Irradiated U937 Cells Trigger Inflammatory Bystander Responses in Human Umbilical Vein Endothelial Cells through the p38 Pathway Author(s): Linlin Xiao, Weili Liu, Jitao Li, Yuexia Xie, Mingyuan He, Jiamei Fu, Wensen Jin, and Chunlin Shao Source: Radiation Research, 182(1):111-121. 2014. Published By: Radiation Research Society DOI: http://dx.doi.org/10.1667/RR13736.1 URL: http://www.bioone.org/doi/full/10.1667/RR13736.1

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RADIATION RESEARCH

182, 111–121 (2014)

0033-7587/14 $15.00 Ó2014 by Radiation Research Society. All rights of reproduction in any form reserved. DOI: 10.1667/RR13736.1

Irradiated U937 Cells Trigger Inflammatory Bystander Responses in Human Umbilical Vein Endothelial Cells through the p38 Pathway Linlin Xiao,a,b Weili Liu,a Jitao Li,a Yuexia Xie,a Mingyuan He,a Jiamei Fu,a Wensen Jinb and Chunlin Shaoa,b,1 a

Institute of Radiation Medicine, Fudan University, Shanghai 200032, China; and b Division of Nuclear Medicine, School of Medical Sciences, Anhui Medical University, Hefei 230032, China

INTRODUCTION Xiao, L., Liu, W., Li, J., Xie, Y., He, M., Fu, J., Jin, W. and Shao, C. Irradiated U937 Cells Trigger Inflammatory Bystander Responses in Human Umbilical Vein Endothelial Cells through the p38 Pathway. Radiat. Res. 182, 111–121 (2014).

Ionizing radiation, increasing age, diabetes mellitus, hypertension, hypercholesterolemia, obesity and smoking are independent risk factors for atherosclerosis that could potentially lead to the occurrence of vascular stenosis, thromboembolism and stroke (1, 2). Radiation-induced atherosclerosis was first reported in 1959 (3) and was suggested to be major cause of radiation-induced cardiovascular disease (CVD), appearing in over half of the cancer survivors who were treated with radiotherapy. Cardiovascular disease has been shown to be the major cause of late radiation-induced morbidity. For example, the risk of stroke was increased in head and neck cancer patients many years after radiotherapy treatment (4, 5). In addition, life span studies of Japanese atomic bomb survivors have demonstrated that radiation exposure significantly increases the risk of CVD (6–9). Further, other epidemiological studies showed that radiation technologists had an increased incidence of atherosclerosis (10), which is a chronic inflammatory disease (11–14) resulting from complex interactions among circulation factors, monocytes and various types of cells in the vessel wall, including endothelial cells (15). The endothelial cell layer acts as a barrier between the blood and vessel wall and endothelium dysfunction such as inflammatory damage is an initiating factor in atherosclerosis (16). Over the past 50 years, numerous studies have confirmed that endothelial cells can be damaged by exposure to ionizing radiation (17). However, additional studies are still needed to investigate crosstalk among different types of cells such as monocytes/macrophages and endothelial cells in the circulatory system after irradiation. It has been previously reported that an irradiated cell can trigger a response to nearby nonirradiated cells, which are now known as radiation-induced bystander effect (RIBE) (18– 21). As an important intrinsic physiological signal, nitric oxide (NO) can be induced from irradiated macrophages (22), and previous studies have demonstrated that nitric oxide generation is an early signaling event of radiationinduced bystander effects (23–25). At low doses, nitric oxide can protect cells from radiation-induced damage, and at high doses nitric oxide can cause DNA damage leading to

Radiation-induced bystander effects are a well-known phenomenon that are observed when treating cancer and other diseases after radiotherapy, and even after occupational exposure to radiation. However, little is known about the crosstalk between irradiated macrophages and endothelial cells that line the circulatory system, which may play a role in the development of atherosclerosis. In the current study, we found that the expression of inducible nitric oxide synthase (iNOS) and the intracellular level of nitric oxide (NO) in gamma-irradiated U937 macrophage cells were significantly increased. When human umbilical vein endothelial cells (HUVECs) were co-cultured with gamma-irradiated U937 cells, additional micronuclei (MN) and apoptosis were induced so that the plating efficiency of the bystander HUVECs decreased and P38 was overexpressed in the bystander HUVECs cells. In addition, the contents of vascular cell adhesion molecule 1 (VCAM-1) and the activities of matrix metalloproteinase-9 (MMP-9) in the culture medium of bystander HUVECs were increased. Furthermore, during cell co-culture the adhesive ability of irradiated U937 cells to the bystander HUVECs increased. When U937 cells were treated with 500 lM S-methylisothiourea sulfate (SMT) (iNOS inhibitor) before irradiation, and HUVECs were treated with 10 lM SB203580 (p38 inhibitor) before cell co-culture or treated with 20 lM cPTIO (NO scavenger) in the co-culture medium, the bystander micronuclei and the amounts of VCAM-1 and MMP-9 in the medium of bystander HUVECs were diminished, and the ability of irradiated U937 cells adhering to HUVECs was also reduced, while the plating efficiency of bystander HUVECs partially recovered. These results demonstrated that irradiated U937 cells appear to release nitric oxide and thereby further trigger apoptosis and inflammatory responses in the bystander HUVECs through a p38dependent pathway. Ó 2014 by Radiation Research Society

1 Address for correspondence: Fudan University, Institute of Radiation Medicine, No. 2094, Xie-Tu Road, Shanghai, Shanghai 200032, China; e-mail: [email protected].

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micronuclei (MN) formation and apoptosis (26) and thus it is possible that nitric oxide at higher concentrations is an inflammatory factor. Inflammation can initiate and maintain the formation of atherosclerosis in coronary and peripheral arteries. One of the earliest events in the development of atherosclerotic lesions is the accumulation of monocytes/macrophages and lymphocytes in the intima of large vessels. Monocytes/ macrophages, which are important immune cells that exist in the blood vessels, do not combine with the endothelial cell layer under normal physiological conditions. However, they have been shown to combine with the endothelium and migrate to the sub-endothelial space where they differentiate into macrophages once the endothelial cells are damaged, leading to a pathological condition such as atherosclerosis and ischemic stroke (27–29). This extravasation is a multistep process (30) consisting of the initial adhesive contact of leukocyte with endothelium, rolling, firm adhesion and transendothelial migration. Accumulation of leukocytes on the vascular endothelium is a hallmark of inflammation. This process of leukocyte-endothelial cell adhesion appears to be regulated by the expression of adhesion molecules. The vascular cell adhesion molecule 1 (VCAM-1) can be expressed in both large and small blood vessels only after endothelial cells are stimulated. The activation of endothelial VCAM-1 contributes to both rolling and firm adhesion (31), which can be regulated by p38/MAPK (mitogen-activated protein kinases) (32). The activation of P38 protein may lead to inflammation and apoptosis in human umbilical vein endothelial cells (HUVECs) (33, 34). Moreover, as a member of immunoglobulin super family, VCAM-1 plays a critical role in leukocyte recruitment to the sites of inflammation, accelerating the progress of atherosclerosis (35). Macrophage homing to the plaque is also responsible for degradation and rupture of atherosclerotic plaque (36). Other crucial players in the development of atherosclerosis include matrix metalloproteinases (MMPs), which are synthesized in latent form and secreted as proenzymes. The activation of MMP-9 could degrade extracellular matrix components and thus is a key mediator for the destabilization of atherosclerotic plaque (37). In the current work, the crosstalk among HUVECs and irradiated macrophage cells was investigated to disclose the potential novel mechanism of radiation-induced atherosclerosis. MATERIALS AND METHODS Cell Culture Primary HUVECs were purchased from ATCC (Manassas, VA) and maintained in Dulbecco’s modified Eagle’s medium (DMEM, HyClone, Beijing, China) containing glucose (4.5 g/L). U937 cells, originally derived from human histiocytic lymphoma and commonly used as a model of macrophages, were obtained from the Shanghai Cell Bank of China and grown in RPMI 1640 (HyClone). Both

culture media contained 100 U/mL penicillin, 100 lg/mL streptomycin and 10% FBS (Gibco Invitrogen, Grand Island, NY). All cells were maintained in a fully humidified atmosphere with 5% CO2 at 378C. Cell Irradiation, Co-Culture and Chemical Treatments U937 cells were irradiated with gamma rays at a dose rate of 0.8 Gy/min using a 137Cs Gammacell-40 irradiator (Nordion International Inc, Kanata, Ontario, Canada). After irradiation, U937 cells were washed three times with PBS and then co-cultured with nonirradiated HUVECs for varying amounts of time (h) in a transwell system (Corning Costar Co, Cambridge, MA). U937 suspension cells (5 3 10 5 ) were grown in an insert dish with a semipermeable polycarbonate membrane bottom and a sufficient number of pores (0.4 lm, 8 3 105 pores/cm2) to allow small molecules to transfer through. HUVECs (2 3 105) were cultured in the companion tissue culture-treated plate. The cell number ratio of HUVECs to U937 cells was set at approximately 3:7 because this proportion has been reported to be an optimum condition for the induction of bystander response (38). In some experiments, 10 h before radiation exposure U937 cells were treated with 500 lM S-methylisothiourea sulphate (SMT, Sigma, St Louis, MO), a highly selective inhibitor of inducible nitric oxide synthase (iNOS) (39). HUVECs were treated 1 h before cell co-culture with 10 lM SB203580 (Sigma), a p38 inhibitor. In some experiments, 20 lM c-PTIO (Sigma), an effective NO scavenger (40) was added into the medium at the beginning of cell co-culture. In addition, to better understand the effect of NO on HUVECs, 5 3 105 cells per dish were treated with a NO donor, sodium nitroprusside (SNP, Beyotime Institute of Biotechnology, Haimen, China), at various doses for 48 h and then the HUVECs were trypsinized for further assay. Colony Formation Assay After the cell co-culture treatment, the HUVECs were harvested and resuspended in DMEM. The 120 cells/100 lL were seeded in 60 mm dishes to form approximately 50–100 colonies (.50 cells each) per dish. After twelve days of incubation, the colonies were fixed with 10% formalin and stained with 1% methylene blue. The plating efficiency was calculated as the number of colonies divided by the number of seeded cells. Micronuclei Assay Micronuclei induction has been widely used as an end point for chromosome damage. Here MN were measured using the cytokinesis-block technique. In brief, after cells were co-cultured, a portion of the harvested HUVECs were incubated for about 6 h to allow cell attachment, then treated with 2 lg/mL cytochalasin B (Sigma) for 30 h and then fixed in situ with methanol/acetic acid (9:1 v/v) for 20 min. Cells were air-dried and then stained with 0.01% acridine orange (Sigma) and observed under a fluorescence microscope (Olympus, Tokyo, Japan). Micronuclei were scored in at least 500 binucleated cells and the MN yield, YMN, was calculated as the ratio of the number of micronuclei to the scored number of binucleated cells. Nitric Oxide Measurement Because the NO free radical has a very short half-life and can be oxidized to form two stable NO products of nitrate (NO3–) and nitrite (NO2–) (41), we quantified the NO free radical indirectly by measuring the concentrations of nitrate and nitrite in the medium of irradiated U937 cells with a NO assay kit (Beyotime Biotechnology), according to the protocol supplied by the manufacturer. The absorbance of cell medium samples was measured at 540 nm using

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FIG. 2. Expressions of p-P38, p-ERK1/2 and p-JNK1/2 in HUVECs co-cultured with 3 Gy c-irradiated U937 cells. Panel A: The representative immunoblots of the expressions of p-P38, p-ERK1/ 2 and p-JNK1/2 in the HUVECs after different times of cell coculture. Panel B: The relative level of p-P38 in the HUVECs after different times of cell co-culture. The data were first compared to corresponding GAPDH and then normalized to the control without coculture. ***P , 0.001 compared with the control without co-culture. FIG. 1. MN induction in HUVECs co-cultured with irradiated U937 cells. Panel A: The dose response of the yield of bystander MN in the HUVECs after 48 h of cell co-culture. Panel B: The time response of the yield of bystander MN in the HUVECs co-cultured with 3 Gy c-irradiated U937 cells. **P , 0.01 and ***P , 0.001 compared with the control without co-culture.

a microplate reader (TECAN Infinite M200 PRO, Ma¨nnedorf, Switzerland). Western Blotting Assay The expressions of iNOS, p-P38, p-JNK and p-ERK were measured with Western blotting. Briefly, U937 cells and HUVECs were treated with RIPA lysis (Beyotime Biotechnology) containing phosphatase inhibitor cocktail (1:100) (Sigma), 2 mM sodium orthovanadate (Sigma) and 1 mM phenylmethanesulfonyl fluoride (PMSF, Sigma) and placed on ice for 10 min. Cell lysate was centrifuged at 12,000 rpm at 48C for 5 min. The supernatant was collected and total protein concentration was quantified using the bicinchoninic acid protein assay kit (Beyotime Biotechnology). An aliquot of 40 lg of protein from each sample was subjected to 10% SDS-polyacrylamide gel for protein separation, and these protein

blots were then electrotransferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA) and immunoblotted with a primary antibody followed by the secondary antibody conjugated to horseradish peroxidase (ICL Inc., Newberg, OR). Immunocomplexes were then visualized with the ECL detection kit (Millipore) and the protein images were captured digitally with the ChemiDoc XRS system (Bio-Rad Laboratories, Hercules, CA). The primary antibodies were diluted as 1:1,000 for phosphorylatedERK1/2 (p-ERK1/2), phosphorylated-JNK1/2 (p-JNK1/2) and phosphorylated P38 (p-P38) (Cell Signaling Technology, Beverly, MA), 1:1,000 for b-tubulin, b-actin, GAPDH (Beyotime Biotechnology) and 1:800 for iNOS (Abcam, Cambridge, MA). U937 Cell Adhesion Assay After irradiation, U937 cells were resuspended in DMEM. U937suspension cells (1.2 3 106) were seeded on a plate well where HUVECs (0.5 3 106) had been seeded 12 h before. These two kinds of cells were co-cultured for 48 h at 378C under standard conditions. The suspended U937 cells were then removed by gently washing the cultures four times with medium. Because the U937 cells that remained adhered over the confluent monolayer of HUVECs had a round shape, they could be easily distinguished from larger fusiform HUVECs under a phase contrast microscope. For each sample, the

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FIG. 3. Radiation-induced NO from U937 cells and the MN induction in HUVECs treated with sodium nitroprusside (SNP) (NO donor). Panel A: The time course of the expression of iNOS in irradiated U937 cells. The relative expression level of iNOS was first, normalized to a-tubulin and then the ratio of each normalized value to the control was calculated. Panel B: The effect of SMT and c-PTIO on the expression of iNOS in the irradiated U937 cells. The relative expression level of iNOS was first, normalized to b-actin and then the ratio of each normalized value to the control value was calculated. Panel C: The times (h) of the concentration of NO in the medium from 3 Gy c-irradiated U937 cells. Panel D: MN induction in HUVECs treated with different doses of SNP for 48 h or treated with 1,000 lM SNP plus 20 lM c-PTIO. ***P , 0.001 compared with the control without irradiation or drug treatment. ###P , 0.001 between indicated groups.

number of adhesive U937 cells was counted in eight different fields (2003) in the middle of the dish and its relative value of each treated culture was normalized to the nonirradiated control.

Flow Cytometry, version 2.8 (Scripps Research Institute, San Diego, CA). For each sample, 20,000 events were collected and analyzed. Gelatin Zymography Assay

Apoptosis Assay Cell apoptosis was measured with the Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) staining assay with a commercial Apoptosis Detection Kit (BD Pharmingen, San Jose, CA). After the co-culture treatment, HUVECs were harvested and washed twice with cold PBS and resuspended in 400 lL of binding buffer containing 5 ll of Annexin V-FITC and 5 ll of PI. After incubation for 30 min at 378C in the dark, the cells were analyzed by a flow cytometer (Guava easyCytee HT System, Millipore, MA). The data were analyzed with the software, Multiple Document Interface (Microsoft Windows) for

After co-culturing with irradiated U937 cells for 48 h, HUVECs were well washed with PBS twice and further cultured in serum-free medium for 12 h. This medium was then collected and the activity of MMP-9 in the medium was measured by a standard gelatin zymography assay. In brief, the nondenatured supernatant proteins in the conditioned medium were loaded on a 10% SDS-polyacrylamide gel, at 48C, containing 0.1% gelatin (Sigma). After electrophoresis, the gels were washed twice for 45 min with the renaturing solution [2.5% (v/v) of Triton X-100 in doubledistilled H2O] to remove SDS and were then incubated in the developing buffer [50 mM Tris-HCl buffer (pH 7.6), 200 mM NaCl, 5 mM CaCl2, 1 mM ZnCl2 and 0.02% Brij-35 in double-distilled H2O] for 40 h at 378C.

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FIG. 4. Influence of SMT, c-PTIO and P38 inhibitor SB203580 on the MN formation of HUVECs that were co-cultured with 3 Gy cirradiated U937 cells for 48 h. In some experiments, U937 cells were treated 10 h before irradiation with 500 lM SMT or treated with 20 lM c-PTIO during co-culture, or HUVECs were treated 1 h before coculture with 10 lM SB203580. ***P , 0.001 compared with the control or between indicated groups. After staining with 0.5% Coomassie brilliant blue R-250 plus 0.1% Naphthol blue black, the gel was treated with destaining buffer (10% acetic acid and 30% methanol in double-distilled H2O) until gelatindegrading proteins were identified as clear sharp zones over the blue background. The nonstaining bands representing the level of gelatinolytic activity were quantified by a densitometer with a digital gel imaging analysis system (Tanon-2500R, Tanon Science & Technology Co., Shanghai, China). Three independent assays were performed. Immunocytochemical Assay of VCAM-1 The expression of VCAM-1 was examined by immunohistochemistry. Briefly, 48 h after co-culture, the HUVECs (5 3 104 cells grown on coverslip) were fixed with 4% paraformaldehyde for 15 min and blocked with the blocking buffer (5% normal goat serum and 0.3 % Triton X-100 in PBS) for 1 h at room temperature to permeabilize the cells and block nonspecific protein–protein interactions. Samples were then incubated overnight with the VCAM-1 primary antibody (1:250, Abcam) at 48C in a humidified chamber. After washing with PBS three times, the cells were incubated with Alexa Fluor 488-conjugated secondary antibody (1:1,000) (Cell Signaling Technology) for 2 h at room temperature in the dark, then 2 lM 4 0 ,6-diamidino-2-phenylindole (DAPI) (Sigma) was used to stain the cell nuclei. After further washing with PBS, the cells on the glass coverslip were embedded in mounting media (Vector Laboratories, Burlingame, CA) and photographed with a fluorescence microscope (Olympus). The FITC and DAPI fluorescence was observed by using WB filter (excitation wavelength 460–490 nm, emission 515 nm) and WU filter (excitation wavelength 330–385 nm, emission 420 nm), respectively.

FIG. 5. Influence of SMT, c-PTIO and P38 inhibitor SB203580 on the induction of apoptosis (panel A) and plating efficiency (panel B) of bystander HUVECs that were co-cultured with 3 Gy c-irradiated U937 cells for 48 h. In some experiments, U937 cells were treated 10 h before irradiation with 500 lM SMT or treated with 20 lM c-PTIO during co-culture, or HUVECs were treated 1 h before co-culture with 10 lM SB203580. ***P , 0.001 compared with the control or between indicated groups. ###P , 0.001 compared with 3 Gy cirradiated cells.

RESULTS

Statistical Analysis

Bystander Responses in HUVECs Co-cultured with Irradiated U937 Cells

The data shown as mean 6 SE were obtained from three independent experiments and analyzed with the software SPSS11.5 (SPSS Inc., Chicago, IL). Data with P , 0.05 are determined to be significantly different between indicated samples.

Figure 1 shows that when nonirradiated HUVECs were co-cultured with irradiated U937 cells, additional MN are induced whose yield increased with radiation dose to a

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FIG. 6. Effects of SMT, c-PTIO and SB203580 on the adhesive ability of U937 cells to HUVECs and the expression of VCAM-1 in the bystander HUVECs that were co-cultured with 3 Gy c-irradiated U937 cells for 48 h. Panel A: Representative cell images showing the adhesion of irradiated U937 cells to HUVECs. Panel B: The semi-quantitative analysis of the number of adherent U937 cells. Data are the mean 6 SEM from at least three independent experiments performed in duplicate. Panel C: The in situ immunocytochemical expression of

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maximum value of 3 Gy, then decreased. At 3 Gy, the bystander MN formation in HUVECs increased with the cell co-culture time up to 48 h, which was consistent with our previous reports (42). Accordingly, 48 h of cell coculture after a 3 Gy irradiation of U937 cells was selected as the ideal condition for the induction of bystander response in HUVECs and thus was chosen for the following mechanistic studies. In addition to DNA damage, we were interested in whether an inflammatory reaction could be induced in the bystander HUVECs. The MAPK signaling pathway has been shown to be very involved in the induction of proinflammatory mediators in endothelial cells. We found that P38 protein was activated in the bystander HUVECs after co-culturing with U937 cells gamma irradiated with a dose of 3 Gy and the phosphorylation level (i.e., p-P38 level) reached at a peak value at 24 h of cell co-culture then declined at 48 h, with the P-38 level remaining higher than the control (Fig. 2). However, the expressions of the pJNK1/2 and p-ERK1/2 proteins in the MAPK pathway were not influenced in the bystander HUVECs after co-culturing with irradiated U937 cells. Generation of NO from Irradiated U937 Cells

We propose that the bystander responses we observed above in the HUVECs, are the result of some reactive factors released from the irradiated U937 cells. We show that an early event of bystander signaling is the induction of NO (Fig. 3A). We show that the expression of iNOS in U937 cells increased after exposure to 3 Gy of radiation, approaching 1.2-fold of the control at 8 h postirradiation, and remained at this high level until 24 h postirradiation (Fig. 3A). When the U937 cells were treated with 500 lM SMT ten hours before irradiation, the expression of iNOS was completely inhibited. However, treatment of U937 cells with 20 lM c-PTIO (NO scavenger) did not influence the expression of radiation-induced iNOS (Fig. 3B). A high expression level of iNOS indicates the generation of NO. Figure 3C shows that the concentration of NO in the medium of irradiated U937 cells increased along with the cell culture time after 3 Gy irradiation, approaching 13.3fold of the control at 48 h postirradiation. This radiationinduced NO is a good candidate for mediating the observed bystander responses, as suggested by our previous reports (43, 44). SNP-Induced Micronuclei in HUVECs

To determine if DNA damage was induced by NO in HUVECs, we treated HUVECs with SNP (NO donor) and found that the yield of MN in HUVECs increased until the dose was more than 500 lM of SNP (Fig. 3D). However, when the dose of SNP increased to 1,000 lM, no additional

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MN were induced, perhaps because most of the cells died at this higher dose. When 20 lM of c-PTIO was added to the culture medium during the SNP treatment, MN induction was clearly reduced to a very low level. Effects of SMT, c-PTIO and SB203580 on Micronuclei Induction in Bystander HUVECs

To further confirm the bystander signals, cells were treated with SMT, c-PTIO and SB203580 regents. Figure 4 shows that, after co-culturing with gamma-irradiated U937 cells, the yield of MN in the HUVECs increased from 0.004 of control to 0.035. When the U937 cells were treated with 500 lM SMT before irradiation or with 20 lM c-PTIO during cell co-culture postirradiation, the incidence of MN in the HUVECs decreased to 0.015 and 0.013, respectively, supporting that NO contributes to the radiation-induced bystander effect of MN formation. We also observed that the treatment of U937 cells with SMT or c-PTIO, only diminished, but did not eliminate, the bystander responses, suggesting that some signaling molecules other than NO might also be involved in the radiation-induced bystander effect. To determine how the bystander cells respond to other signal factors, the HUVECs were treated with 10 lM SB203580 for 1 h before co-culturing with gammairradiated U937 cells. Under this condition, the incidence of MN in the bystander HUVECs was reduced to 0.023, indicating that the p38 pathway in HUVECs plays an important role in perceiving the bystander signals. Effects of SMT, c-PTIO and SB203580 on the Induction of Apoptosis and Plating Efficiency of Bystander HUVECs

We observed that apoptosis was also induced in the bystander HUVECs and the apoptotic rate increased from 5.3% in the control to 17.0% after co-culturing with gamma-irradiated U937 cells. When the U937 cells were treated with 500 lM SMT before irradiation or treated with 20 lM c-PTIO during cell co-culture after irradiation, the apoptotic rate in the bystander HUVECs was decreased to control level. When the HUVECs were treated with 10 lM SB203580 before co-culture, the apoptotic rate in the bystander HUVECs decreased to 9.15%, although this was still higher than the control (Fig. 5A). The significant increase of DNA damage therefore also appears to cause cell-killing in the bystander HUVECs. It was found that, after co-culturing with U937 cells irradiated with 3 Gy of gamma ray, the plating efficiency of HUVECs decreased from 51.7% in the control to 34.5%, indicating a strong lethal bystander effect. When the U937 cells were treated with 500 lM SMT or 20 lM c-PTIO to reduce the bystander signaling factor of NO, the bystander cell-killing effect was significantly reduced and the plating efficiency of

VCAM-1 in the bystander HUVECs. In some experiments, U937 cells were treated 10 h before irradiation with 500 lM SMT or treated with 20 lM c-PTIO during co-culture or HUVECs were treated 1 h before co-culture with 10 lM SB203580. ***P , 0.001 compared with the control or between the indicated groups.

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expression was measured immunocytochemically in the bystander HUVECs. Figure 6C shows that after co-culturing with irradiated U937 cells, the expression of VCAM-1 in the cell membrane of HUVECs was obviously enhanced and it was partly attenuated by SMT and c-PTIO and completely inhibited by SB203580, consistent with the above results of U937 cell adhesion. Effects of SMT, c-PTIO and SB203580 on MMP-9 Generation from Bystander HUVECs

It has been known that VCAM-1 can accelerate the progress of atherosclerosis (35) where MMP-9 may be involved. We show by gelatin zymography assay that the activity of MMP-9 was much higher than MMP-2 in HUVECs (data not shown), suggesting that MMP-9 may play a more important role in the cell response. After coculturing with irradiated U937 cells, the activity of MMP-9 in the supernatant of bystander HUVECs was obviously increased to 1.35-fold of control (Fig. 7). This increase of MMP-9 was completely eliminated by the treatment of irradiated U937 cells with SMT and c-PTIO, and partly inhibited by the treatment of HUVECs with SB203580. DISCUSSION

FIG. 7. Effects of SMT, c-PTIO and SB203580 on the MMP-9 activity in the medium of the bystander HUVECs Panel A: A typical image of gelatin zymography assay for the MMP-9 activity. Panel B: The relative expression level of MMP-9 activity. In some experiments, U937 cells were treated 10 h before irradiation with 500 lM SMT or treated with 20 lM c-PTIO during co-culture or HUVECs were treated 1 h before co-culture with 10 lM SB203580. ***P , 0.001 compared with the control without irradiation. ###P , 0.001 compared with SMT þ 3 Gy or c-PTIO þ 3 Gy treated cells.

bystander HUVECs had mostly recovered. When the HUVECs were treated with 10 lM SB203580 before cell co-culture treatment, the plating efficiency also partly recovered to 38.9% (Fig. 5B), indicating again that appears to be p38 is an important intrinsic cellular sensor to bystander signals. Role of Nitric Oxide and p38 Pathway in Cell Adhesion of Bystander HUVECs

Monocyte adhesion to endothelial cells is an essential event in the initiation of atherosclerosis development (45). Leukocyte–endothelial monolayer adhesion assay is a well-documented in vitro model for studying the interaction between leukocytes and endothelial cells (46). As shown in Fig. 6A and B, after 3 Gy exposure, the adhesive ability of U937 cells to HUVECs was markedly increased by about 3 times. This increase was shown to be partially attenuated by SMT and cPTIO, while SB203580, the inhibitor of p38, inhibited the adhesion of irradiated U937 cells to HUVECs completely. VCAM-1 is an important factor in leukocyte recruitment to the sites of inflammation, and therefore VCAM-1

Radiation increases the risk of cardiovascular disease and stroke. The mechanism of radiation-induced atherosclerosis, however, is not well understood. Most of previous studies have focused on the direct radiation damage of endothelial cells, but little investigation has been done on the role of macrophages in the process of atherosclerosis. As a model cell line, U937 cells derived from human histiocytic lymphoma were widely used as macrophages in the study of the mechanism of atherosclerosis (47–49) and have also been used to study the behavior and differentiation of monocytes as normal cells (50). Macrophages operate as immune cells and when the body is irradiated, may produce large amount of inflammatory cytokines and that further impact bystander and even distant endothelial cells, which ultimately may lead to chronic selfperpetuating inflammation. Radiation-induced bystander effects have potential implications in human health. Our current study found that iNOS was activated in the irradiated U937 cells and that a copious amount of NO was produced. NO can be generated from the guanido-group of L-arginine by three isotypes of NOS. Two constitutive NOS, calcium-dependent NOS (cNOS or NOS1) and endothelial NOS (eNOS or NOS3), generate small quantities of NO sufficient for cellular signaling. However, the third isotype, calcium-independent NOS, i.e., iNOS, is typically expressed in response to proinflammatory stimuli (51). NO can react with reactive oxygen species (ROS) to form peroxynitrite that may lead to nitrosylation or nitration on tyrosine residues of proteins (52, 53) and thus alter the activities of these proteins. The small quantity of NO generated through eNOS has a protective effect in atherosclerotic lesions (54). Conversely, the inflammatory environment in human atherosclerotic lesion results in

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overexpression of iNOS in macrophages and foam cells so that a large amount of NO as well as ROS are produced. Overexpression of iNOS, a common phenomenon during chronic inflammatory condition, has been shown to generate sustainable amounts of NO and its mutagenic reactive intermediates may cause DNA damage or impair DNA repair. Increase of NO/iNOS has been suggested as a major mechanism by which cytokines mediate cardiac contractile dysfunction and develop cardiovascular disease. NO is also a diffusible messenger that plays an important role in cell growth, differentiation and apoptosis. Our study showed that after irradiation the expression of iNOS in the U937 cells was increased so that the yield of MN in the bystander HUVECs increased. Since the bystander MN in the HUVECs was partly reduced by SMT and c-PTIO, NO could be an important contributor to the radiation-induced bystander effect. But besides NO, some other bystander signaling factors such as ROS, transforming growth factors-b1 (TGF-b1), tumor necrosis factor-a (TNF-a) may also be partially involved. However, which pathway does the bystander signaling factor observed in the HUVECs utilize? It has been reported that P38 in the MAPK family can be activated by cellular stresses including irradiation, heat shock, high osmotic stress, lipopolysaccharide, protein synthesis inhibitors, proinflammatory cytokines and certain mitogens (55). Exposure of cells to ionizing radiation and other toxic stresses induces simultaneous activation of multiple MAPK pathways (56, 57). SNP, a NO donor, could induce apoptosis in different types of cells by activating MAPKs (58, 59). Our study showed that P38 was activated in the bystander HUVECs after co-culturing with irradiated U937 cells and the activation of P38 increased with the cell culture time postirradiation, during this period, the concentration of NO in the supernatant of irradiated U937 cells was also gradually increased. Meanwhile, we also showed that the P38 inhibitor SB203580 decreased radiation-induced bystander effect. These results indicate that there appears to be a relationship between P38 activation and NO release. Interestingly, Hoving et al. found that NO-donating aspirin as a new drug could be used for the treatment of age-related atherosclerosis but not radiation-induced atherosclerosis in apoE null mice (60). A large amount of NO released from irradiated U937 cells may be involved in the radiation-induced atherosclerosis compared to age-related atherosclerosis. Accordingly, NO released from irradiated U937 cells could activate p38 MAPK pathway and further regulate the generation of radiation-induced bystander effect in HUVECs. P38 activation is thought to be involved in the apoptosis of endothelial cells (61, 62). The P38 inhibitor, SB203580, partly decreased the endothelial cell apoptosis, in our studies, and is a key event in the pathogenesis of arthrosclerosis. Because apoptosis has both pro-adhesive and pro-coagulant properties in endothelial cells, it is regarded as an important determinant in atherosclerosis progression (63). Apoptosis may coincide with the activation of stress-dependent protein

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kinases including p38 MAPK (64). When the endothelial cells were damaged, a cascade of inflammatory responses, could be induced, that result in the accumulation and migration of monocytes, differentiation and overproliferation of monocytes, and the release of MMPs. Nitrosative and oxidative stresses have been shown to initiate the upregulation of inflammatory adhesion molecules (65). It is known that an early step in the atherogenic process is the transmigration of blood monocytes. The monocytes can pass through the injured or dysfunctional endothelium into the extravascular space and then differentiate into macrophages (12). These processes are partly regulated by chemotactic factors, such as endothelial VCAM-1, which directly takes part in the development of atherosclerosis as an inflammatory factor (66). The induction of adhesion molecules in vascular endothelial cells and the subsequent recruitment of circulating monocytes are proinflammatory events that promote atherogenesis and plaque instability (67). We show here that the expression of VCAM-1 was increased in the bystander HUVECs and that the number of irradiated U937 cells adhering to HUVECs increased. This U937 cell adhesion was completely inhibited by SB203580, suggesting that the expression of VCAM-1 in the bystander HUVECs was regulated by the p38 pathway. In conclusion, our data demonstrated that the irradiated U937 cells appear to release a high concentration of NO by activating iNOS that triggers inflammatory responses in the bystander HUVECs through the p38 pathway. These findings indicate that NO can induce different effects in radiationinduced atherosclerosis and age-related atherosclerosis. Our data also suggests that NO, as a signaling molecule of radiation-induced bystander effect, may be involved in radiation-induced atherosclerosis. Accordingly, nontarget effects may enhance the incidence of radiation-induced atherosclerosis. ACKNOWLEDGMENTS We thank the National Nature Science Foundation of China (grant numbers 11179002, 81270001, 31070758), the MOST project (2012YQ030142) and the Doctoral Program of Higher Education (20120071110057) for funding this work. Received: March 12, 2014; accepted: May 8, 2014; published online: June 24, 2014

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