ERK signaling pathway and ROS generation

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PM2.5 induces vascular permeability increase through activating MAPK/ERK signaling pathway and ROS generation Yan-Min Longa, Xue-Zhi Yangb, Qing-Qing Yanga, Allen C. Clermontc, Yong-Guang Yina,b, Guang-Liang Liua,d, Li-Gang Hua,b, Qian Liua,b, Qun-Fang Zhoua,b, Qian S. Liub, Qian-Chi Mab, Yu-Chen Liua, Yong Caia,b,d,* a Hubei Key Laboratory of Environmental and Health Effects of Persistent Toxic Substances, Institute of Environment and Health, Jianghan University, Wuhan, Hubei 430056, China b State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China c Joslin Diabetes Center, Harvard Medical School, Boston, Massachusetts 02215, USA d Department of Chemistry and Biochemistry, Southeast Environmental Research Center, Florida International University, University Park, Miami, Florida 33199, USA

G R A P H I C A L A B S T R A C T

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Editor: Debora R

Although in-vivo exposure of PM2.5 has been suggested to initiate a disorder on vascular permeability, the effects and related mechanism has not been well defined. In this work, an obvious increase on vascular permeability has been confirmed in vivo by vein injection of PM2.5 into Balb/c mouse. Human umbilical vein vascular endothelial cells and the consisted ex-vivo vascular endothelium were used as model to investigate the effects of PM2.5 on the vascular permeability and the underlying molecular mechanism. Upon PM2.5 exposure, the vascular endothelial growth factor receptor 2 on cell membrane phosphorylates and activates the downstream mitogen-activated protein kinase (MAPK)/ERK signaling. The adherens junction protein VE-cadherin sheds and the intercellular junction opens, damaging the integrity of vascular endothelium via paracellular pathway. Besides, PM2.5 induces the intracellular reactive oxygen species (ROS) production and triggers the oxidative stress including activity decrease of superoxide dismutase, lactate dehydrogenase release and permeability increase of cell membrane. Taken together, the paracellular and transcellular permeability enhancement jointly contributes to the significant increase of endothelium permeability and thus vascular permeability upon PM2.5 exposure. This work

Keywords: Fine particulate matter (PM2.5) Vascular permeability Vascular endothelial growth factor receptor Mitogen-activated protein kinase signaling Oxidative stress

⁎ Corresponding author at: Department of Chemistry and Biochemistry, Southeast Environmental Research Center, Florida International University, University Park, Miami, Florida 33199, USA. E-mail address: cai@fiu.edu (Y. Cai).

https://doi.org/10.1016/j.jhazmat.2019.121659 Received 26 July 2019; Received in revised form 6 November 2019; Accepted 9 November 2019 0304-3894/ © 2019 Elsevier B.V. All rights reserved.

Please cite this article as: Yan-Min Long, et al., Journal of Hazardous Materials, https://doi.org/10.1016/j.jhazmat.2019.121659

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provides an insight into molecular mechanism of PM2.5 associated cardiovascular disease and offered a real-time screening method for the health risk of PM2.5.

1. Introduction

mortality.

Epidemiological studies have revealed that PM2.5 exposure is closely associated with cardiovascular mortality and morbidity (PenardMorand et al., 2005; Kunzli et al., 2010). It has been reported that the risk of PM2.5 related to fatal coronary heart disease was 1.42 with each 10 μg/m3 increase in women (Chen et al., 2005). A 1.28 % increase in the risk of heart failure was shown to be associated with a per- 10 μg/ m3 increase of PM2.5 (Dominici et al., 2006). The results of sampling surveys from the American Cancer Society showed that the mortality risk resulting from ischemic heart disease would increase from 0.24 to 0.60 when the PM2.5 concentration increased by 10 μg/m3 (Kunzli et al., 2010; Pope et al., 1995). The disorder of vascular permeability causes the penetration and accumulation of fat and cholesterol molecules on the vascular wall (Forastiere et al., 2005; Sima and Simionescu, 2009; Phinikaridou et al., 2012) which can significantly decrease blood flow and reduce the supply of the blood and oxygen, causing life-threatening cardiovascular diseases (CVDs) such as thrombosis and etc. In fact, a few in-vivo results demonstrate that chronic PM2.5 exposure induce an obvious increase in blood pressure and vascular inflammation (Nemmar et al., 2009, 2011; Sun et al., 2005), which implies that PM2.5 can cause changes in vascular permeability. However, the effects of PM2.5 on vascular permeability and its related mechanisms have not been clearly defined. Recently, a few studies focusing on this topic have reported that PM2.5 can stimulate endothelial cells to increase vascular permeability through the elevation of the transcriptional activity of HI-5α (Dai et al., 2016). PM2.5 can disrupt the respiratory chain in vivo to induce O2radicals inside endothelial cells. Induction of oxidative stress mediates cytokine secretion which leads to dysfunction of the vascular endothelium and affects vascular permeability (Mo et al., 2009; Tseng et al., 2016). In another report, oxidative stress also promotes cytoskeleton rearrangement of the endothelial cells, thereby causing barrier dysfunction of the endothelium layer (Wang et al., 2017). However, the question on how PM2.5 acts on endothelial cells and leads to a vascular permeability disorder remains unanswered. In this work, an in-vivo exposure model based on the Balb/c mouse has been established and the permeability of vessels in the lung was quantitatively analyzed with Evans blue infiltration. The endothelium consists of a monolayer endothelial cells which line the inner vascular wall and primarily regulate vascular permeability. Human umbilical vein vascular endothelial cell (HUVEC) is a commonly used primary pooled endothelial cell for studies because of their viability. HUVECs and the composed ex-vivo vascular endothelium were taken as the model to investigate the potential effect of PM2.5 on vascular permeability and the underlying molecular mechanisms. A transwell format was utilized to quantitatively assess the change in ex-vivo endothelial permeability upon PM2.5 exposure under diverse conditions. Junction protein expression and integrity of the consisted junctions were examined in detail to probe the mechanism of increased paracellular permeability through western blotting analysis and immunofluorescent imaging. In addition, oxidative stress induced by PM2.5 exposure of HUVECs was evaluated by examination of ROS generation, SOD activity and lactate dehydrogenase (LDH) release to explore changes in transcellular permeability. The results indicate that PM2.5 exposure can increase both paracellular and transcellular permeability via contact activation of vascular endothelial growth factor receptor (VEGFR-2) and induction of oxidative stress which leads to an increase in endothelial and vascular permeability. This work provides an insight into the molecular mechanism of PM2.5 causing cardiovascular morbidity and

2. Materials and methods 2.1. Reagents and instruments The endothelial cell medium (ECM), fetal bovine serum (FBS), endothelial cell growth factors and antibiotic reagents were purchased from ScienCell Research Laboratories (San Diego, USA). Bovine serum albumin (BSA), alamar blue reagent, FITC-dextran (70 kDa), 2′, 7′-dichlorodihydrofluorescein diacetate (DCFH-DA) and U0126 monoethanolate (≥98 %) were provided by Sigma-Aldrich (St. Louis, USA). ECL western blotting substrate and antibody for VEGFR 2, DC101 were obtained from ThermoScientific (Pierce, USA) and GeneTex Corporation (Neobioscence, Beijing, China), respectively. The primary antibodies used in western blotting were obtained from Cell Signaling Technology (CST, USA) and abcam (Shanghai, China). The protein quantification, SOD activity and LDH leakage were evaluated using Enhanced BCA Protein Assay Kit (Beyotime, Beijing, China), Superoxide Dismutase Activity Assay Kit (Colorimetric, abcam, UK), and LDH Cytotoxicity Assay Kit (Beyotime, Beijing, China) respectively. The reference solutions used for element analysis of PM2.5 were multi-metal solution (GSB04-1767-2004, Thermo, USA) and inorganic mercury solution (GBW(E)080124, National Institute of Metrology, Beijing, China). The instruments used in this work included medium-volume aerosol sampler (2031, Qingdao Laoshan Institute of Applied Technology, Qingdao, China), DRI Thermal/Optical Carbon Analyzer (Model 2001, Atmoslytic Inc., Calabasas, USA), inductively coupled plasma mass spectrometer (iCAP RQ ICP-MS, Thermo, USA), multi-mode plate reader (SYNERGY 2, Bio-Tek, Winooski, USA), Millicell ERS-2 voltohmmeter (Merck Millipore, USA), inverted fluorescence microscope (Vert.A1, Zeiss, GER), laser scanning confocal microscope (SP8, Leica, GER) and ChemiDoc Imaging System (BIO-RAD, USA). 2.2. PM2.5 collection and extraction PM2.5 sampling was conducted by cutting off the air PM of < 2.5 μm in size with medium-volume air sampler (LaoYing-2034, China) at a flow rate of 100 L/min. The sampler was in the Research Center for EcoEnvironmental Sciences, Chinese Academy of Sciences, Beijing, China. The sampling lasted for 24 h starting at 10:00 am each day during the 3rd to 7th, November in 2016. The particles trapped on the polypropylene filter (5.0 μm, φ200 mm, Qingdao Laoshan Institute of Applied Technology, Qingdao, China) were used for toxicity study, and those on quartz filters (5.0 μm, φ200 mm, Qingdao Laoshan Institute of Applied Technology, Qingdao, China) were used for organic carbon (OC)/elemental carbon (EC) analysis. The PM2.5-adsorbed polypropylene filter paper was cut into several pieces and soaked in 75 % ethanol for a few seconds to swelling the membrane. The soaked fragments were put into sterilized ultrapure water and the PM was transferred into water through sonication. The PM2.5 suspension was lyophilized and weighed for use. 2.3. Chemical analysis of PM2.5 The PM2.5-adsorbed polypropylene filter was digested by mixed acids of HNO3/HClO4 (4:1, v/v) at 100 °C for metals qualification with ICP-MS (Jin et al., 2019; Hu, 2015). An 0.5 cm2 of quartz filter was cut and analyzed for OC and EC 2

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Male Balb/c mice of 6–8 weeks used for the in-vivo assay were obtained from Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China). The average body weight of mouse was 19 g. The mice were randomly divided into three exposure groups consisting of control, low-dose and high-dose groups. Three or four mice were assigned to each group. The related experimental protocol was approved by the Animal Care and Use Committee of Research Centre for EcoEnvironmental Sciences (RCEES), Chinese Academy of Sciences and the proof/certificate of approval is available upon request.

transendothelial electrical resistance (TEER) was monitored as the indicator of endothelium formation since the day cells were seeded. When the cells differentiate and connect with cell-cell junctions, the flow of current across the cell layer will be restricted and the TEER value will increase. Until the rigid monolayer endothelium forms, the value stops increasing and stabilizes. The TEER value increased from 62.7 ± 1.6 Ω•cm2 at seeding, and leveled off at 112.9 ± 2.4 Ω•cm2, suggesting an intact endothelium is formed. Then, the culture medium was replaced with 100 μL and 600 μL of starving medium. The PM2.5, respective inhibitor and fluorescent probe of FITC-dextran (70 kDa, λex/λem 495 nm/525 nm) at 5 mg/mL were added into every insert. At the exposure time of 6, 12, and 24 h, 50 μL of media in bottom well was transferred onto a 96-well plate and mixed with another 50 μL of PBS. The fluorescent signal of FITC-dextran penetrated under different stimuli was determined for quantitative analysis of the endothelial permeability.

2.5. Evans blue infiltration assay

2.8. Western blot assay

Evans blue (EB) is a blue-colored dye showing a high affinity to the serum albumin. The extravasation of dye from the vessels into the cellular tissue is quantified to evaluate the vascular permeability (Xu et al., 2001; Long et al., 2015). The mice of control, low-dose and highdose groups were injected with PBS saline, PM2.5 suspension of 1.27 mg/kg and 6.34 mg/kg through tail vein, respectively. After 48-h circulation of PM2.5, Evans blue dye was infused at 50 mg/kg via tail vein and allowed to circulate for one hour. The mice were subsequently sacrificed by cervical dislocation. 5 mL of PBS and 5 mL of 10 % formalin were perfused at a physiological pressure to remove the residue blood in the whole vasculature. The middle lobe of right lung was collected and dried under vacuum. The dried tissues were weighed and extracted overnight with 300 μL of formamide at 72 °C. 100 μL of the supernatants containing infiltrate dye were loaded onto the 96-well plate and submitted to absorbance determination at 620 nm (Evans blue) and 740 nm (background) on the multi-mode plate reader. The concentration of Evans blue infiltrated was determined in terms of the adjusted absorbance (Ab620nm-Ab740nm) of each sample in reference to that of the dye standards. The leakage is expressed as the concentration of infiltrate Evans blue normalized by the corresponding tissue weight.

The cells were seeded in 6-well plate and endured PM2.5 exposure for appropriate durations after cells grew 70–80% confluence according to the section 2.6. At the end of exposure, the cells were washed twice with ice-cold DPBS containing Ca2+/Mg2+ (Gibco, USA) and scratched from the plate after adding lysis buffer containing 1 % cocktail, 1 mmol/L Na3VO4, 1 mmol/L NaF, and 0.6 mmol/L H2O2 (Long et al., 2015; Orsenigo et al., 2012). The cell was lyzed on ice for 1 h and the protein supernatant was collected after centrifuge at 13,000 rpm for 15 min. The total protein was quantified with the enhanced BCA protein assay kit. Further mixing with 4× Laemmli buffer containing 4 % βmecaptoethanol, the equal protein sample was loaded on the SDS-PAGE gel (4–20 % gradient, Bio-Rad, USA) and fully separated under electrophoresis. Then, the separated proteins on gel were transferred to a cellulose acetate membrane under constant current of 270 mA for 1 h. The membrane with protein was blocked with 5 % skimmed milk (Invitrogen, USA) at room temperature for 1 h, and then immunoblotted overnight at 4 °C with respective first antibody of phosphorylated VEGFR 2 (1:1000, CST, USA), phosphorylated MEK1/2 (1:1000, CST, USA), phosphorylated ERK1/2, ERK1/2 (1:1000, CST, USA), VE-cadherin (1:150, abcam, UK) and β-actin (1:10000, Hangzhou HuaAn Biotechnology Co. Ltd., China). After incubation with the peroxidaseconjugated secondary antibodies, the target proteins on the membrane were developed with ECL reagents and quantified with ChemiDoc Imaging System.

content using the thermal/optical reflectance (TOR) method with a DRI Thermal/Optical Carbon Analyzer according to the Interagency Monitoring of Protected Visual Environments (IMPROVE) protocol (Wu et al., 2016). 2.4. Animals and in-vivo exposure

2.6. Cell culture and exposure HUVECs were seeded and cultured in a 10-cm dish pre-coated with 0.1 % gelatin (Gibco, USA) at 37 °C under 5 % CO2. The culture medium for cell growth contained ECM solution supplemented with 5 % FBS, 1 % antibiotics and 1 % endothelial cell growth factors. Each cell study was conducted with three independent experiments. The in-vitro exposure were conducted in the starving medium of ECM containing 1 % BSA and four levelled PM2.5 of 0, 2, 20, and 100 μg/mL for control, low, medium and high dose groups, respectively. 100 μg/mL of DC101 or 10 μmol/L of U0126 monoethanolate was pre-treated with HUVECs for 1 h before PM2.5 exposure to examine the effects of VEGFR 2 and MAPK/ERK signaling activation on the VEcadherin shedding and resultant endothelium permeability. N-acetyl-Lcysteine (NAC) of 5 mmol/L was introduced to verify the effects of oxidative stress on endothelium permeability. At the end of each exposure, the corresponding assay was performed in detail as follows.

2.9. Immunostaining of VE-cadherin on HUVECs The HUVECs were seeded and cultured in 0.1 % gelatin pre-coated confocal dishes (35 mm, NEST, Wuxi, China). When getting 70–80% confluence, the cells were stimulated with PM2.5 suspension according to the protocol in section 2.6. At the end of the exposure, the cells were washed with DPBS containing Ca2+/Mg2+ and fixed with ice-cold methanol (Sinopharm, Shanghai, China) for 5 min. The fixed samples were blocked with DPBS containing 5 % BSA at room temperature for 30 min after triple rinse. Then, the cells were incubated with human VEcadherein antibody at the dilution ratio of 1:150 in DPBS containing 2 % BSA overnight at 4 °C. After triple rinse, the cells were stained with dylight680 tagged anti-rabbit antibody at the dilution ratio of 1:200 (Abbkine, Redlands, USA) in DPBS with 2 % BSA at room temperature. The cell nuclei was stained with the probe of Hoechst33342 (Beyotime, Beijing, China) for 5 min at 37 °C. The incubation involving fluorescent dye should avoid light. Finally, the samples were imaged under confocal fluorescence microscope for visualizing the VE-cadherin distribution on cell border. The cellular junction integrity upon diverse stimuli was quantitatively assessed by scoring the continuity of VE-cadherin according to the following rules (Murakami et al., 2009): grade 1 for 0–25%

2.7. Permeability determination of the endothelium consisting of monolayer HUVECs HUVECs were seeded at the density of 50,000 cells/cm2 and cultured on the polyester insert of a 24-well transwell format (0.4-μm, Corning, USA) which was pre-coated with 0.1 % gelatin. 200 μL and 1 mL of culture medium were added into the upper insert and bottom well, respectively, for cell growth and changed every two days. The 3

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continuous staining on the cell border; grade 2 for 25–50% continuous staining; grade 3 for 50–75% continuous staining; and grade 4 for 75–100% continuous staining. Thirty cells in each exposure group were randomly selected for the scoring statistics of junction integrity.

Ab is absorbance. The sample SOD activity was expressed as the rate normalized by the corresponding protein concentration. 2.10.3. Lactate dehydrogenase (LDH) release HUVECs in 96-well plates were exposed to PM2.5 with appropriate concentrations for 6, 12 and 24 h respectively. At the end of exposure, the LDH penetrating across the cell membrane was measured using the assay kit according to users’ manual. Briefly, 120 μL of exposure supernatant was transferred to another 96-well plate together with 60 μL of LDH working solution, and incubated in dark for 1 h at room temperature. The absorbance at both 490 nm (sample response) and 650 nm (reference) were measured. The adjusted absorbance (Ab490nmAb650nm) of sample versus that of the control is used to plot the graph.

2.10. The determination of cellular oxidative stress When the cells were seeded and grew until 60–70% confluence, the exposure was conducted according to the protocol described in section 2.6. The cellular oxidative stress upon PM2.5 exposure was evaluated using three assays of intracellular ROS generation, SOD activity and lactate dehydrogenase (LDH) release described in detail as follows. 2.10.1. Intracellular ROS generation The cells were seeded in the 96-well plate at the density of 10,000cells/well and endured exposure of PM2.5 until reaching 60–70% confluence. At the end of 6, 12 and 24-h exposure, the cells were washed with PBS and incubated with the 10 μmol/L of DCFH-DA probe in dark at 37 °C for another 30 min. After discarding the unreacted probes, the fluorescent intensity of oxidized product was determined at 525 nm (λex = 484 nm) after twice wash with PBS. The concentration of the oxidized products was in proportion to the ROS generated. Therefore, the intracellular ROS level was quantified with the concentration of the oxidized products (Yin et al., 2013).

2.11. Statistical analysis All the results within this work were obtained based on at least three independent experiments and the values were given by the mean value ± standard deviation. The statistical analysis was conducted using one-way analysis of variance (ANOVA) with the software of Origin 8.5 and the significant difference was given based on the value of P less than 0.05 or 0.01. 3. Results and discussion

2.10.2. SOD activity The cells were seeded in the 6-cm culture dish and employed to the exposure of PM2.5 at four levels of 0, 2, 20 and 100 μg/mL. As for the inhibition, the cells were pre-incubated with 5 mmol/L of NAC for 1 h before PM2.5 exposure. After 24-h incubation of PM2.5, the cells were lyzed on ice for 1 h with the lysis buffer containing 0.1 M pH 7.4 Tris/ HCl, containing 0.5 % Triton X-100, 5 mM β-mecaptoethanol, 0.1 mg/ mL PMSF. The lysate protein were collected via centrifugation at 4 °C and quantified by BCA protein assay. Then, the SOD activity for each sample was measured according to the manufacture’s protocol of the assay kit. In brief, the protein solution of each sample was mixed with an appropriate amount of WST working solution, enzyme working solution and dilution buffer, and the absorbance at 450 nm was determined with plate reader after incubation at 37 °C for 30 min. The SOD activity rate was calculated according to the following equation:

SOD activity rate =

3.1. Chemical characterizations of sampled PM2.5 PM2.5 particles with extremely small-sized core of carbon or silicon can strongly adsorb and concentrate organic (e.g. polycyclic aromatic hydrocarbons (PAHs) and volatile organic compounds (VOC)) and inorganic contaminants (e.g. lead (Pb), cadmium (Cd) and arsenic (As)). It has been generally recognized that the absorbates on the particles largely dictate the toxicity of PM2.5. Epidemiological and in-vivo studies have revealed that adsorbed organic pollutants like PAHs are closely associated with the development or exacerbation of CVDs by triggering endothelium inflammation (Zheng et al., 2019; Holme et al., 2019). Metal and metalloid components like Pb, Cd, As and etc., could bind with protein or induce ROS to reduce NO release and initiate oxidative stress, thus causing the endothelium dysfunction (Zheng et al., 2019; Bao and Shi, 2010; Nagarajan et al., 2013; Kesavan et al., 2014). In this work, the OC/EC was determined to roughly estimate the organic compounds absorbed on the PM. The OC/EC ratio of sampled PM2.5 was

(Absample − Abenzyme blank) (Absample blank − Abenzyme blank+sample blank)

Fig. 1. The Evans blue leakage into lung tissue of Balb/c mice (A) following tail vein injection of PBS for control group, PM2.5 of 6.34 mg/kg for high-dose group and PM2.5 of 1.27 mg/kg for low-dose group, respectively. **P < 0.01 compared to the control by ANOVA. Inset: example image of one lung lobe from the exposed mice after infiltration of Evans blue probe. Con: control, LD: low dose, and HD: high dose. Dose and time course for the penetration of FITC-dextran (70 kDa) fluorescent probe through a monolayer of HUVECs (B). **P < 0.01, **aP < 0.01 and **bP < 0.01 by ANOVA compared to the group of 6-h, 12-h and 24-h incubation with PBS only. 4

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measured to be 5.1. This value is comparable to the average level of ∼5 during haze days in Beijing, but relatively higher than ∼3 during nonhaze days in the same district (Hu, 2015; Zhang et al., 2015). This could be caused by the increased OC from secondary organic carbon and coal combustion for heat supply in the winter season. The metal concentration was also determined and the results are summarized in Table S1. The mass concentrations of the carcinogenic metals and metalloid like As, Cd, Pb and Hg were in the range of 2.8∼5077.7 μg/g, which are comparable to the previous reports for the same city (Jin et al., 2019). The above characterization results suggest sampled PM2.5 potentially pose an adverse impact on endothelial function and therefore the vascular permeability.

demonstrated that PM2.5 damaged the integrity of vessels in lung and caused the EB infiltration and accumulation in the nearby tissue. It confirms that the PM2.5 exposure through systematic instillation can elevate the vascular permeability in vivo. The vascular endothelium consisting of monolayer endothelial cells acts as a natural barrier that tightly maintains the dynamic balance of bilateral transportation between intravascular blood and the interstitium (Dejana, 2004; Dejana et al., 2009). The integrity of the endothelial layer primarily controls the permeability of the blood vessels. To explore the effects of PM2.5 exposure on endothelium integrity, an ex-vivo endothelial model was constructed by growing a compact monolayer of HUVECs on the membrane of a 24-well transwell insert. The penetration flux through the endothelium upon different stimulations was measured by the probe of FITC-dextran (70 kDa) across the insert into the bottom well. Since the blood volume for a 19 g mouse is approximately 1.2 mL, the equivalent concentrations of PM2.5 from the in vivo studies of 1.27 and 6.34 mg/kg were calculated as 20 and 100 μg/mL, respectively. Thus, the middle and high concentrations used in the ex vivo and subsequent in-vitro assays were set as 20 and 100 μg/mL. A PM2.5 concentration of 2 μg/mL was used for the low concentration for the ex-vivo and in-vitro assays. As shown in Fig. 1B, the flux of infiltrated probe increased for PM2.5 concentrations of 2, 20 and 100 μg/mL from 1.3 to 2.5, 1.5 to 2.8, and 2 to 3 times (P < 0.01) of the control, respectively, at 6 h compared to 24 h. This result demonstrates that PM2.5 exposure induces an increase in endothelium permeability in a time- and concentration-dependent manner. Furthermore, this also suggests that PM2.5 exposure affects vascular function in the lung in vivo via damaging the endothelium integrity.

3.2. PM2.5 exposure elevates the vascular permeability in vivo by impairing the endothelium monolayer Previous reports have demonstrated that in-vivo exposure to PM2.5 can initiate vascular inflammation of the lung (Nemmar et al., 2009, 2011; Sun et al., 2005) by impairing vascular integrity resulting in increased permeability. Therefore, the permeability of vessels in the lung was examined in response to in vivo exposure to PM2.5 over 2 days in the mouse. PM2.5 can penetrate across the air-blood barrier into the blood circulation and damage the cardiovascular system. Thus, the instillation of PM2.5 through tail vein was utilized as the in-vivo exposure route herein to investigate the effects of PM2.5 on the vascular permeability of lung in mouse. It has been reported that the PM2.5 concentration in the severe haze weather of Shenyang in China reached maximum value of 1.4 mg/m3 (Department of Ecology and Environment of Liaoning Province, 2019), based on which the PM2.5 concentration for high-dose exposure was calculated. Since the respiratory tidal volume for an adult mouse is approximately 0.150.18 mL/breath, the total respiratory volume over 2 days would be 84 L. The maximum amount of inhaled PM2.5 by a mouse over 2 days is approximately 0.12 mg. Thus, the equivalent single instillation for the high-dose group is estimated to be 6.34 mg/kg based on the average body weight of 19 g for this experiment. Considering the range of doses used in related studies (Aztatzi-Aguilar et al., 2016; Liu et al., 2018, 2015; Zhao et al., 2017), one fifth of the maximum dose, 1.27 mg/kg, was injected as the exposure concentration for the low-dose group. As shown in Fig. 1A, the blue color staining in lung lobe was darker with increasing PM2.5 concentration. The quantitative data demonstrated that the EB leakage of mice in low-dose and high-dose group was 2.42 and 4.32 folds of control group, respectively. This result

3.3. PM2.5 induces adherens junction shedding and the intercellular junction open In order to understand the underlying mechanism for the endothelial permeability increase induced by PM2.5, the viability of HUVECs after particle exposure has been examined. The results shown in Figure S1 demonstrated that the PM2.5 did not cause obvious cellular toxicity at three stimulation levels. The morphology of HUVEC monolayer prior to and post exposure to PM2.5 was inspected with an inverted microscope under bright field. As shown in Fig. 2A–D, aggregation of the particles was observed up to several microns in size with an increasing number of aggregates (marked with red arrows) with increasing concentration of PM2.5 exposure. HUVECs contracted and separated from each other in response to increasing concentrations of

Fig. 2. Microscopic images of HUVECs under bright field (A–D) and the expression of the adherens junction protein, VE-cadherin, on the HUVECs membrane (E) after PM2.5 exposure for 24 h at the concentrations of 0, 2, 20 and 100 μg/mL. **P < 0.01 compared to that in absence of PM2.5 by ANOVA. The red arrows in the images indicate aggregation of particles on the cell membrane. Yellow arrows mark intercellular gaps. 5

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PM2.5 from 2 to 100 μg/mL compared to PBS alone. Moreover, the Zscan confocal images under bright field (Figure S2) showed that the particles were internalized into cytoplasm of cells after 24-h exposure of 100 μg/mL PM2.5, at the same time the cellular substructure and membrane has been damaged to some extent. The integrity of the cellular junctions of HUVECs in the monolayer was compromised as seen by the formation of intercellular gaps (marked with yellow arrows) (Fig. 2B–D). The intercellular gaps appeared to expand in response to increases in PM2.5 concentration from 2 to 100 μg/mL when compared to control cells (Fig. 2A). Previous work has reported that the adjacent endothelial cells closely connect to each other via junctional proteins to form a tight monolayer of endothelium that regulates dynamic vascular permeability (Ohsugi et al., 1997; Taddei et al., 2008). As shown in Fig. 2A–D, the intercellular junctions between endothelial cells upon stimulation by PM2.5 become disrupted which leads to the formation of intercellular gaps. These gaps increase the paracellular permeability and may contribute to the enhanced permeability observed in the endothelium monolayers. It is known that tight junctions (TJs) and adherens junctions (AJs) are vital components of cell-cell junctions and mainly control the rigidity of the endothelium layer. More specifically, the AJ promotes the homophilic binding between the vascular endothelial cells, and plays a significant role in organizing the TJ structure (Ohsugi et al., 1997; Taddei et al., 2008). Thus, the transmembrane protein, VE-cadherin, which is expressed on the cell membrane of adherens junction, was assessed by examining the intercellular junctions at the molecular level. The results in Fig. 2E showed a significant dissociation of VE-cadherin by decreasing expression to 96.6 %, 60.3 %, and 40.6 % of the control with increasing exposure to PM2.5 at 2 and 20 and 100 μg/mL, respectively. This result indicates loss of VE-cadherin after exposure to PM2.5 which would destabilize endothelial junctions and support the

microscopic imaging results observed in Fig. 2B–D which show gap formation between cells. 3.4. PM2.5 activates VEGFR 2 and MAPK/ERK signaling to induce VEcadherin shedding Vascular endothelial growth factor receptors (VEGFR) are tyrosine kinase receptors located at the endothelial cell membrane that mediates cellular functions such as survival, vasculogenesis and permeability. VEGFR consists of an extracellular domain, a transmembrane domain, and a kinase domain in the cytoplasm of endothelial cells (Seetharam et al., 1995; Waltenberger et al., 1994; Esser et al., 1998). Activation of VEGFR has been shown to initiate several pathways that regulate vascular permeability (Seetharam et al., 1995; Cunningham et al., 1995; Guo et al., 1995). Specifically, the VEGFR subtype 2 can be activated through physical contact with stimuli or binding with pro-angiogenesis factors such as VEGF isoforms (Waltenberger et al., 1994; D’Angelo et al., 1995). Activation of VEGFR 2 via contact autophosphorylation upon attachment to the cell membrane of HUVECs by PM2.5 (Fig. 2 and S2) may contribute to further regulation of endothelium permeability. Autoactivation of VEGFR 2 by PM2.5 exposure was examined by blotting phosphorylated VEGFR 2 (p-VEGFR) on HUVECs after 30 min exposure to PM2.5 at concentrations of 0, 2, 20, and 100 μg/mL. As shown in Fig. 3A, the band for p-VEGFR has been detected, which was blotted with the antibody of phosphor-VEGF receptor 2 (Tyr996) specific for the autophosphorylation site in the kinase insert domain (Tyr996) (Doughervermazen et al., 1994; Meyer et al., 1999). The intensity of the p-VEGFR 2 band increased with increasing concentration of PM2.5 (Fig. 3A). The expression level upon exposure of PM2.5 at 2, 20 and 100 μg/mL, was 1.5, 1.6 and 2.4 times that of the control, respectively (Fig. 3B) while the autophosphorylation of VEGFR 2 was totally

Fig. 3. Western blotting of phosphorylated VEGFR 2 (A), phosphorylated MEK1/2, phosphorylated ERK, ERK (C), and VE-cadherin (E) expression on HUVECs and the respective statistical results (B, D and F) under diverse stimuli as indicated. *P < 0.05 and **P < 0.01 compared to that in absence of PM2.5 by ANOVA. 6

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from the cell membrane through activation of MAPK/ERK signaling mediated by VEGFR 2 phosphorylation. The effects of PM2.5 exposure on the adherens junction protein VEcadherin and intercellular junctions of HUVECs were visualized by immunostaining of VE-cadherin with antibody tagged with red-colored fluorescent dylight800. The cell nuclei were stained with DNA dye Hoechst33342 as indicated by blue. The confocal images in Fig. 4A showed that VE-cadherin was located continuously along the cell membrane and within the cellular boundary of the control group. As the concentration of PM2.5 increased, a loss of VE-cadherin was observed through obvious breaks and hollow sites as marked by yellow arrows in Fig. 4C and D. Upon addition of inhibitor U0126, the junction was restored to some extent as indicated by disappearance of hollow sites and the even distribution of junction on the cell borders (Fig. 4E). Grading of the junction integrity by assessment of VE-cadherin continuity (Murakami et al., 2009) showed a restoration with co-treatment with U0126 inhibitor with PM2.5 at 100ug/mL towards control. The average integrity score of HUVEC monolayer under PM2.5 exposure at 0, 2, 20 and 100 μg/mL were 3.5 ± 0.6, 3.2 ± 1.0, 2.7 ± 1.2 and 2.3 ± 1.1, respectively (Fig. 4F). The grade of U0126 pretreatment group was similar to control at 3.2 ± 1.0, confirming the protective effects of U0126 on VE-cadherin shedding and further junctional damage. These results, together with the western blots in Fig. 3, indicate that VEGFR 2 was phosphorylated upon PM2.5 physical contact which subsequently activated the MAPK/ERK cascade through successive MEK and ERK phosphorylation. ERK phosphorylation induced shedding of the downstream adherens junction protein VE-cadherin, resulted in the opening of intercellular junctions and the increase of endothelium permeability through paracellular leakage.

inhibited in the presence of DC101, a specific antibody of VEGFR 2, at all three exposure levels of PM2.5. These results confirm that the PM2.5 contact induces the activation of VEGFR 2 on the HUVECs membrane. The activation of VEGFR 2 recruits cytoplasmic proteins and stimulates associated transduction pathways which include the mitogenactivated protein kinase cascade (MAPK)/ERK signaling and phosphoinositide-3 kinase/Akt cascade (Sarkar et al., 2014; Wu et al., 2000). In particularly, ERK protein can directly interact with the Cterminal region of junction proteins and regulate their phosphorylation and shedding to maintain the integrity of endothelial barrier (Anderson and Van Itallie, 1995; Basuroy et al., 2006). Thus, MAPK/ERK signaling activation and ERK phosphorylation was speculated to be involved in the regulation of cell junction integrity and the increase in permeability observed in monolayer HUVECs upon PM2.5 exposure. To address this question, the expression of phosphorylated MEK (p-MEK) and phosphorylated ERK (p-ERK) on HUVECs membrane, two key molecules of the MAPK/ERK signaling, were assayed by western blotting. As shown in Fig. 3C and D, the increased phosphorylation of MEK and ERK occurs after HUVEC exposure to PM2.5 at 2, 20, and 100 μg/mL. In order to investigate the association between the activation of VEGFR 2 and MAPK/ERK signaling with the shedding of the adherens junction protein VE-cadherin and junction opening, the antibody DC101 and inhibitor U0126 monoethanolate (specifically blocking MEK1/2 phosphorylation, labeled as U0126) were introduced into PM2.5 exposure. Cells pretreated with U0126 expressed low levels of pMEK and p-ERK protein, which were similar to the untreated control group. VE-cadherin levels on HUVECs membrane pretreated with DC101 recovered to 81.0 %, 73.3 % and 71.0 % of the controls at concentrations of 2, 20, and 100 μg/mL of PM2.5 exposure, and the U0126 pretreatment recovered the level to 80.0 %, 79.3 % and 83.4 % of the control, respectively (Fig. 3E and F). The lack of recovery at the low concentration of PM exposure may be due to a low level of stimulation or the inhibitory capacity of antagonists at this concentration. Thus, the PM2.5 induces the shedding of junction protein VE-cadherin

3.5. PM2.5 elevates the permeability of cell membrane through oxidative stress As discussed in Section 3.1, the sampled PM2.5 contained several

Fig. 4. The confocal images (A–E) and statistical analysis (F) of the paracellular junction of the HUVECs monolayer by immunostaining the adherens junction protein of VE-cadherin with dylight800 labeled antibody under different exposure conditions as indicated. The cell nuclei were stained with blue-colored dye of Hoechst33342. Yellow arrows mark the intercellular gap between the HUVECs. **P < 0.01 compared to that in absence of PM2.5 by ANOVA. 7

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protein VE-cadherin shedding which opens junctions open and increases paracellular permeability of the endothelium. In addition, PM2.5 exposure induces intracellular ROS production and oxidative stress affecting membrane permeability which increases endothelium permeability through the transcellular pathway. The elevated endothelial permeability upon PM2.5 exposure leads to the resultant vascular permeability, which provides an insight into the molecular and pathological mechanism on the relevance of CVDs and PM2.5 exposure.

kinds of metal and metalloid like Cd, Pb, Hg and As, which indicates that PM2.5 exposure may induce oxidative stress through the generation of exogenous and endogenous reactive oxygen species (ROS) in cells. In particular, the electron transfer to oxygen molecule from the persistent free radical evolving from the interaction between metal and PM, can produce exogenous ROS (Vejerano et al., 2018; Khachatryan et al., 2011; Fang et al., 2014, 2015). Moreover, particles inside the cells and the metal/metalloid released are capable of inducing endogenous ROS by binding with sulfur-containing proteins or other perturbed pathways (Zheng et al., 2019; Bao and Shi, 2010; Nagarajan et al., 2013; Kesavan et al., 2014). The abnormal cellular morphology induced by PM2.5 exposure in Fig. 2B–D and Figure S2 seemed to support this speculation. After exposure to PM2.5, HUVECs produced intracellular ROS following a concentration- and time-dependent mode (Fig. 5A). It may involve the impairment of redox enzyme system consisting of redox-active enzymes, such as superoxide dismutase (SOD) and catalase. In severe cases, ROS leads to lipid peroxidation and breakdown of the plasmic membrane structure, thus elevating transcellular permeability. Herein, the effects of PM2.5 exposure on SOD activity and LDH leakage have been determined as the index for evaluating oxidative stress. The results shown in Fig. 5B and C suggest that as the concentration of particles increased from 0 to 100 μg/mL, SOD activity declined and LDH leakage significantly increased, which is associated with the occurrence of oxidative stress into HUVECs. Notably, PM2.5 exposure may induce an increase in permeability of the endothelium through transcellular pathway besides paracellular mechanism.

4. Conclusions This work demonstrated that PM2.5 exposure increased vascular permeability, through a molecular mechanism involving two pathways a paracellular permeability increase initiated by VEGFR 2 activation and a transcellular permeability increase mediated through oxidative stress. In the paracelluar pathway, PM2.5 activated the transmembrane VEGFR 2 via physical activation, stimulated the downstream signaling of MAPK/ERK including MEK and ERK phosphorylation, and then shed the junction protein of VE-cadherin, causing opening of junctions and paracellular permeability elevation. For the other pathway, the PM2.5 treatment of HUVECs produced excessive ROS and triggered the oxidative stress to attack the plasmic membrane, leading to the significant membrane damage. Therefore, the integrity of the endothelium consisting of monolayer HUVECs is damaged and cell permeability increases, which causes an increase in the vascular permeability. The results from this work focus on the effects of PM2.5 upon vascular permeability may provide some insight into the mechanisms of cardiovascular diseases associated with PM2.5 contamination. Moreover, the transwell format and the related paracellular flux analysis would offer effective and fast means for real-time assessing the risk of potential cardiovascular diseases toxicity rising from the PM2.5 exposure. Although, the defined relationship between the chemical composition of PM2.5 and vascular toxicity has only been broadly examined in this work, and future work will elucidate the specific contaminants that lead to inflammation, cellular oxidative stress and vascular permeability.

3.6. The antagonists of U0126 and NAC recover the permeability of monolayer HUVECs To further confirm the contribution of both paracellular (MAPK/ ERK) and transcellular (ROS) pathways to the increase in endothelium permeability, the flux of FITC-dextran (70 kDa) probe penetrating across the HUVECs monolayer was measured upon exposure to PM2.5 at 0, 2, 20 and 100 μg/mL following pretreatment with MAPK/ERK inhibitor U0126 at 10 μmol/L or the antioxidant, N-acetyl-L-cysteine (NAC) at 5 mmol/L. Pretreatment with U0126 significantly decreased the leakage of FITC-dextran (Fig. 6A) to the values of 0.52, 0.44 and 0.91 folds as compared to exposure of PM2.5 alone at 2, 20, and 100 μg/ mL for 24 h (Fig. 1B). In addition, the pretreatment of NAC at 5 mmol/L ameliorated the increase in permeability (Fig. 6B), in which the probe leakage dropped and the value was 0.55, 0.82 and 0.89 folds of that for the HUVECs monolayer under exposure of PM2.5 alone at concentrations of 2, 20 and 100 μg/mL for 24 h duration (Fig. 1B). The antagonistic effects of the inhibitors on FITC-dextran leakage confirmed the permeability increase of the endothelium observed after PM2.5 exposure occurs through both paracellular and transcellular pathways. The overall mechanisms were sketched as the Scheme 1. The physical contact of PM2.5 with cells activated the VEGFR 2 on the membrane of HUVECs cells, and stimulated the MAPK/ERK signaling through successive MEK and ERK phosphorylation, causing the adherens junction

Declaration of Competing Interest The authors declare no competing interests. Acknowledgements This work was jointly supported by National Natural Science Foundation of China (91543103, 91743203, 21407063 and 21677063) and State Key Laboratory of Environmental Chemistry and Ecotoxicology, RCEES, CAS (KF2015-08). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.jhazmat.2019.121659.

Fig. 5. Intracellular ROS production (A), SOD activity (B) and LDH leakage (C) of HUVECs upon the exposure of PM2.5 at four levels of 0, 2, 20 and 100 μg/mL for different durations as indicated. *P < 0.05 compared to that in absence of PM2.5 by ANOVA. 8

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Fig. 6. The flux of FITC-dextran (70 kDa) penetrating across the monolayer HUVECs upon the exposure of PM2.5 at 0, 2, 20 and 100 μg/mL in presence of 10 μmol/L U0126 (A) and 5 m mol/L NAC (B) as indicated.

Scheme 1. The proposed mechanism of PM2.5 inducing vascular permeability increase through paracellular leakage of endothelium by successively activating VEGFR and MAPK/ERK signaling together with transcellular leakage of endothelium by lipid peroxidation induced by the oxidative stress.

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