Accepted Manuscript Uric acid promotes chemokine and adhesion molecule production in vascular endothelium via nuclear factor-kappa B signaling W.Y. Liang, X.Y. Zhu, J.W. Zhang, X.R. Feng, Y.C. Wang, M.L. Liu PII:

S0939-4753(14)00279-8

DOI:

10.1016/j.numecd.2014.08.006

Reference:

NUMECD 1343

To appear in:

Nutrition, Metabolism and Cardiovascular Diseases

Received Date: 21 April 2014 Revised Date:

22 July 2014

Accepted Date: 27 August 2014

Please cite this article as: Liang WY, Zhu XY, Zhang JW, Feng XR, Wang YC, Liu ML, Uric acid promotes chemokine and adhesion molecule production in vascular endothelium via nuclear factor-kappa B signaling, Nutrition, Metabolism and Cardiovascular Diseases (2014), doi: 10.1016/ j.numecd.2014.08.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Uric acid promotes chemokine and adhesion molecule production in vascular endothelium via nuclear factor-kappa B signaling

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Liang, W.Y.a, Zhu, X.Y.b, Zhang, J.W.a, Feng, X.R.a, Wang, Y.C.a, Liu, M.L.a

Department of Geriatrics, Peking University First Hospital, Beijing, China.

b

Department of Cardiology, Peking University Shougang Hospital, Beijing, China.

Corresponding author Liu, M.L.

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a

Department of Geriatrics, Peking University First Hospital, No. 8, Xishiku Street, Xicheng District, Beijing 100034, China.

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Phone: +86 10 83572022

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Email: [email protected]

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ACCEPTED MANUSCRIPT Abstract Background and Aims: Hyperuricemia is an important risk factor for atherosclerosis, yet the potential mechanisms are not well understood. Migration and adhesion of

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leukocytes to endothelial cells play key roles in initiation and development of atherosclerosis. We investigated monocyte–endothelial cell interactions and potential signaling pathways under uric acid (UA)-stimulated conditions.

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Methods and Results: Primary human umbilical vein endothelial cells (HUVECs)

Experimental

hyperuricemia

rat

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were cultured and exposed to different concentrations of UA for various periods. models

were

established.

Expression

of

chemoattractant protein-1 (MCP-1), interleukin 8 (IL-8), vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) were

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evaluated. Monocyte–endothelial cell interactions were elucidated by chemotaxis and adhesion assays, and nuclear factor-kappa B (NF-κB) pathway was studied using fluorescent microscopy and electrophoretic mobility shift assay. Results showed that

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high concentration of UA stimulated generation of chemokines and adhesion

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molecules in ex vivo and in vivo experiments. Migration and adhesion of human monocytic leukemia cell line THP-1 cells to HUVECs were promoted and activated NF-κB was significantly increased. UA-induced responses were ameliorated by organic anion transporter inhibitor probenecid and NF-κB inhibitor BAY11-7082. It was also observed that human endothelial cells expressed urate transporter-1, which was not regulated by UA. Conclusion: High concentration of UA exerts unfavorable effects directly on vascular 2

ACCEPTED MANUSCRIPT endothelium via the NF-κB signaling pathway, the process of which requires intracellular uptake of UA.

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Keywords: Uric acid; Hyperuricemia; Atherosclerosis; Chemokines; Adhesion molecules; NF-κB.

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Abbreviations: UA, uric acid; HUVECs, human umbilical vein endothelial cells;

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URAT1, urate transporter-1; NF-κB, nuclear factor-kappa B; EMSA, electrophoretic mobility shift assay; eNOS, endothelial nitric oxide synthase; CRP, C-reactive protein; MCP-1, monocyte chemotactic protein-1; IL-8, interleukin 8; VCAM-1, vascular cell adhesion

molecule-1;

ICAM-1,

adhesion

molecule-1;

PBS,

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phosphate-buffered saline.

intercellular

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ACCEPTED MANUSCRIPT Introduction Epidemiological studies have addressed the strong association between hyperuricemia and atherosclerotic vascular diseases [1-4]. In some cases, hyperuricemia is strikingly

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defined as a cardiorenal toxin [5] or a component of the cardiovascular continuum [6]. Serum uric acid (UA) level may be a potent predictor of cardiovascular outcome and mortality in both genders [7,8].

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UA used to be considered an antioxidant to scavenge singlet oxygen and free

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radicals [9]. In the atherosclerotic pro-oxidative milieu, the original antioxidant properties of UA may paradoxically become pro-oxidant [10,11]. Previous studies have reported that a high UA concentration (12 mg/dL for 24 h) significantly decreased endothelial nitric oxide synthase (eNOS) activity and nitric oxide

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production in human umbilical vein endothelial cells (HUVECs) by reducing the binding of eNOS and calmodulin [12], and attenuating arginine uptake [13]. UA also induced production of C-reactive protein (CRP) in human vascular smooth muscle

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cells and HUVECs, and the increase of CRP may partake actively in plaque formation

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and cardiovascular morbidity [14]. Another in vitro study suggested that UA stimulated the increase of chemoattractant protein-1 (MCP-1) in vascular smooth muscle cells by activating nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase signaling molecules [15]. The pathogenesis of atherosclerosis is a chronic inflammatory process. Vascular endothelial cells, activated at sites of inflammation, interact with different leukocyte subtypes through chemokines, adhesion molecules and other cofactors. MCP-1, 4

ACCEPTED MANUSCRIPT interleukin 8 (IL-8), vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) are crucial for firm adhesion and migration of leukocytes into the sub-endothelium. This process is thought to play a key role in

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early atherosclerotic plaque development as well as in plaque progression. Pharmacological inhibition or genetic knockdown of these pro-inflammatory molecules result in reduced lesion size, monocyte–macrophage infiltration, and

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slowed disease progression in experimental models of atherosclerosis [16,17].

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In the present study, we investigated UA-induced injuries in vascular endothelium. Our results support the notion that regular inspection and treatment of hyperuricemia are recommended in patients predisposed to atherosclerotic vascular diseases.

Cell cultures

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Methods

HUVECs were isolated by collagenase A perfusion as previously described [18]

and

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cultured in EGM-2 medium (Lonza, Walkersville, MD, USA) according to the

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recommendation [22]. Tissue collection was approved by the Institutional Ethics Committee of Peking University First Hospital. Human monocytic leukemia cell line THP-1 (ATCC, Rockefeller, MD, USA) was grown in medium RPMI 1640, and kept in standard incubation conditions (37°C, humidified atmosphere containing 5% CO2).

Reagents Uric acid (Sigma, St. Louis, MO, USA) was added to pre-warmed culture medium at 5

ACCEPTED MANUSCRIPT 2–18 mg/dL. The medium was stirred, re-warmed (37°C, ~30 min) and filtered. Other reagents included probenecid and oxonic acid (Sigma), CCK-8 and BCECF-AM [3’-O-Acetyl-2’,7’-bis(carboxyethyl)-4 or 5-carboxyfluorescein, diacetoxymethyl (Dojindo,

Kumamoto,

Japan)

and

BAY11-7082

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ester]

[(E)3-(4-methylphenylsulfonyl)-2-propenenitrile] (Beyotime, Shanghai, China).

Antibodies used were rabbit anti-URAT1 antibody (Alpha Diagnostic, San Antonio,

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TX, USA) [19], rabbit anti-MCP-1, anti-IL-8 and anti-ICAM-1 antibodies (Abcam,

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Cambridge, UK), mouse anti-VCAM-1 and anti-α-tubulin antibodies (Abcam), rabbit anti-NF-κB p65 antibody (Cell Signaling Technology, Beverly, MA, USA), and HRP-conjugated and TRITC-conjugated secondary antibodies (ZSGB-BIO, Beijing,

Cell viability assay

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China).

HUVECs at exponential growing phase were seeded at 5×103 cells per well in a

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96-well plate. After treatment, ten microliters of CCK-8 was added to each well, and

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the plates were incubated for 2 h at 37°C. The absorbance at 450 nm was measured using a microplate reader (Biotek Synergey H1, Winooski, VT, USA).

RNA extraction and quantitative real-time PCR Total RNA was extracted using Trizol reagent and reverse-transcribed into cDNA. Quantitative real-time PCR was performed using an ABI PRISM 7500 Sequence Detection System and the intercalating dye SYBR Green I (Applied Biosystems, 6

ACCEPTED MANUSCRIPT Foster City, CA, USA). Primers used were: sense 5’-TGTGCCTGCTGCTCATAG and anti-sense 5’-CTTGCTGCTGGTGATTCTTC for MCP-1 cDNA; sense 5’-ACACTGCGCCAACACAGAAAT and anti-sense

5’-CCCTTGACCGGCTGGAGATT and anti-sense

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5’-CCTCTGCACCCAGTTTTCCTT for IL-8 cDNA; sense

5’-CTGTCACTCGAGATCTTGAGG and anti-sense

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5’-CTGGGGGCAACATTGACATAAAGTG for ICAM-1 cDNA; sense

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5’-CCTGCAGTGCCCATTATGA for VCAM-1 cDNA; and sense 5’-TCTTTTGCGTCGCCAGCCGAG and anti-sense

5’-CAGAGTTAAAAGCAGCCCTGGTGAC for GAPDH cDNA. Thermal cycling settings were pre-denaturing at 95°C for 10 min, followed by 40 cycles of 95°C for 30

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s, 60°C for 30 s and 72°C for 35 s. Melting curves and electrophoresis of the amplicons in 2% agarose gel were both used to confirm the absence of nonspecific

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ELISA

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amplification. The relative cDNA amount was derived using the 2-∆∆Ct method [29].

Cell culture supernatants were collected and centrifuged to remove cell debris. MCP-1, IL-8, VCAM-1 and ICAM-1 in the supernatant were determined using ELISA kits (eBioscience, San Diego, CA, USA) following the manufacturer’s manuals.

Western blotting

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ACCEPTED MANUSCRIPT Protein samples (50 µg) were separated on 4–12% Bis-Tris gel (Invitrogen, Carlsbad, CA, USA) under reducing conditions as described [30]. The blots were probed with primary antibodies: anti-URAT1 (1:200), anti-MCP-1 (1:1500), anti-IL-8 (1:50),

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anti-VCAM-1 (1:1000), anti-ICAM-1 (1:600), or anti-α-tubulin (1:2000) antibody, followed by HRP-conjugated secondary antibody (1:7500), and finally developed

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using SuperSignal enhanced chemiluminescent substrate solution (Pierce).

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Chemotaxis assay

Monocyte chemotactic activity was performed using Millicell insert with PET membrane (pore size: 8 µm) (Millipore, Billerica, MA, USA). HUVECs were treated in a 24-well plate (lower chamber). 4×105 THP-1 cells were pipetted into the insert

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(upper chamber). After co-culture for 3 h, the insert was washed and fixed and stained with 0.1% crystal violet. Stained cells were counted in three random fields at ×200

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magnification.

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Adhesion assay

HUVECs were treated as described in the chemotaxis assay. An equal amount of THP-1 cells pre-labeled with 5 µM BCECF-AM for 30 min in RPMI 1640 was added to each well. After co-culture for one hour, each well was washed three times with warm PBS, fixed with 4% paraformaldehyde in PBS for 20 min at 4°C, and washed again. Digital images were captured over three regions in each well at ×200 magnification using a fluorescence microscope (Olympus IX-71, Melville, NY, USA). 8

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Fluorescent microscopy After treatment, cells were fixed in 4% paraformaldehyde for 15 min, then washed

with

rabbit

anti-p65

antibody

(1:200)

overnight

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twice with PBS and blocked in 10% goat serum for 60 min. The slides were incubated at

4°C

followed

by

TRITC-conjugated goat anti-rabbit antibody for 1h at room temperature. Nuclei were

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confocal microscopy (Olympus FV1000).

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visualized by staining with DAPI. Digital images were captured by laser scanning

Electrophoretic mobility shift assay (EMSA)

Nuclear proteins were isolated using NE-PER nuclear and cytoplasmic extraction

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reagents (Pierce). The nucleotide sequence for consensus NF-κB binding site was 5’-AGTTGAGGGGACTTTCCCAGGC [20]. EMSA was performed using a DIG Gel Shift Kit (Roche, Mannheim, Germany). Ten micrograms of nuclear protein extract

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was incubated with 30 fmol DIG-labeled double-strand NF-κB DNA probe in binding

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buffer. The protein–DNA complexes were separated in 5% polyacrylamide gel and transferred onto Hybond-N+ nylon membranes (Amersham, Buckinghamshire, UK). Detection was performed following the manual.

Animal models Male Wistar rats weighing 120±8g were randomly divided into normal group, in which rats were allowed free access to regular chow and tap water, and 9

ACCEPTED MANUSCRIPT hyperuricemia group, in which rats were fed, by gavage, with oxonic acid 2g/kg b.w./day. After six weeks, serum UA concentration was measured by kinetic uricase method and aorta arch tissue was fixed in 10% buffered formalin, embedded in

Immunofluorescence histochemistry

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Animal Care Committee of Peking University First Hospital.

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paraffin, and sectioned. Animal experimental procedures were approved by the

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Rat aorta sections were incubated with anti-MCP-1 (1:200), anti-IL-8 (1:20), anti-VCAM-1 (1:200) or anti-ICAM-1 (1:100) antibody, and the appropriate TRITC-conjugated secondary antibody. Sections were observed using a fluorescence

Statistical analysis

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microscope (Olympus IX-71).

Data are presented as mean ± standard deviation. Student’s t-test was used for

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comparison of quantitative real-time PCR data. One-way analysis of variance

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(ANOVA) was conducted for other experiment results. Post-hoc analyses using LSD and SNK algorithm were performed to allocate the source of significance. When variances were unequal, Tamhane’s T2 and Dunnett’s T3 tests were used instead. A two-tailed P-value