Modulatory Effects of Egg White Ovotransferrin-Derived Tripeptide

Sep 20, 2016 - Modulatory Effects of Egg White Ovotransferrin-Derived Tripeptide IRW (Ile-Arg-Trp) on Vascular Smooth Muscle Cells against Angiotensin...
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Modulatory effects of egg white ovotransferrin-derived tripeptide IRW (IleArg-Trp) in vascular smooth muscle cells against angiotensin II stimulation Wang Liao, Subhadeep Chakrabarti, Sandra T. Davidge, and Jianping Wu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b03513 • Publication Date (Web): 20 Sep 2016 Downloaded from http://pubs.acs.org on September 21, 2016

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Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Agricultural and Food Chemistry

Modulatory effects of egg white ovotransferrin-derived tripeptide IRW (Ile-Arg-Trp)

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on vascular smooth muscle cells against angiotensin II stimulation

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Wang Liao, † Subhadeep Chakrabarti, †,∥,⊥ Sandra T. Davidge, ‡,§,∥,⊥ Jianping Wu *,†,∥

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Department of Agricultural, Food & Nutritional Science, ‡ Department of Obstetrics &

Gynecology,

§

Department of Physiology,



Cardiovascular Research Centre, ⊥ Women and

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Children’s Health Research Institute

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University of Alberta, Edmonton, Alberta, T6G 2P5, Canada

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Corresponding author

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*E-mail: [email protected]. 4-10 Ag/For Centre, University of Alberta, Edmonton, AB T6G

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2P5, Canada. Phone: (780) 492-6885. Fax: (780) 492-4265.

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ABSTRACT

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The renin angiotensin system (RAS) is a key mediator of blood pressure regulation. Angiotensin

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II (Ang II), the active component of RAS, is a potent vasoconstrictor that also causes abnormal

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proliferation, oxidative stress and inflammation in vascular smooth muscle cells (VSMCs) that

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contribute to atherosclerotic changes. Egg white ovotransferrin-derived tripeptide IRW (Ile-Arg-

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Trp) was previously shown to exert antihypertensive effect by reducing Ang II synthesis as well

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as endothelial cell inflammation and endothelial dysfunction. However, the effects of IRW on

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VSMCs are still unclear. In the present study, we evaluated the anti-proliferative, anti-oxidant,

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and anti-inflammatory effects of IRW on VSMCs in the presence of Ang II stimulation. It was

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found that IRW treatment could attenuate Ang II-stimulated proliferation, superoxide production

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and inflammation in VSMCs. These beneficial effects appeared to involve modulation of the NF-

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κB pathway. These findings could further our understanding on the antihypertensive mechanism

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of IRW beyond vascular endothelium.

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KEYWORDS: IRW, VSMC, proliferation, oxidative stress, inflammation

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Journal of Agricultural and Food Chemistry

INTRODUCTION

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Hypertension is a global health challenge, accounting for about 6% of the total deaths

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worldwide.1 Hypertension is also a major risk factor for renal and cardiovascular diseases.2 The

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renin angiotensin system (RAS) is one of the major regulators of blood pressure and fluid and

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electrolyte homeostasis,3 in which, angiotensin II (Ang II) is the main effector peptide of

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vasoconstriction.4 Hence, angiotensin converting enzyme (ACE) inhibitor (to reduce the

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production of Ang II), or angiotensin II type I receptor (AT1R) blocker (to inhibit the binding of

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Ang II to its target tissues) are the first-line drugs for hypertension therapy.5

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Ang II is more than a potent vasoconstrictor. It also plays multiple roles in controlling the

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functional and structural integrity of the vascular wall.6 In vascular smooth muscle cells

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(VSMCs), over stimulation of Ang II could lead to aberrant proliferation,7 excessive superoxide

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production8 and inflammation,6 which would further promote vascular remodelling and

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inflammation, playing pivotal roles in atherosclerotic diseases.4 The anti-proliferative, anti-

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oxidant and anti-inflammatory actions of ACE-inhibitory agents have been documented.

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However, as pharmacological drugs require long-term (often life long) therapy with considerable

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side-effects, there is an unmet need for safer alternatives from naturally based sources.12,13

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In the past three decades, food protein-derived antihypertensive peptides have received

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substantial interest as natural alternatives to clinical drugs.14 Antihypertensive peptides derived

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from egg white, milk and soy proteins have been reported and the in vivo effects of some of these

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peptides have been validated on spontaneously hypertensive rats (SHRs).15-19 Functional food

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and nutraceuticals developed from these peptides may provide new opportunities for treatment

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and prevention of hypertension. IRW (Ile-Arg-Trp), an egg white ovotransferrin-derived

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tripeptide, was identified and characterized as a potent ACE inhibitor.20 The anti-hypertensive

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activity of IRW is associated with amelioration of endothelial inflammation and oxidative

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stress,21 enhancement of nitric oxide-mediated vasodilation17 and upregulation of angiotensin

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converting enzyme 2 (ACE2) expression.22 However, beneficial effects of IRW on VSMCs, if

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any, have not been investigated previously.

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Given this background, we aimed to investigate effects of IRW on proliferation, oxidative

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stress and inflammation in VSMCs under Ang II stimulation in this study.

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MATERIALS and METHODS

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Reagents. Dulbecco’s phosphate buffered saline (PBS), Ang II and dithiothreitol (DTT) were

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from Sigma Aldrich (St Louis, MO, USA). Dulbecco’s modified Eagle medium (DMEM) and

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fetal bovine serum (FBS) were purchased from Gibco/ Invitrogen (Carlsbad, CA, USA). Type 1

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Collagenase used for cell splitting was from Worthington Biochemical Corporation (Lakewood,

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NJ, USA). Penicillin-Streptomycin as well as Gentamicin was from Life Technologies (Carlsbad,

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CA, USA). Triton-X-100 was from VWR International (West Chester, PA, USA). The tripeptide

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IRW was synthesized by Genscript (Piscataway, NJ, USA). Peptide sequence and purity (99.8

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%) were validated by HPLC-MS/MS. The peptide was dissolved in PBS at a stock concentration

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of 10 mM, aliquoted and stored at -20οC until use.

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Cell culture. Rat aortic VSMC cell line A7r5 was purchased from ATCC (cat# ATCC CRL-

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1444, Manassas, VA, USA). The cells were grown in DMEM supplemented with 10% FBS and

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antibiotics (Penicillin-Streptomycin and Gentamicin) until they reached 80% confluence. For

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actual experiments, the confluent cells were placed in a quiescing medium (DMEM + 1% FBS +

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antibiotics) and then treated with 50 µM of IRW 1 h prior to the addition of 1 µM of Ang II for

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different time periods. Cells between passages 4 and 11 (since receiving) were used for all

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experiments.

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Cell proliferation assay. Cell proliferation was evaluated by bromodeoxyuridine (BrDU)

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incorporation assay. A7r5 cells were seeded into a 48 well plate and grown in DMEM with 10%

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of FBS for overnight. After the medium was replaced by quiescing medium, 50 µM of IRW was

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added 1 h prior to stimulation of Ang II (1 µM). The co-treatment period with IRW and Ang II

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was 23 h. Afterwards, BrDU reagent (Invitrogen) diluted in quiescing medium at a final

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concentration of 1% was added for 1 h incubation at 37 ℃. Then the cells were fixed by 70%

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ethanol (20 min, room temperature). The

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hydrochloric acid (20 min, room temperature) followed by permeabilization by 0.1% Triton-X-

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100 in PBS (5 min, room temperature) and finally, blocking was done in 1% bovine serum

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albumin (BSA) in PBS (60 min, room temperature). Mouse monoclonal antibody against BrDU

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(1:1000; Cell Signaling, Beverly, MA, USA) was diluted in PBS with 0.1% BSA and incubated

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at 4 °C overnight. Upon washing, cell was treated with anti-mouse secondary antibody

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(Molecular Probes, Eugene, OR, USA) for 30 minutes in the dark. Nuclei were stained with the

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Hoechst33342 nuclear dye (Molecular Probes). Visualization was performed by Olympus IX81

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fluorescent microscope (Carson Scientific Imaging Group; Markham, ON, Canada). For each

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data point, 3 fields were randomly selected and the number of nuclei counted. The percentage of

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nuclei positive for BrDU stain was noted in each field and the mean value was calculated.

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antigen was exposed by incubation with 1M

Superoxide detection. Cellular superoxide generation was detected by dihydroethidium (DHE)

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staining similar to our previous work.21 Reactive oxygen species (ROS) could react with DHE to

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form ethidium, which then binds to nuclear DNA and release nuclear fluorescence.23 A7r5 cells

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were seeded into 48 well plate and grown in DMEM with 10% of FBS until confluent. After the

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medium was replaced by quiescing medium, 50 µM of IRW was added 1 h prior to stimulation

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of Ang II (1 µM). The co-treatment period of IRW and Ang II was 30 min. Then cells were

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treated with 20 µM of DHE and incubated in dark for 30 min. Afterwards, the cells were washed

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3 times. Fluorescence signal was detected by Olympus IX81 fluorescent microscope (Carson

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Scientific Imaging Group). For each data point, images from 3 random fields were taken. Mean

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fluorescence intensity (MFI)

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(http://imagej.net/Welcome). MFI/cell was calculated based on the cell number in each field.

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Results were presented as % of the untreated group.

of

each

image was

quantified

by ImageJ

software

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Western blot. At the end of experiment, the A7r5 cells were lysed in boiling hot Laemmle’s

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buffer containing 50 mM DTT (a reducing agent) and 0.2% Triton-X-100 to prepare samples for

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western blotting. These cell lysates were then run in 9% sodium dodecyl sulfate polyacrylamide

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gel electrophoresis (SDS-PAGE), transferred to nitrocellulose membranes and immunoblotted

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with antibodies. Protein bands for cyclooxygenase 2 (COX2) (rabbit monoclonal antibody from

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Abcam, cat# ab52237), inducible nitric oxide synthase (iNOS) (mouse monoclonal antibody

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from BD Transduction Laboratories, cat# 610432) and inhibitory κBα (IκBα) (Rabbit polyclonal

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antibody from Cell Signaling, cat# 9242) and AT1R (rabbit polyclonal antibody from Santa Cruz

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Biotechnology, cat# sc-1173) were normalized to the loading control α-tubulin (rabbit polyclonal

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antibody from Abcam, cat# ab15246). Phospho-p65 (p-p65) (rabbit polyclonal antibody from

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Cell Signling, cat# 3033) were normalized to the total p65 (mouse monoclonal antibody from

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Santa Cruz Biotechnology, cat# sc-8008). Anti-tubulin was used at 0.4 µg/ml, while all other

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antibodies were used at 0.5-1 µg/ml. Goat anti-rabbit IRDye 680RD or Donkey anti-mouse

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800CW from Licor Biosciences (Lincoln, NE, USA) was used as the secondary antibody. Protein

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bands were detected by Licor Odyssey BioImager (Licor Biosciences) and quantified by

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densitometry using Image Studio Lite 5.2 (Licor Biosciences). Cell lysates from untreated cells

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were loaded on every gel and all data were expressed as % of the corresponding untreated.

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Statistics. All data have been presented as mean ± SEM (standard error of mean) of 4 to 6

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independent experiments. Data were analyzed by one way analysis of variance (ANOVA) with

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Tukey’s post-hoc test. The PRISM 5 statistical software (Graph Pad Software, San Diego, CA)

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was used for all analyses. P