–14 days to promote MAEC

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outgrowth prior to passaging. MAECs were used at passage 2 to 3 and were stained with anti-CD31 to confirm endothelial cells phenotype (Data not shown). All cell experiments

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were performed under serum-free condition.

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Inhibitors were purchased from Sigma-Aldrich (St. Louis, MO), including SB203580 (p38

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MAPK), PD98059 (ERK1/2), LY294002 (PI3K) and Staurosporine (PKC). Recombinant human tumor necrosis factor α (TNF-α) was purchased from BD Biosciences. Mouse

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monoclonal anti-ANX1 antibody and rat monoclonal anti-CD34 antibody were purchased from BD Biosciences. Rabbit polyclonal anti-ANX1 antibody and rat monoclonal anti-CD31 antibody were purchased from Invitrogen Corporation. Rabbit monoclonal anti-Scavenging Receptor class B type I (SR-BI) antibody and goat polyclonal anti-SM22 antibody were purchased from Abcam. Rabbit polyclonal anti-ICAM-1, anti-VCAM-1, anti-E-selectin antibodies, mouse monoclonal anti-β-actin antibody, FITC -goat-anti-rabbit IgG, FITC-goat-anti-mouse IgG and PE-goat-anti-rat IgG were purchased from Boster (Wuhan, China). Rabbit monoclonal anti-phospho-ERK1/2, anti-phospho-p38 MAPK and anti-phospho-Akt antibodies were purchased from Cell Signaling Technology (Danvers, MA). 8 / 42

ACCEPTED MANUSCRIPT Anti-ERK1/2 antibody, anti-p38MAPK antibody, anti-Akt antibody and goat IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Cy3-donkey-anti-goat IgG were

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purchased from Beyotime Biotechnology (Shang Hai, China). HRP-goat-anti-rabbit IgG and

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HRP-goat-anti-mouse IgG were purchased from MBL (Nagoya, Japan)

Isolation of Lipoproteins

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Fresh, fasting plasma was separated by centrifugation from peripheral blood obtained from

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healthy subjects. This part was approved by the local ethics committee. HDL (d=1.063–1.210 g/ml) was isolated by sequential ultracentrifugation as described elsewhere.[18] Resulting

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preparations of lipoproteins were dialyzed against four changes of phosphate-buffered saline

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(PBS) (pH=7.4) containing 1 mM EDTA and 100 μM diethylenetriamine pentaacetic acid

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(DTPA) (Sigma, USA), sterilized with 0.22-μm filter, stored away from light at 4 °C and used within 2 months. HDL concentration was determined by immunoturbidimetrical detection of

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apolipoprotein A-I (Roche Diagnostic).

Western Blot

After treatment, cells were washed with PBS and lysed in RIPA buffer (50 mM Tris (pH 7.4), 0.5% NP-40, 150 mM NaCl, 0.1% SDS, 2 mM EGTA, 2 mM EDTA, protease inhibitors and protein phosphatase inhibitors (used to detect phosphorylated proteins)). Cell lysates were collected and quantified using the Bradford protein assay (Bio-Rad). Equal amounts of total protein were loaded onto SDS-PAGE gels and blotted onto nitrocellulose membranes. Membranes were blocked in 5% non-fat milk for 2 hours, and sequentially incubated with 9 / 42

ACCEPTED MANUSCRIPT respective primary antibodies for 4 hours and appropriate HRP-conjugated secondary antibodies for 2 hours at room temperature. The specific immune-reactive protein bands were

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detected by ECL kit (Pierce, USA) and analyzed using Quantity One 1-D Analysis Software

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(Bio-Rad, CA).

Real time PCR Analysis

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Total RNA was extracted with TRIzol (Invitrogen, USA). Concentration and purity of RNA

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were determined by absorbance at 260 and 280 nm. RNA was used as template for cDNA synthesis with M-MLV kit (KEYGEN) and assessed by SYBR real-time PCR. Quantitative

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real-time PCR was performed on DNA Engine Opticon System (MJ research Inc, USA) using

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Power SYBR ® Green PCR Master Mix kit in triplicate.

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The PCR primers synthesised by Sangon (China) were as follows: ANXA1 [5’-GAGAAATGCCTCACAGCTATCGT-3’ (forward);

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5’-AGTTTCCTCCACAAAGAGCCAC-3’ (reverse)], GAPDH [5’-CGGAGTCAACGGATTTGGTCGTAT-3’ (forward); 5’-AGCCTTCTCCATGGTGGTGAAGAC-3’ (reverse)]. GAPDH was used as a housekeeping gene to normalize expression. Threshold cycle number (Ct) was normalized into relative using 2-△△Ct method, △△Ct = (Ct, ANXA1 -Ct, GAPDH) treatment - (Ct, ANXA1-Ct, GAPDH) control.

Immuno-fluorescent Staining For immunocytochemistry and immunohistochemistry, immuno-fluorescent staining was 10 / 42

ACCEPTED MANUSCRIPT performed as described previously.[19] Briefly, following proteins were detected. Mouse monoclonal anti-ANXA1 antibody (diluted 1:1000) for HUVECs; rabbit polyclonal

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anti-ANXA1antibody (diluted 1:200), rat monoclonal anti-CD34 (1:100), rat monoclonal

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anti-CD31 antibody (1:100), goat anti-SM22 antibody (diluted 1:200) and goat IgG (diluted 1:200) for animal tissues. Goat IgG or PBS was used as negative control. Nuclear localization was counterstained with DAPI. Images were obtained under a laser-scanning confocal

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microscope (TCS SP5; Leica, German). The relative fluorescent intensity of ANXA1 in

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HUVECs was determined by Leica QWin Analysis Software.

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siRNA Knockdown of ANXA1 or SRBI

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ANXA1 siRNA, SRBI siRNA and scramble siRNA were synthesized by Shanghai

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GenePharma Co (Shanghai, China). HUVECs were plated onto 12-well plates and allowed to grow to sub-confluent. Cells were transiently transfected with the siRNA by lipofectamine

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RNAi MIX reagent (Invitrogen, Carlsbad, CA) in OPTI-MEM medium (Gibco) for 6 h, incubated in ECM with 5% FBS for 30 h, and treated by HDL (100 μg/ml) or PBS (equal volume) with or without the presence of TNF-α (2 ng/ml) for an additional 12 h. Cells were then used for further experiments.

Over-expression of ANXA1 Empty vectors and adenovirus ANXA1 (Ad-ANXA1) were synthesized and supported by SinoGeno Max Co (Beijing, China). HUVECs were plated onto 12-well plates and allowed to grow to sub-confluent. Cells were transiently transfected with empty vectors and Ad-ANXA1 11 / 42

ACCEPTED MANUSCRIPT in ECM for 6 h, incubated in ECM with 5% FBS for 30 h, and treated by HDL (100 μg/ml) or PBS (equal volume) with or without the presence of TNF-α (2 ng/ml) for an additional 12 h.

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Cells were then used for further experiments.

Monocyte Adhesion Assay

THP-1 cells were labeled with the fluorescent dye Calcein-AM (10 μM) in RPMI 1640 for 20

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min. HUVECs were seeded into 12-well plates, allowed to grow to 80% confluence, and

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treated with test reagents for 12 h. For co-culture experiment, THP-1- labeled cells (5×105 cells/well) were overlaid on the confluent monolayer of HUVECs. After incubating for 1 h at

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37 °C, non-adherent THP-1 cells were gently washed three times with PBS. Images of 6

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random high power (100×) fields were obtained at 485 nm excitation and 538 nm emission

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under fluorescence microscopy (Leica, German). The number of adherent THP-1 cells was

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determined by Leica QWin Analysis Software.

HUVECs were transfected with siRNA of ANXA1 to attenuate the level of ANXA1, and transfected with Ad-ANXA1 to over-express the level of ANXA1. Following treatment of test reagents for 12 h, monocyte adhesion assay was performed as described above.

HUVECs were treated with test reagents for 12 h. Thereafter, HUVECs were pre-incubated with neutralizing anti-ANXA1 monoclonal antibodies (20 μg/ml) or anti-IgG antibody (20 μg/ml) at 37 °C for 60 min. Monocyte adhesion assay was performed as described above.

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ACCEPTED MANUSCRIPT Animal Experiments Six-week old male BALB/c nude mice (n = 24) were maintained in the Laboratory Animal

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Center of Peking University Health Science Center, and housed in laminal-flow cabinets

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under specific pathogen-free (SPF) conditions. Mice were randomly divided into four groups. Mice received human HDL (10 mg/kg) by tail intravenous injection every other day or recombinant human TNF-α (0.1 mg/kg) by daily intra-peritoneal injection. Vehicle group

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received PBS by tail intravenous injection every other day, HDL + TNF-α group was

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pretreated with HDL for 24 h before given TNF-α. After 3 days’ treatment, animals were euthanized with 1% pelltobarbitalum natricum and thoracic aorta was isolated for fixation in 4%

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paraformaldehyde solution. To preserve tissues for histological characterization, surgically

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resected tissues were embedded in optimal cutting temperature (OCT) compound.

Immunohistochemical Analysis

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OCT-embedded tissue sections were cut into standard 7-μm sections. After the antigen retrieval and endogenous peroxidases inactivation, these sections were incubated at 37 °C with rabbit polyclonal anti-ANXA1 antibody (dilution 1:1000) for 2 h in a humidified chamber and HRP-conjugated-anti-rabbit antibody for 40 min at 37 °C. Images of 15 random high power (400×) fields were taken with under light microscope (Olympus, Leeds Precision Instruments). Relative ANXA1expression on intima of thoracic aorta was determined by Leica QWin Analysis Software.

PepTag Assay for Nonradioactive Detection of PKC Activity 13 / 42

ACCEPTED MANUSCRIPT PKC activity was determined by non-radioactive detection kit of protein kinase (PepTag Corporation, USA) according to the manufacturer's instructions. Briefly, PKC in HDL-treated

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or apoAI-treated cell lysates was separated by column of DEAE cellulose, and incubated with

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the brightly colored, fluorescent peptide substrates. The phosphorylation by PKC of its specific substrate altered the peptide’s net charge from +1 to –1, which allowed the phosphorylated and non-phosphorylated versions of the substrate to be rapidly separated on

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an agarose gel.

Cell Surface enzyme linked immunosorbent assay

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HUVECs transfected with ANXA1 siRNA or scramble siRNA for 20 h were seeded by the

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same amount onto 96-well plates with 100% confluence. Cells were washed twice and

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incubated in serum-free ECM with test reagents for 12 h. Thereafter, samples were washed with PBS and fixed with methanol at room temperature. The plates were blocked with 2%

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BSA in PBS for 2 h at 37 °C. Cell surface expression of target molecules was determined by incubating primary antibodies for VCAM-1, ICAM-1 or E-selectin for 3 h at 37 °C and sequential secondary HRP-conjugated goat anti-rabbit IgG antibody for 2h at 37 °C as described previously.[20] Quantification was performed by determination of colorimetric conversion at OD at 450 nm of 3, 3’, 5, 5’-tetramethylbenzidine using TMB peroxidase EIA substrate kit (Bio-Rad).

Quantification of secreted MCP-1, IL-8, VCAM-1, and E-selectin HUVECs were transfected with ANXA1 siRNA, scramble siRNA, empty vectors or 14 / 42

ACCEPTED MANUSCRIPT Adenovirus ANXA1 vectors for 20 h and allowed to grow until 80% confluence. Cells were washed twice and incubated in serum-free ECM with different test reagents for 12 h.

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Thereafter, the media were harvested and spun down at 12, 000 g, 10s. The supernatant was

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collected and quantified using MCP-1, IL-8, VCAM-1, or E-selectin enzyme linked immunosorbent assay (ELISA) kits (Boster, China) according to the instructions. Finally, the concentrations of MCP-1, IL-8, VCAM-1, or E-selectin were normalized to total cellular

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protein.

Statistical Analysis

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All data were presented as the means ± SEM. They were performed at least 3 different

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separate experiments. Data were analyzed using one-way analysis of variance followed by

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Bonferroni’s test for samples. All analyses were performed using GraphPad Prism (GraphPad

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Software, USA), and values were considered significant at P< 0.05.

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ACCEPTED MANUSCRIPT

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HDL Up-regulated Expression of ANXA1 in Endothelial Cells

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Results

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Experiments were performed on HUVECs. It was found that HDL up-regulated ANXA1 expression in a dose-dependent (Fig. 1A-B) and time-dependent manners (Fig. 1C-D). 100ug/ml HDL increased ANXA1 expression by 80% (P<0.001). Also, real time PCR

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detection of ANXA1 showed that mRNA level of ANXA1 was up-regulated by HDL (Fig.

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1E). Subsequent immune-fluorescence assay demonstrated that HDL could up-regulate

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ANXA1 in HUVECs (Fig. 1F and G).

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HDL Prevented the Decrease of ANXA1 in TNF-α-activated Endothelial Cells in vitro

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and in vivo

Several studies identified ANXA1 as a mediator of anti-inflammation, especially in immune

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cells, such as neutrophils, monocytes and mast cells.[21] Therefore, this study further explored the effect of HDL on ANXA1 expression in TNF-α-activated vascular endothelial cells. It was found that TNF-α dose-dependently reduced ANXA1 expression in HUVECs (Fig. 2A-B). HDL prevented decrease of ANXA1 by TNF-α (P<0.05). Besides, there was no significant difference in ANXA1 expression between TNF-α and HDL+ TNF-α after knockdown of ANXA1, which demonstrated that HDL didn’t exhibit above effect (Fig. 2C-D). Subsequently, male BALB/c nude mice were used to detect the effect of HDL and TNF-α on ANXA1expression in vivo. As shown in Fig. 2E and 2F, HDL (10 mg/kg) induced ANXA1expression on the intima of thoracic aorta (n = 6; P<0.001). TNF-α (0.1 mg/kg) 16 / 42

ACCEPTED MANUSCRIPT reduced ANXA1 expression (n = 6; P<0.001), HDL prevented decrease of ANXA1 by TNF-α (n = 6; P<0.001). In addition, we selected the representative groups to detect ANXA1 in

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vascular endothelial cells of thoracic aorta using confocal imaging. ANXA1was detected in

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endothelium (Fig. 2G) and smooth muscle cells (see supplemental figure II). Goat IgG was negative control for SM22. Supplemental figure IIC showed that connective tissue auto-fluorescence cannot be detected under these parameters. These representative pictures

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indicated that the vascular endothelial ANXA1 was up-regulated by HDL in vivo (Fig. 2G).

HDL-induced ANXA1 Exerted an Inhibitory Effect on Monocytes Adhesion to

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Endothelial Cells

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Both exogenous recombinant ANXA1 and monocytes-derived ANXA1 were involved in

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inhibiting the firm adhesion of monocytes to endothelium.[5, 22] It is well known that HDL has endothelial protection including anti-inflammatory function. Therefore, monocyte

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adhesion assay was performed to investigate the effect of HDL-up-regulated ANXA1 in activated endothelial cells. As shown in figure 3B-C, HDL inhibited TNF-α-stimulated THP-1 cells adhesion by 93±10.7% (P<0.001), while HDL only inhibited TNF-α-stimulated THP-1 cells adhesion by 65.9±24.6% (P<0.05) after knockdown of ANXA1. Knockdown of ANXA1 attenuated HDL-mediated inhibition of THP-1 cells adhesion by 45.2±13.7% (P<0.05; Fig. 3B-C) in comparison to negative control. According to figure 3D-E, HDL inhibited TNF-α-stimulated THP-1 cells adhesion by 85±6.0% (P<0.001) after pre-incubation with anti-IgG anti-body, while HDL could not inhibit TNF-α-stimulated THP-1 cells adhesion with statistical significance after pre-incubation with anti-ANXA1 blocking antibody. 17 / 42

ACCEPTED MANUSCRIPT HUVECs-pre-incubated with anti-ANXA1 blocking antibody attenuated HDL-mediated inhibition of THP-1 cells adhesion by 78.7±16.3% (P<0.001; Fig. 3D-E) in comparison to

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pre-incubation with anti-IgG antibody. Furthermore, we used transfection of Ad-ANXA1 to

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over-express endothelial ANXA1. We found that HDL inhibited TNF-α-stimulated THP-1 cells adhesion by 91.5±6.5% (P<0.001) after transfection of empty vector, and HDL also inhibited TNF-α-stimulated THP-1 cells adhesion by 88.0±9.0% (P<0.001) after transfection

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of Ad-ANXA1. Besides, despite transfection of Ad-ANXA1 enhanced HDL-mediated

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inhibition of THP-1 cell adhesion by 11.2±6.9% at P=ns (Fig. 3F-G) in comparison to transfection of empty vector, over-expression ANXA1 inhibited the THP-1 cells adhesion to

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the TNF-α activated HUVEC by 22.4±4.8% (P<0.001), which demonstrated that ANXA1

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could prevent inflammation response of TNF-α activated endothelial cells.

HDL Up-regulated ANXA1 Partially through SR-BI and Several Signaling Pathways

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To investigate how HDL up-regulated ANXA1 expression in endothelial cells, we explored the role of main HDL receptor SR-BI during this process. SR-BI genotype of three-month old wild type (SR-BI+/+) and SR-BI-/- mice was assayed by PCR (see supplemental figure I). MAECs were treated with PBS, HDL and TNF-α receptively (Fig. 4A-B). We found that HDL (100ug/ml) up-regulated ANXA1and TNF-α (2ng/ml) down-regulated ANXA1 in wild type MAEC. As for SR-BI-/- MAEC, TNF-α still down-regulated ANXA1, while HDL lost the effect of up-regulating ANXA1. Furthermore, HUVECs were transfected with siRNA of SR-BI to knock down the level of SR-BI. Similar results were obtained (Fig. 4C-D). Therefore, this study demonstrated that HDL-induced ANXA1 in endothelial cells were 18 / 42

ACCEPTED MANUSCRIPT mediated by SR-BI. Subsequently, a detailed analysis of cell signaling in HUVECs was performed to assess activation levels of Akt, PKC, ERK and p38MAPK pathways with HDL

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treatment as shown in Fig. 4E-H. Briefly, HDL activated the phosphorylation of AKT and

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PKC pathways which reached the peak at 30 min, and also activated the phosphorylation of ERK and p38MAPK pathways which reached the peak at 15 min. In addition, the ANXA1 induction by HDL was reduced after pre-incubation with signaling pathway inhibitors, 30.7%

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for the PKC inhibitor Staurosporine at P<0.05; 42.4% for the Erk1/2 inhibitor PD98059 at

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P<0.01; 39.9% for the p38 inhibitor SB203580 at P<0.01; 29.1% for the phosphoinositide 3-kinase/AKT (PI3K/AKT) inhibitor LY294002 at P<0.05 (Fig. 5A-B). Finally, after the

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pre-incubation for 6 h with above inhibitors, HDL inhibition of THP-1 cell adhesion to

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TNF-α-activated HUVECs was impaired by different degrees, 88.3±15.1% for Staurosporine

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at P<0.001; 35.5±11.7% for PD98059 at P=ns; 47.9±9.9% for SB203580 at P<0.05;

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53.5±12.1% for LY294002 at P<0.01 (Fig. 5C).

Apo AI up-regulated ANXA1 and Activated Similar Signaling Pathways Apo AI, the most abundant apolipoprotein in HDL, comprises 60% to 70% of the total HDL protein mass. As shown in figure 6A-B, 100ug/ml apo AI increased ANXA1 expression by112.95±43.38 % (P<0.001). Besides, apoA-I activated the phosphorylation of ERK, p38MAPK, AKT and PKC pathways whose efficiencies were a little lower than those activated by HDL (Fig. 6C-F). Finally, this study detected ANXA1 expression on the intima of thoracic aorta (n = 6) from wild type C57BL/6 mice or apoA1-/- C57BL/6 mice. All pictures were taken under confocal imaging with constant parameters. CD31 was used to 19 / 42

ACCEPTED MANUSCRIPT mark the endothelium. It was shown that endothelial ANXA1 on the intima of thoracic aorta

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from apo A1-/- mice was lower than that from wild type mice (Fig. 6G).

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HDL-induced ANXA1 Partially Inhibited Cell Surface VCAM-1, ICAM-1 and E-selectin, and Secretion of MCP-1, IL-8, VCAM-1 and E-selectin.

Based on previous studies, VCAM-1, ICAM-1, E-selectin, MCP-1 and IL-8 were essential for

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monocytes adhesion to endothelial cells, [23, 24] and TNF-α significantly induced the

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production of these inflammatory factors. As shown in Fig. 7A-C, HDL inhibited up-regulation of cell surface VCAM-1 (45.0 %, P<0.05), ICAM-1 (56.2%, P<0.05) and

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E-selectin (77.4 %, P<0.05) by TNF-α. However, HDL couldn’t exhibit above effects with

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statistical significance after knockdown of ANXA1 in HUVECs. Subsequently, we detected

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the level of MCP-1, IL-8, VCAM-1 and E-selectin in media by ELISA. As shown in Fig. 7D-G, HDL inhibited secretion of MCP-1(30%, P<0.05), IL-8(38.4%, P<0.05),

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VCAM-1(42.2%, P<0.01) and E-selectin (36.7%, P<0.05) by TNF-α in HUVECs transfected by scramble siRNA. However, HDL couldn’t exhibit above effects with statistical significance after knockdown of ANXA1. As shown in Fig. 7H-K, HDL inhibited secretion of MCP-1(33.8%, P<0.001), IL-8(37.1%, P<0.001), VCAM-1(37.7%, P<0.001) and E-selectin (41.8%, P<0.001) by TNF-α in HUVECs transfected by empty vector. After transfection of Ad-ANXA1 in HUVECs, HDL still inhibited secretion of MCP-1 (34.8%, P<0.01), IL-8 (39.6%, P<0.05), VCAM-1(31%, P<0.01) and E-selectin (40.8%, P<0.05) by TNF-α. Therefore, Our findings demonstrated that ANXA1 mediated the anti-inflammatory function of HDL in TNF-α activated endothelial cells by inhibiting cell surface VCAM-1, ICAM-1 and 20 / 42

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E-selectin, as well as attenuating secretion of MCP-1, IL-8, VCAM-1 and E-selectin.

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ACCEPTED MANUSCRIPT Discussion

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ANXA1, an anti-inflammatory mediator, was recently reported to play protective role in the

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progression of atherosclerosis, such as promoting plaque stability and counteracting arterial

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leukocyte recruitment.[8, 25] This study identified that HDL could up-regulate ANXA1 in endothelial cells in vitro and in vivo, which raised a novel insight of its anti-inflammation in

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endothelial protection and further clarified the protective mechanism of ANXA1 in atherosclerosis.

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Based on previous studies, endothelial ANXA1 was impaired during endothelial dysfunction

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and vascular inflammation.[6, 7] Besides, according to Danielle B et al, there were different

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protein compositions between endothelial microparticles (EMPs) generated without treatment (control EMPs) and those generated with TNF-α treatment (TNF-α EMPs).[26] Despite 432

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common proteins in both kinds of EMP populations, ANXA1 was one of the 231 proteins which were different between control EMPs and TNF-α EMPs. Therefore, this study explored

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ANXA1 expression in TNF-α- activated endothelial cells which induced endothelial dysfunction. In the present study, we first demonstrated that TNF-α decreased endothelial ANXA1 in vitro and in vivo, which may help researchers clarify why ANXA1 exists in control EMPs while not in TNF-α EMPs. In addition, accumulated studies reported that Annexin A5 (ANXA5), another member of annexin superfamily, exerted athero-protective roles through inhibiting pro-inflammatory effects, vascular protective effects and induction of T-cell activation, which played key roles in the development of CVD.[27-29] This work indicated that HDL significantly reversed the trend of TNF-α d6ecreasing ANXA1 in endothelial cells, inhibiting monocyte-endothelial cells adhesion. It is the first time to report 22 / 42

ACCEPTED MANUSCRIPT the role of HDL-induced ANXA1 in TNF-α- activated endothelial cells, enriching the anti-inflammatory mechanism of HDL. These findings revealed that ANXA1 also excerted

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role of annexin superfamily members in protection of CVD.

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athero-protective roles through inhibiting pro-inflammatory effects, which further clarified the

HDL exerts its endothelial protective effect mainly through SR-BI which is located on cell membrane. [30] In this study, HDL didn’t exhibit up-regulation of endothelial ANXA1 after

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deletion or knockdown of SR-BI, indicating that HDL up-regulation of ANXA1 was mediated

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by SR-BI. Based on previous studies, HDL exerted its protective effects through stimulating several signaling pathways, including PI3K/Akt, Erk1/2 and p38MAPK activation.[31, 32]

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and ANXA1 expression was related to activation of PKC pathway.[33] In this study, HDL

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activated the phosphorylation of PI3K/Akt, Erk1/2, p38MAPK and PKC. Special inhibitors of

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above signaling pathways impaired up-regulation of endothelial ANXA1 by HDL, as well as attenuated the inhibitory effect of HDL on monocytes adhesion to TNF-α-activated

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endothelial cells. These observations indicated that HDL up-regulated ANXA1 in a multi-pathway manner. However, further experiments are needed to delineate which signaling pathway plays the primary role during up-regulation of ANXA1 by HDL. Apo AI is the most important structural protein of HDL, and apo AI knockout mice have abnormal (apoE-enriched) HDL. Consequently, the effect of apo AI on endothelial ANXA1 was detected. It was found that apo AI also increased endothelial ANXA1 expression. Apo AI could activate similar signaling pathways to HDL, which was consistent with previous study.[34] Besides, level of endothelial ANXA1 on the intima of thoracic aorta from apo AI-/mice was less than that from wild type mice. These new findings indicated that HDL as well 23 / 42

ACCEPTED MANUSCRIPT as apo AI augmented ANXA1expression in endothelium. In the process of monocyte-endothelial cell interaction and subsequent monocyte recruitment

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into vascular tissue, chemokines (e.g. MCP-1 and IL-8) are key mediators. Previous studies

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revealed that HDL prevented the expression of VCAM-1, ICAM-1, E-selectin, IL-8 and MCP-1.[15, 35, 36] In addition, it was reported that ANXA1 peptide Ac2-26 suppressed TNF-α-induced inflammatory response via inhibition of VCAM-1 and ICAM-1 in endothelial

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cells.[37] In the present study, HDL-induced ANXA1 inhibited cell surface VCAM-1,

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ICAM-1 and E-selectin, and secretion of MCP-1, IL-8, VCAM-1 and E-selectin in TNF-αactivated endothelial cells. Our novel results indicated that ANXA1 was a crucial mediator for

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anti-inflammation, which enriched protective mechanism of HDL. Of course, previous studies

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demonstrated that monocyte-derived ANXA1 could mobilize and compete with VCAM-1 (the

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endothelial integrin counter-receptor) [22] or serum amyloid protein A,[38] contributing to inhibition of monocyte adhesion. Therefore, further researches are needed to explore whether

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anti-inflammatory mechanism of endothelium-derived ANXA1 is different from or similar to that of monocyte-derived ANXA1. A growing number of studies have explored the role of ANXA1 in atherosclerotic plaques.[8, 39] Collectively, our finding has uncovered a novel HDL/SR-BI/ANXA1 dependent anti-inflammation mechanism in endothelial cells (figure 8). This study may provide a novel understanding of HDL vascular protection and draw a novel perspective of ANXA1 in endothelial homeostasis and atherosclerosis in the future clinical research.

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ACCEPTED MANUSCRIPT Acknowledgements

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The authors would like to thank Professor George Liu (Peking University Health Science

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Center) for providing the SR-BI-/- mouse. This project was supported by Grant 2011CB503900 from “973” National S&T Major Project; by Grant 81172500, 81170101, 81370235, 81322005 from the National Natural Science Foundation of China. This project is

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also supported by the University of Michigan-Peking University Health Science Center Joint

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Institute.

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Disclosures

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None

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inflammation of advanced atherosclerotic lesions in apoE(-/-) mice, Journal of cellular and molecular medicine, 18 (2014) 2117-2124.

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ACCEPTED MANUSCRIPT Figure legend Figure 1 HDL induced expression of ANXA1 in endothelial cells. (A-B) HUVECs were treated

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with different doses of HDL (0, 25, 50, 100 and 150 µg/ml) for 12 h. The expression of ANXA1

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was assayed by western blot and evaluated by density analysis. (C-D) HUVECs were incubated with HDL (100 μg/ml) at different time points (0, 1, 4, 12 and 24 h). The expression of ANXA1 was assayed by western blot and evaluated by density analysis. (E) HUVECs were incubated with

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HDL (100 μg/ml) at different time points (0, 1, 4 and 12h). The mRNA level of ANXA1 was

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assayed by real time PCR. (F-G) HUVECs were incubated with HDL (100 μg/ml) at different time points (0, 12 and 24 h). The expression of ANXA1 (green) was assayed by Immunofluorescence

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and confocal imaging. The DNA-binding dye DIPA (blue) was used for nuclear localization.

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Quantitative results showed intensity of ANXA1. Bars = 50 μm. Data were mean±SEM of three

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independent experiments (*, P<0.05; **, P<0.01; ***, P<0.001; one-way ANOVA).

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Figure 2 HDL up-regulated ANXA1 in TNF-α-activated endothelial cells in vitro and in vivo. (A-B) HUVECs were treated with different doses of TNF-α (0, 0.1, 0.5, 1 and 2 ng/ml) for 12 h. The expression of ANXA1 was assayed by western blot and evaluated by density analysis. (C-D) HUVECs were transfected with scramble siRNA (negative control) or siRNA of ANXA1 (ANXA1-RNAi), they were treated with PBS, TNF-α (2 ng/ml) or HDL (100 ug/ml) + TNF-α (2 ng/ml). The expression of ANXA1 was detected by western blot and evaluated by density analysis. Data are mean±SEM of three independent experiments. (*, P<0.05. one-way ANOVA) (E-F) Vehicle and HDL were administrated by tail vein injection. TNF-α was administrated by intra-peritoneal injection. ANXA1 expression in the intima of thoracic aorta was detected by 29 / 42

ACCEPTED MANUSCRIPT immunohistochemical assay. Quantitative results showed fold change of ANXA1 in comparison to vehicle group. (n=6, ***, P<0.001; one-way ANOVA, Bars = 30 μm) (G) Vehicle and HDL were

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administrated by tail vein injection. The endothelial cell marker CD34 (red) and the expression of

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ANXA1 (green) were assayed by confocal imaging. The DNA-binding dye DIPA (blue) was used for nuclear localization. Bars = 10 μm.

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Figure 3 HDL-induced ANXA1 inhibited THP-1 cells adhesion to TNF-α-activated HUVECs.

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(A-C) HUVECs were transfected with negative control or ANXA1-RNAi. Monocyte-endothelial cells adhesion assays were performed. (D-E) HUVECs were pretreated with specific anti-ANXA1

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mAb or control IgG for 60 min to block ANXA1. Monocyte-endothelial cells adhesion assays

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were performed. (F-G) HUVECs were transfected with empty vector or adenovirus-ANXA1

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(Ad-ANXA1). Monocyte-endothelial cells adhesion assays were performed. Representative pictures were chosen. And quantitative results showed fold change of cell adhesion in comparison

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to PBS- treated. Data were mean±SEM of three independent experiments (*, P<0.05; ***, P<0.001; ns, no significance, one-way ANOVA).

Figure 4 Receptor and signaling pathways which were involved in HDL-induced ANXA1 in endothelial cells. (A-B) MAECs were derived from 3 month-old wild type (SRBI+/+), SRBI-/mice, which were treated with PBS, HDL (100 µg/ml) and TNF-α (2 ng/ml) respectively. ANXA1 levels were detected by western blot and evaluated by density analysis. (C-D) HUVECs were transfected with negative control or SR-BI-RNAi, which were treated with PBS, HDL (100 µg/ml) and TNF-α(2 ng/ml) respectively. ANXA1 levels were detected by western blot and evaluated by 30 / 42

ACCEPTED MANUSCRIPT density analysis. (E-H) HUVECs were incubated with HDL (100 μg/ml) at different time points (0, 5, 15, 30, 60 and 120 min). The phosphorylation of ERK, p38MAPK and Akt signaling pathways

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were detected by western blot and evaluated by density analysis. Activation of PKC was analyzed

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by PepTag Assay. Data were mean±SEM of three independent experiments (*, P<0.05; **, P<0.01; ***, P<0.001; ns, no significance. one-way ANOVA).

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Figure 5 The effects of inhibitors of different signaling pathways on ANXA1 expression and

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monocyte-endothelial cells adhesion. (A-B) HUVECs were pretreated with the Staurosporine (Stau, 10 nM), PD98059 (PD, 50 μM), SB203580 (SB, 5 μM) or LY294002 (LY, 25 μM) for 6

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hour, then were incubated with HDL (100 μg/ml) for 12 h. ANXA1 expression was assayed by

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western blot and evaluated by density analysis in comparison to HDL-treated group. (C) HUVECs

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were pretreated with the Staurosporine (Stau, 10 nM), PD98059 (PD, 50 μM), SB203580 (SB, 5 μM) or LY294002 (LY, 25 μM) for 6 hour, then were incubated with HDL (100 μg/ml) and TNF-α

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(2 ng/ml) for 12 h. Monocyte-endothelial cells adhesion assays were performed. Data were mean ±SEM of three independent experiments (*, P<0.05; **, P<0.01; ***, P<0.001; ns, no significance; one-way ANOVA).

Figure 6 ApoA1 components in HDL up-regulated ANXA1 expression. (A-B) HUVECs were treated with PBS, HDL (100 µg/ml) and apoA1(100µg/ml). ANXA1 expression was detected by western blot and evaluated by density analysis. (C-F) HUVECs were incubated with apoA1 (100 μg/ml) at different time points (0, 5, 15, 30, 60 and 120 min). The phosphorylation of ERK, p38MAPK and Akt signaling pathways were detected by western blot and evaluated by density 31 / 42

ACCEPTED MANUSCRIPT analysis. Activation of PKC was analyzed by PepTag Assay. (G) ANXA1 expression in the thoracic aorta of wild type and apoA1-/- mice was detected. The endothelial cell marker CD31

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(red) and the expression of ANXA1 (green) were assayed by confocal imaging. The DNA-binding

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dye DIPA (blue) was used for nuclear localization. Bars =100 μm. Data are mean±SEM of three independent experiments (*, P<0.05; **, P<0.01; ***, P<0.001; one-way ANOVA).

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Figure 7 The effects of ANXA 1 on cell surface VCAM-1, ICAM-1, E-selectin and secretion

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of MCP-1, IL-8, VCAM-1, ICAM-1 in TNF-α-activated HUVECs. Confluent monolayers of endothelial cells were incubated with vehicle (PBS) or HDL (100 μg/ml) in the presence or

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absence of TNF-α (2 ng/ml) for 12 h, then several inflammatory factors on cell surface or in media

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were detect by ELISA. (A-C) HUVECs were transfected with negative control or ANXA1-RNAi.

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Cell surface VCAM-1, ICAM-1 and E-selectin were measured by Cell ELISA. (D-G) HUVECs were transfected with negative control or ANXA1-RNAi. MCP-1, IL-8, VCAM-1 and E-selectin

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in media were measured by ELISA. (H-K) HUVECs were transfected with empty vector or Ad-ANXA1. MCP-1, IL-8, VCAM-1 and E-selectin in media were measured by ELISA. Data were mean±SEM of three independent experiments (*, P<0.05; **, P<0.01; ***, P<0.001; ns, no significance; one-way ANOVA).

Figure 8 Model for the molecular mechanism of HDL-induced ANXA1, inhibiting monocyte adhesion. HDL activated p38MAPK, PI3K/Akt, ERK1/2 and PKC signaling pathways through SR-BI, up-regulating endothelial ANXA1. HDL-induced ANXA1 inhibited cell surface VCAM-1,

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Highlights 1. HDL up-regulated endothelial annexin A1 via SR-BI and activation of ERK, p38MAPK, Akt and PKC signaling pathways. 2. Endothelial ANXA 1 inhibited monocyte adhesion. 3. High density lipoprotein-induced annexin A1 not only inhibited cell surface VCAM-1, ICAM-1, and E-selectin but also suppressed secretion of MCP-1, IL-8, VCAM-1 and E-selectin, which alleviated inflammatory response.

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