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Received Date : 20-Sep-2013 Revised Date : 20-Feb-2014 Accepted Date : 26-Feb-2014 Article type : Original Research

Calreticulin Protects Rat Microvascular Endothelial Cells against Microwave Radiation-induced Injury by Attenuating Endoplasmic Reticulum Stress

Wei-Hong Li1,2,3), Yu-Zhen Li1,3), Dan-Dan Song1,3), Xiao-Reng Wang1,3), Mi Liu1,3), Xu-Dong Wu4), and Xiu-Hua Liu1,3)*

1)

Department of Pathophysiology, Chinese PLA General Hospital, Beijing, China

2)

Department of Pathophysiology, College of Basic Medical Sciences, Taishan Medical University, Taian,

China 3)

State Key Laboratory of Kidney Disease (Chinese PLA General Hospital, 2011DAV00088), Beijing,

China 4)

Department of Out-patient, Chinese PLA General Hospital, Beijing, China

Running title: Exogenous calreticulin and endothelial cells

*Correspondence:

Xiu-Hua Liu, M.D, Ph.D. Department of Pathophysiology, Chinese PLA General Hospital, 28 Fuxing Road, Beijing 100853, People’s Republic of China. Tel: 86-10-6693-9763

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/micc.12126 This article is protected by copyright. All rights reserved.

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Fax: 86-10-6693-6375 E-mail: [email protected]

ABSTRACT

Objective: The present study was designed to evaluate whether exogenous calreticulin (CRT) was beneficial for alleviating microwave radiation (MR)-induced injury by suppressing endoplasmic reticulum (ER) stress in rat myocardial microvascular endothelial cells (MMECs). Methods: MMECs were pretreated with CRT (25 pg/mL) for 12 h, followed by the exposure to 2.856 GHz radiation at a mean power density of 30 mW/cm2 for 6 min. MR-induced injury in MMECs was evaluated by lactate dehydrogenase (LDH) leakage, apoptosis and cell viability analysis. The expression of glucose-regulating protein 78 (GRP78), CRT, C/EBP homologous protein (CHOP), Bcl-2 and Bax were examined by Western blot analysis to reflect ER-stress response and ER stress-related apoptosis. Results: MR induced marked MMECs injury, as shown by increased LDH leakage and apoptosis rate and decreased cell viability. MR also induced excessive ER stress, characterized by increased expression of GRP78 and CRT, and ER stress-related apoptotic signaling as well, as shown by the up-regulation of CHOP and Bax and the down-regulation of Bcl-2. Exogenous CRT pretreatment remarkably attenuated MR-induced cell apoptosis and LDH leakage, ER stress and activation of the ER stress-related apoptotic signaling. Conclusions: Exogenous CRT attenuates MR-induced ER stress-related apoptosis by suppressing CHOP-mediated apoptotic signaling pathways in MMECs.

Keywords: microwave radiation, calreticulin, endothelial cells, endoplasmic reticulum stress, apoptosis

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List of Abbreviations

CHOP: C/EBP homologous protein CRT: calreticulin ECs: endothelial cells ER: endoplasmic reticulum ER stress: endoplasmic reticulum stress GRPs: glucose-regulated proteins GRP78: glucose-regulated proteins 78 LDH: lactate dehydrogenase LRP1: lipoprotein receptor-related protein 1 MMECs: myocardial microvascular endothelial cells MR: microwave radiation S-D: Sprague-Dawley UPR: unfolded protein response

INTRODUCTION

Microwaves are electromagnetic waves with frequencies between 300 MHz and 300 GHz. They are

usually categorized as low-power, moderate-power and high-power microwaves according to their power ranges. Microwave radiation (MR) has been reported to induce adverse effects in a variety of organs. It has been reported that MR can injure the testis and hippocampus of rats, and the major pathological changes are hemangiectasis and interstitial edema [29, 33]. These results suggest that MR causes parenchymal organ injury, which is accompanied by microvascular damage. Our previous study found that high-power-microwave radiation at 2.856GHz induced severe damage of the micrangium in rat

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mesentery and mouse auricle, and the damage is mainly manifested as increased permeability of the vessel wall, capillary hemorrhaging, leukocyte rolling and swimming out of the vessels and micrangium constriction (X. H. Liu, unpublished observations). Our previous results indicate that microvascular endothelial dysfunction is involved in the mechanism of MR injury. However, the precise cellular and molecular mechanisms by which MR induces microvascular endothelial cells (ECs) injury have not been elucidated. The endoplasmic reticulum (ER) is an elaborate organelle that plays crucial roles in cell homeostasis

and survival, including the regulation of protein folding, lipid biosynthesis, calcium and redox homeostasis and apoptosis [24]. Alterations in ER homeostasis due to increased protein synthesis, accumulation of mis-folded proteins or disturbances in calcium or redox in ER result in ER stress [23]. It is well known that the ER stress response, also known as the unfolded protein response (UPR), is an adaptive mechanism by which cells react to perturbations in ER homeostasis. The initial objective of the UPR is to re-establish homeostasis and alleviate ER stress by inhibiting general protein translation and increasing the expression of ER-resident chaperones such as calreticulin (CRT) and glucose-regulated proteins (GRPs). However, if the stress is prolonged or severe, the adaptive response fails to re-establish ER homeostasis and the organelle spreads apoptotic signals via the up-regulation of pro-apoptotic transcription factor C/EBP homologous protein (CHOP) and caspase-12 [7, 12]. CHOP, also known as growth arrest and DNA damage-inducible gene 153 (GADD153), is a key signaling component of ER stress-associated apoptosis that is present at low levels under physiolgical condition but is robustly expressed in response to ER stress [26]. Recent studies showed that the factors that induce ER stress in ECs mainly include oxidative stress inducer such as H2O2 [30], peroxidation products [27], ER-stress inducers such as tunicamycin and thapsigargin [13], alteration of shear stress [1], hyperglycemia [25] and hypoxia [14]. However, there are no reports on whether MR can induce excessive ER stress and apoptosis in ECs. CRT is a highly conserved major calcium (Ca2+)-binding chaperone in the ER. A great deal of

attention has been directed toward understanding the role of CRT as a Ca2+-binding protein and a

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molecular chaperone in the ER. Several studies have verified that CRT is involved in and influences the cell injury induced by oxidative stress or hypoxia/reoxygenation, and it affects a cell’s susceptibility to apoptosis, with inconsistent results. Hung et al. [10] found that CRT overexpression protects LLC-PK1 cells against H2O2-induced cell injury. We found that hypoxic preconditioning up-regulates CRT and protects cardiomyocytes from hypoxia/reoxygenation [16, 32]. However, CRT-overexpressed H9c2 cells are highly susceptible to oxidative stress-induced apoptosis [11]. Recent studies have shown that CRT is localized to the cytoplasm, outer cell surface and extracellular compartments of a variety of cell types, and it regulates a variety of diverse and important biological processes from these non-ER compartments, including enhancing cell proliferation, adhesion and migration and inhibiting apoptosis [6]. An in vitro study confirmed that exogenous CRT induces increased proliferation of cultured microvascular ECs [18], suggesting that non-ER CRT may promote endothelial repair. However, there are no reports on whether non-ER CRT is involved in the protection of ECs against MR injury. We hypothesize that exogenous CRT pretreatment attenuates MR-induced microvascular ECs injury by suppressing ER stress-related apoptosis. In this study, MR injury was induced in myocardial microvascular endothelial cells (MMECs), and lactate dehydrogenase (LDH) leakage, apoptosis and cell viability were evaluated. The expression of GRP78, an ER stress marker, was examined to reflect the effect of exogenous CRT on ER-stress response in cultured MMECs treated with MR. Moreover, we explored whether exogenous CRT could attenuate MR-induced ER stress-related apoptosis by suppressing CHOP-mediated pathway in cultured MMECs.

MATERIALS AND METHODS

Chemicals M199 medium (31100019) was obtained from Gibco Co. (Carlsbad, CA, USA). Trypsin was obtained from Amresco Inc. (Solon, OH, USA). Collagenase I (C0130), endothelial cell growth supplement (ECGS, E0760), ethylenediaminetetraacetic acid (EDTA), hydroxyethyl piperazine ethanesulfonic acid (HEPES),

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phenylmethylsulfonyl fluoride (PMSF, P-7626), TritonX-100, sodium dodecyl sulfate (SDS, L-3771), acrylamide, bis-acrylamide, dithiothreitol (DTT), leupeptin, tetramethylethylenediamine (TEMED, T-7024) and ammonium persulfate (APS) were obtained from Sigma-Aldrich Corporation (St. Louis, MO, USA). Human CRT recombinant protein (10-288-22432F) was obtained from GenWay Biotech, Inc. (San Diego, CA, USA). The compound 3-(4,5-dimethylthiazol-2-y-l)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was obtained from Genview (Houston, TX, USA). Rabbit polyclonal antibodies against CRT (SPA-600) and GRP78 (SPA-826) were obtained from Stressgen Co. (San Diego, CA, USA); rabbit polyclonal antibodies against glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Sc-25778), CHOP (Sc-575), Bax (Sc-493) and Bcl-2 (Sc-783), mouse monoclonal antibody against caspase-3(Sc-56055) and integrin β1 (Sc-9970), goat polyclonal antibodies against CRT (Sc-7431) and enhanced chemiluminescence immunodetection kits (Sc-2048) were obtained from Santa Cruz Biotech, Inc. (Santa Cruz, CA, USA). Donkey anti-goat-Alexa Fluor-568 (A-11057) and donkey anti-mouse-Alexa Fluor-488 (A-21202) were obtained from Invitrogen Corporation (Carlsbad, CA, USA). Horseradish peroxidase-conjugated AffiniPure goat anti-rabbit IgG (111-035-003) and goat anti-mouse IgG (115-035-003) were obtained from Jackson ImmunoResearch Co. (West Grove, PA, USA). Protease cocktail tablets were obtained from Roche Co. (Basel, Switzerland).

Cell culture and experimental protocol Male and female Sprague-Dawley (S-D) rats weighing 80–100 g (4 weeks old) were used for the MMECs culture protocol. All procedures were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) and approved by the local animal care and use committee. The culture and identification of MMECs from S-D rats were described previously [31]. Briefly, the rats were killed by intraperitoneal injection of a lethal dose of pentobarbital (100 mg/kg body weight), and the left ventricles were fully minced and digested with 0.1% collagenase I for 10 min at 37°C in a shaking water bath. Then, 0.1%

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trypsin was added for incubation for another 10 min at 37°C. The digested solution was filtered through a 100-μm mesh filter, and the filtrate was suspended in M199 medium containing 2 mmol/L L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, 25% fetal calf serum, 40 U/mL heparin and 0.02 g/L ECGS. Then, the suspension was cultured in a humidified cell culture incubator with 5% CO2 at 37°C, and the medium was changed every 3 days. The cells were characterized based on their typical cobblestone morphology and the presence of VE-cadherin (CD144), an EC-specific marker and determinant of microvascular integrity in vivo described previously [31]. All studies were performed on MMECs between passages three and five. Cells were plated at

5×104/cm2 (except for the immunofluorescence assay for the cellular localization of CRT, for which the density was 1×104/cm2 to obtain individually plated cells) and allowed to rest overnight before the application of stress. The MMECs were divided randomly into the following groups for treatment: (1) control group: under sterile conditions, cells on culture plates were sham-exposed to MR in the radiation darkroom environment for 6 min, then cells were cultured normally in a 5% CO2 incubator at 37°C for 24 h; (2) MR group: under sterile conditions, cells on culture plates were exposed to 2.856 GHz radiation at a mean power density of 30 mW/cm2 for 6 min according to the previous study [28] and the results of preliminary experiment, then cells were cultured normally for 24 h; (3) CRT+MR group: cells were pretreated with CRT (25 pg/mL) for 12 h according to the previous study [18] and the results of preliminary experiment before being subjected to the previously described MR treatment; and (4) CRT group: cells were incubated with CRT (25 pg/mL) for 12 h and then cultured normally for 24 h. The 2.856 GHz high-power MR simulation device was provided by the Institute of Radiation Medicine of the

Academy of Military Medical Sciences in China. The mean power density of microwaves is 30 mW/cm2, and the radiation distance was 1 m. The environmental temperature in the radiation darkroom was 25±0.5°C, and the humidity was 65±5%. To monitor the temperature inside the MR system, we used an optical fiber thermometer (Luxtron M3300, LumaSense Technologies), attesting a temperature stability of 25±0.5°C during the whole exposure duration, and the temperature difference between sham- and MR-exposed cultures never exceeded 0.1°C. Therefore, the reported results are of non-thermal.

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LDH activity analysis LDH activity in the culture medium was measured to estimate LDH leakage under different treatment conditions. LDH activity in the medium was measured spectrophotometrically by the use of the LDH assay kit (Jiancheng Bioengineering Inst., Nanjing, China) according to the manufacturer’s instructions.

Apoptosis analysis For the apoptosis analysis, cells were collected using 0.2% trypsin and stained using the Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit (KeyGEN Bioengineering Inst., Nanjing, China), and apoptosis rate was analyzed using the BD FACScalibur flow cytometer (BD Biosciences, San Jose, CA, USA). Each experiment was repeated four times.

MTT assay Cell viability was detected using the MTT assay according to the following method. Cells were seeded at a density of 1,500/well in 96-well plates (each group has 6-8 parallel wells), treated with CRT (25 pg/mL) for 12 h and subsequently exposed to 2.856 GHz microwaves at a mean power density of 30 mW/cm2 for 6 min. Then, cells were cultured normally for 24 h. Next, MTT was added to a final concentration of 0.5 mg/ml, and the samples were incubated at 37°C for 4 h. After removing the supernatant, 150 μL dimethylsulfoxide (DMSO) was added to dissolve the formazan crystals, and the optical density (OD) was detected at 492 nm using an Infinite F200 microplate reader (Tecan, Switzerland). Cell viability was expressed as the percentage of the control group (100%).

RNA extraction and quantitative real-time PCR analysis Total RNA was extracted using the TRNzol-A+ total RNA extraction reagent (TianGen, Beijing, China), and reverse transcription was performed with a cDNA first-strand synthesis kit (Quanshijin, Beijing, China) following the manufacturer’s instructions. The CRT primers used for real-time PCR were synthesized

by

Sanboyuanzhi

Biotech

Co.

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(Beijing,

China)

as

follows:

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5'-CAAGGATATCCGGTGTAAGGA-3' (forward) and 5'-CATAGATATTCGCATCGGGG-3' (reverse). Real-time PCR was performed with an ABI 7500 Fast Real-time PCR System (Applied Biosystems, Foster City, CA, USA) using the TaqMan® Gene Expression Master Mix and TaqMan® fluorescence probe (Applied Biosystems, Foster City, CA, USA). The relative mRNA levels were calculated using the 2–

△△Ct

method and normalized against the housekeeping gene GAPDH. Each experiment was repeated

three times.

Western blot analysis Protein was extracted from the MMECs as described previously [17]. Equal amounts of protein (60 μg/lane) were subjected to SDS-PAGE (10% resolving gel) and transferred onto a nitrocellulose membrane by electroblotting for immunoblot analysis. After incubation with blocking buffer (Tris-buffered saline containing 0.1% Tween 20 and 10% nonfat dry milk) for 2 h at room temperature, the membranes were incubated with the primary antibodies anti-CRT (1:1,000), anti-GRP78 (1:1,000), anti-CHOP (1:200), anti-caspase-3 (1:200), anti-Bax (1:200), anti-Bcl-2 (1:200) and anti-GAPDH (1:500) overnight at 4°C. After washing four times with Tris-buffered saline containing 0.1% Tween 20, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:1,000) for 1 h at room temperature. The immunoblots were revealed using an enhanced chemiluminescence kit. The integrated optical density (IOD) of immunoreactive bands was measured using Image-Pro Plus software (version 6.0, Media Cybernetics, LP, USA) and normalized to that of the housekeeping protein GAPDH. Each experiment was repeated three times.

Immunofluorescence assay Cells were seeded at a density of 1×104/cm2 on glass coverslips in 24-well plates (each group has 6-8 parallel wells) and allowed to rest overnight, then treated with CRT (25 pg/mL) for 12 h and subsequently exposed to 2.856 GHz microwaves at a mean power density of 30 mW/cm2 for 6 min. Then, cells were

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cultured normally for 24 h. Next, the treated cells were fixed in 4% paraformaldehyde for 15 min, washed with phosphate-buffered saline (PBS) and blocked in 1% BSA for 50 min. Non-permeabilized cells were incubated with the primary antibodies goat anti-CRT (1:100) and mouse anti-Integrin β1 (1:100) for 1 h at room temperature. After washing three times with PBS, the cells were incubated with the secondary antibodies donkey anti-goat-Alexa Fluor-568 (1:600) and donkey anti-mouse-Alexa Fluor-488 (1:400) for 1 h at room temperature. After washing three times with ice-cold PBS, stained cells were mounted in mounting medium with DAPI (Vector Laboratories, Burlingame, CA, USA) and viewed under a confocal laser scanning microscope (FV1000, Olympus, Tokyo, Japan). The integrated light intensity of immunoreactive CRT was measured using Image-Pro Plus software (version 6.0, Media Cybernetics, LP, USA) to estimate the fluorescence intensity of CRT in MMECs under different treatment conditions. Each experiment was repeated three times.

Statistical analysis The results are presented as the mean±SD. Significant differences among groups were assessed by one-way analysis of variance (ANOVA) followed by Student–Newman–Keuls post-tests. P-values less than 0.05 were considered statistically significant.

RESULTS

1. Exogenous CRT attenuated MR-induced MMECs injury 1. 1. Apoptosis rate Apoptosis rate detected by flow cytometry analysis is shown in Figure 1. The apoptosis rate was increased with MR compared to the control treatment (4.97±0.35% vs. 1.58±0.24%, P

Calreticulin protects rat microvascular endothelial cells against microwave radiation-induced injury by attenuating endoplasmic reticulum stress.

This study was designed to evaluate whether exogenous CRT was beneficial for alleviating MR-induced injury by suppressing ER stress in rat MMECs...
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