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Arsenite Induces Endothelial Cell Permeability Increase through a Reactive Oxygen Species-Vascular Endothelial Growth Factor Pathway Lingzhi Bao and Honglian Shi* Department of Pharmacology and Toxicology, School of Pharmacy, UniVersity of Kansas, Malott Hall 5044, 1251 Wescoe Hall DriVe, Lawrence, Kansas 66045, United States ReceiVed June 1, 2010

As a potent environmental oxidative stressor, arsenic exposure has been reported to exacerbate cardiovascular diseases and increase vascular endothelial cell monolayer permeability. However, the underlying mechanism of this effect is not well understood. In this paper, we test our hypothesis that reactive oxygen species (ROS)-induced vascular endothelial growth factor (VEGF) expression may play an important role in an arsenic-caused increase of endothelial cell monolayer permeability. The mouse brain vascular endothelial cell bEnd3 monolayer was exposed to arsenite for 1, 3, and 6 days. The monolayer permeability, VEGF protein release, and ROS generation were determined. In addition, VE-cadherin and zonula occludens-1 (ZO-1), two membrane structure proteins, were immunostained to elucidate the effects of arsenite on the cell-cell junction. The roles of ROS and VEGF in arsenite-induced permeability was determined by inhibiting ROS with antioxidants and immuno-depleting VEGF with a VEGF antibody. We observed that arsenite increased bEnd3 monolayer permeability, elevated the production of cellular ROS, and increased VEGF release. VEcadherin and ZO-1 disruptions were also found in cells treated with arsenite. Furthermore, both antioxidant (N-acetyl cysteine and tempol) and the VEGF antibody treatments significantly lowered the arsenite-induced permeability of the bEnd3 monolayer as well as VEGF expression. VE-cadherin and ZO-1 disruptions were also diminished by N-acetyl cysteine and the VEGF antibody. Our data suggest that the increase in VEGF expression caused by ROS may play an important role in the arsenite-induced increase in endothelial cell permeability. Introduction Human beings are widely exposed to arsenic through environmental and occupational sources (1). Drinking water contaminated by arsenic remains a major public health problem. Individuals exposed to arsenic are prone to develop skin, bladder, liver, and lung cancers (2), black foot disease, and type II diabetes mellitus (3-5). Epidemiological studies have demonstrated that chronic arsenic exposure is linked to elevated risks of cardiovascular diseases including atherosclerosis (6-8), ischemic heart diseases, and hypertension (9). Although the mechanisms of arsenic-induced cancer have been widely studied, comparatively little attention has been paid to arsenic-induced vascular diseases. An increase of vascular permeability (leakage of solutes from the vascular confines) has been implicated in the pathophysiology of many diseases such as acute lung injuries, ischemia reperfusion injuries, atherosclerosis, and diabetes (10). Recently, arsenic has been reported to impair the endothelial permeability both in vitro and in vivo (11, 12). However, the mechanism of arsenic-induced vascular permeability increase has not been well clarified. Arsenic is a known oxidative stressor (13). Experimental results have shown that reactive oxygen species (ROS)1 are produced in different kinds of cells exposed to arsenic at various concentrations. For example, arsenite induces detectable levels of superoxide anion radical (O2•-) in U937 cells at a concentra* To whom correspondence should be addressed. Tel: 785-864-6192. Fax: 785-864-5219. E-mail: [email protected].

tion of 1-10 µM (14), human vascular smooth muscle cells at 7-16 µM (15), and human-hamster hybrid cells at 50 µM (16). At environmentally relevant concentrations or at nonlethal concentrations (not greater than 5 µM), arsenic has been shown to stimulate O2•- and H2O2 formation in vascular endothelial cells (17). Vascular endothelial growth factor (VEGF) is first described as a vascular permeability factor due to its ability to stimulate a rapid reversible increase in microvascular permeability without damaging the endothelial cell (18). Furthermore, VEGF can lead to endothelial cell leakage by disrupting gap junction, adherens junctions (AJ), and tight junctions (TJ) (19). ROS has been implicated in triggering expression of VEGF in many cell types including microvascular endothelial cells (20-24). All of these observations suggest that ROS may be involved in arsenic-induced permeability increases, possibly through regulating VEGF expression. However, there is no evidence demonstrating a direct link between the permeability increases and the ROS-mediated VEGF increase in endothelial cells exposed to arsenic. We hypothesize that arsenic-induced 1 Abbreviations: CM-H2DCFHDA, 2′,7′-dichlorodihydrofluoresceindiacetate; ATF4, activating transcription factor 4; AJ, adherens junctions; AMPK, AMP-activated protein kinase; AFU, arbitrary fluorescence unit; BBB, blood-brain barrier; DPI, diphenyleneiodonium chloride; DMEM, Dulbecco’s modified Eagle’s medium; DMSO, dimethyl sulfoxide; FBS, fetal bovine serum; FITC, fluorescein isothicyanate; GSH, glutathione; HO1, heme oxygenase 1; LDH, lactate dehydrogenase; NAC, N-acetyl-cysteine; ROS, reactive oxygen species; RT, room temperature; NaAsO2, sodium arsenite; TJ, tight junctions; VEGF, vascular endothelial growth factor; ZO1, zonula occludens-1.

10.1021/tx100191t  2010 American Chemical Society Published on Web 10/18/2010

Arsenic Induces Permeability through a ROS-VEGF Pathway

increases of permeability may result from up-regulation of VEGF by ROS. To test this hypothesis, we studied the effects of sodium arsenite (NaAsO2) on the permeability, ROS generation, VEGF release, and cell-cell junction structure, using a brain microvascular endothelial cell line (bEnd3). The selection of this cell line was based on a previous publication that has well characterized bEnd3 as an in vitro blood-brain barrier (BBB) model (25). Several of the most recent publications have also characterized the bEnd3 endothelial monolayer as an appropriate model of in vitro BBB (26, 27). In addition, the cell line has been used to investigate the effect of matrix metalloproteinase on tight junction protein disruption (28), C5ainduced BBB integrity (29), and glutamate-induced BBB disruption (30). We found that arsenite exposure induced a concentration- and time-dependent increase of endothelial cell monolayer permeability. Using the antioxidant N-acetyl-cysteine (NAC), superoxide scavenger tempol, and immune-depleting VEGF with a VEGF antibody, we provide direct evidence that ROS-mediated VEGF up-regulation contributes to arseniteinduced increases of permeability.

Materials and Methods Materials. 2′,7′-Dichlorodihydrofluoresceindiacetate (CMH2DCFHDA), Alexa Fluoro 488-conjugated goat antirabbit secondary antibody, Alexa Fluoro 488-conjugated rabbit antigoat secondary antibody, and a primary antibody for zonula occludens-1 (ZO-1) were purchased from Invitrogen (Eugene, OR). Fluorescein isothicyanate (FITC)-labeled dextran, dimethyl sulfoxide (DMSO), lucigenin, NADPH, diphenyleneiodonium chloride (DPI), 0.1% poly-L-lysin, tempol, digitonin, and NAC were obtained from Sigma Chemical Co. (St. Louis, MO). Primary antibodies for VEGF and VE-cadherin were from Santa Cruz Biotechnology (Santa Cruz, CA). Normal rabbit control IgG was purchased from R&D system (Minneapolis, MN). Fetal bovine serum (FBS) was purchased from Hyclone Laboratories, Inc. (South Logan, UT). Endothelial cell growth medium and mouse VEGF ELISA kit were purchased from Cell Application, Inc. (San Diego, CA). Dulbecco’s modified Eagle’s medium (DMEM), penicillin, and streptomycin were obtained from Gibco (Grand Island, NY). A lactate dehydrogenase (LDH) cytotoxicity assay kit was purchased from Takara Bio Inc. (Shiga, Japan). Cell culture inserts were purchased form BD Falcon (Burnham, NJ). Cell Culture. Immortalized mouse brain microvascular endothelial cells bEnd3 (American Type Culture Collection, Manassas, VA) were cultured according to the supplier’s instructions in DMEM supplemented with 4.5 g/L glucose, 3.7 g/L sodium bicarbonate, 4 mM glutamine, 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin at pH 7.4. Human vein umbilical endothelial cells (HUVEC) were obtained from Cell Application, Inc., and cultured in a complete endothelial cell growth medium according to the provider’s instruction. bEnd3 cells and HUVECs were maintained in 25 cm2 tissue culture flasks in a humidity chamber at 37 °C in an atmosphere of 95% air and 5% CO2. Confluent 25 cm2 flasks were trypsinized, and cells were seeded (31) to cover glass, cell culture insert, 24-well tissue culture plates or 96-well tissue culture plates depending on experimental requirements. Permeability Assay. Endothelial permeability was assessed by the passage of FITC-conjugated dextran through the bEnd3 cell or HUVEC cell monolayer (32). Briefly, cells were seeded on the 0.4 µm pore polyester membrane of cell culture inserts in 24-well plates. After they reached confluence, cells were treated with arsenite at various concentrations in a fresh medium. NAC (2 mM), tempol (500 µM), VEGF antibody (0.8 µg/mL), or rabbit IgG (0.8 µg/ mL) control antibody was added 30 min prior to the addition of arsenic and presented in the arsenite treatment period wherever applicable. This VEGF antibody can be used as a blocking antibody according to the supplier’s instruction. At the end of each treatment,

Chem. Res. Toxicol., Vol. 23, No. 11, 2010 1727 cells were washed with fresh medium, and 300 µL of FITC-dextran (average molecular weight 40000, final concentration of 1 mg/mL) was added to each insert. After 4 h of incubation with FITC-dextran, the fluorescence intensity of medium in the plate well was analyzed by a microplate fluorescent reader (Biotek synergy 2 multimode) using an excitation wavelength at 488 nm and an emission wavelength at 518 nm. ROS Measurement. The fluorescent probe, CM-H2DCFDA, was employed to quantify the levels of ROS (33). Cells were incubated in 24-well plates and grown to confluence. Then, cells were treated by arsenite with or without cotreatment of NAC (2 mM) or tempol (500 µM) for 1-6 days. At the end of each treatment, cells were washed with PBS solution complemented with 1% FBS at 37 °C for 2 min, stained with 20 µM CM-H2DCFHDA for 40 min in an incubator, and then washed with cold PBS containing 1% FBS twice. To determine the DCF in cells, 0.02% digitonin (PH 4.1) was added to release intracellular DCF. Subsequently, the medium was decanted and centrifuged for 5 min at 700g. The fluorescence of DCF in supernatant was measured with the microplate fluorescent reader using an excitation wavelength at 488 nm and an emission wavelength at 518 nm. Quantification of VEGF Release. Following arsenite treatments with or without cotreatment of NAC (2 mM) or tempol (500 µM), the culture media were collected and centrifuged to remove the nonadherent cells. The level of VEGF protein was then assessed using a mouse VEGF ELISA kit. Average OD values were acquired at a wavelength 450 nm, with the correction wavelength set at 540 nm, using the microplate reader. The levels of VEGF protein were normalized to the total cellular protein and expressed as pg/mg. Immunostaining of VE-Cadherin and ZO-1. Cells were grown on 18 mm cover glass coated with 0.1% poly-L-lysin. After they reached confluence, cells were treated with arsenite for 6 days. NAC was added 30 min prior to the addition of arsenic and presented in the period of arsenite treatments wherever applicable. The medium was changed every other day. After treatments, cultures were fixed with 4% paraformaldehyde for 20 min at room temperature (RT) and permeabilized with 0.3% triton X-100 for 15 min. To block nonspecific binding, cells were exposed to blocking solution (PBS containing 0.05% triton X-100 and 0.25% BSA) for 40 min at RT. For VE-cadherin staining, cells were incubated with goat anti-VEcadherine polyclonal antibody overnight at 4 °C followed by incubation with Alexa Fluoro 488-conjugated rabbit antigoat secondary antibody for 1 h at RT in dark. For ZO-1 staining, cells were incubated with rabbit anti-ZO-1 polyclonal antibody overnight at 4 °C followed by incubation with Alexa Fluoro 488-conjugated goat antirabbit secondary antibody for 1 h at RT in dark. Cells were further counterstained with Hoechst 33342 for 10 min at RT and washed. Slides were photographed with a Leica DMI4000 B microscope. The specificity of the immunoreaction for VE-cadherin and ZO-1 was verified by control studies showing the absence of immunolabeling when the primary antibody was omitted. Gaps formed between adjacent cells were counted and presented as the number of gaps per 100 cells. Cell Viability Assay. The release of LDH from cells was measured for cell viability. Cells were plated at a density of 5 × 104 cells/well in 96-well tissue culture plates. Cells grown to confluence in each well were treated with 0-20 µM sodium arsenite and cultured at 37 °C in 5% CO2 for 1, 3, or 6 days. The medium was changed every other day during treatments. At the end of the treatments, the cell-free culture medium (100 µL) was collected and then incubated with 100 µL of the reaction mixture from the LDH cytotoxicity assay kit for 20 min at RT. The optical density of the solution was then measured at 490 nm (655 nm as reference) on a microplate reader (Biotek synergy 2 multimode). Detection of NADPH Oxidase Activity. The NADPH oxidase activity was detected by lucigenin chemiluminescence assay in the presence of its substrate NADPH as described previously (34). After exposed to 0-20 µM arsenite for 24 h, cells were rinsed thoroughly with PBS, removed by scraping, resuspended in 500 µL of PBS, and placed in Eppendorf tubes. Cells were lysed by being frozen at -80 °C overnight. Samples were aliquotted (100 µL) in

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Figure 1. Arsenite increased bEnd3 cell monolayer permeability. Cells in culture inserts were grown to monolayer and exposed to 0-20 µM arsenite for 1, 3, and 6 days. Fluorescence of FITC-dextran leaked from insert to plate well was assessed to determine the permeability. Data were normalized by the value of control (0 µM arsenite) of days 1, 3, and 6, respectively. Results were presented as means ( SDs, n ) 3-5 independent experiments; *, p < 0.05 vs control (0 µM arsenite) of days 1, 3, and 6, respectively.

quadruplicate and placed in a 96-well microplate. Immediately before measurement, lucigenin (5 µM) was added to the sample followed by adding of 100 µM NADPH and was mixed. Luminescence was measured using a microplate reader (Biotek synergy 2 multimode) with a 1 min interval for each sample. Statistical Analysis. All data were pooled from at least three independent experiments. Results were presented as means ( SDs. The significance of variance was determined by ANOVA analyses with Newman-Keuls post-test for multiple comparisons. A p value less than 0.05 between groups was considered statistically significant.

Results Arsenite Induced Increase of Cell Monolayer Permeability in bEnd3 Cells. The effect of arsenite on cell monolayer permeability was assessed by FITC-dextran leakage assay. This method has been widely used as an in vitro model to detect cell monolayer permeability. A confluent monolayer of bEnd3 grown on inserts was treated with 0-20 µM arsenite for a period of up to 6 days. The permeability of the monolayer was determined by measuring the content of FITC-dextran leaked from the insert to the medium of the plate well. The basal leak of FITC-dextran (control bEnd3 cells) was 308.6 ( 19.6 AFU (arbitrary fluorescence unit). For easy comparison, the percentage change of permeability was calculated from this basal value. As Figure 1 shows, the cell monolayer permeability increased with arsenite concentration and exposure time. At the concentration of 2 µM, arsenite caused a 7% increase in permeability in 6 days. Cells treated with 5 µM arsenite had a 15% permeability increase at day 6. The permeability of the cells treated with 10 µM arsenite increased 14, 27, and 53% at days 1, 3, and 6, respectively. The permeability of cells treated with 20 µM arsenite for 1, 3, and 6 days increased 22, 60, and 95%, respectively. Arsenite Increased ROS Production. It is known that arsenic induces ROS generation in many kinds of cells (13). To test our hypothesis that ROS is involved in arsenite-induced permeability increase, we evaluated the levels of ROS in bEnd3 cells at the end of exposures lasted for 1, 3, and 6 days. Figure 2 illustrates that the treatment of cells with 10 µM arsenite induced a 36-40% increase in the level of ROS at the end of

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Figure 2. Arsenite elevated cellular ROS generation. Following arsenite (10 µM) treatment with or without NAC (2 mM) or tempol (500 µM) for 1, 3, and 6 days, cells were washed and stained with 20 µM CMH2DCFHDA. The fluorescence of DCF was measured with a microplate fluorescent reader. Data were normalized by the value of control (0 µM arsenite) of days 1, 3, and 6, respectively. Results were presented as means ( SDs, n ) 3-5 independent experiments; *, p < 0.05 vs control (0 µM arsenite) of days 1, 3, and 6, respectively; #, p < 0.05 vs As + NAC; and Φ, p < 0.05 vs As + tempol.

each exposure, as compared to the control (P < 0.05). Interestingly, the level of ROS did not seem dependent on the exposure time but remained at a relatively stable and higher level during the exposures of 1-6 days. In addition, the increase of ROS induced by arsenite was further supported by the inhibitory effects of the antioxidant NAC (2 mM) and the superoxide anion radical scavenger tempol (500 µM). The fluorescence intensity increase induced by arsenite was diminished to the control level by NAC and tempol at all of the tested time points. Arsenite Up-regulated VEGF Expression. ROS has been reported to increase VEGF expression in many cell types including microvascular endothelial cell (20-22). However, it is not known whether or not arsenite increases VEGF expression though increasing ROS production. To establish a direct link between arsenite-induced ROS production and arsenite-induced VEGF expression, we determined the effects of arsenite on the VEGF protein level. After cells were treated with 10 µM arsenite for 1, 3, and 6 days, the VEGF release was analyzed by ELISA. Figure 3 shows that arsenic induced a significant increase of VEGF protein release at each exposure time point when compared to the control VEGF level. The VEGF protein concentration significantly increased from 87 to 187, 194, and 196 pg/mg protein in cells treated with arsenite for 1, 3, and 6 days, respectively. Similar to the ROS level, there was no significant difference in the VEGF level among different treatment periods (e.g., 1, 3, and 6 days). Figure 3 also demonstrates that suppressing ROS levels with NAC and tempol significantly diminished arsenite-induced VEGF release. Both Antioxidant and VEGF Antibody Treatments Reduced Arsenite-Induced Permeability in bEnd3 Cells. To test our hypothesis that arsenite-induced permeability is mediated by the increases in the ROS level and in the VEGF expression, we investigated the effects of antioxidants (NAC and tempol) and a VEGF antibody on the arsenite-induced permeability. Figure 4A shows that both NAC and tempol efficiently decreased arsenite-induced permeability. NAC and tempol decreased arsenite-induced permeability from 156 to 117 and 110% of control, respectively. Figure 4B clearly demonstrates that the VEGF antibody significantly suppressed arsenite-

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Figure 3. Arsenite increased cellular VEGF release. Following arsenite (10 µM) treatment with or without NAC (2 mM) or tempol (500 µM) for 1, 3, and 6 days, the culture media were collected and centrifuged to remove the nonadherent cells. The level of VEGF protein was then assessed using a mouse VEGF ELISA kit, normalized to total cellular protein, and expressed as pg/mg protein. Results were presented as means ( SDs, n ) 3-5 independent experiments; *, p < 0.05 vs control (0 µM arsenite) of days 1, 3, and 6, respectively; #, p < 0.05 vs As + NAC; and Φ, p < 0.05 vs As + tempol.

induced permeability. The arsenite-induced bEnd3 monolayer permeability decreased from 156 to 113% of control when treated with 0.8 µg/mL VEGF antibody. The same concentration of rabbit IgG control antibody showed no effect on the permeability change. Arsenite Induced Permeability Increase in Human Endothelial Cells That Could Be Inhibited by Tempol and VEGF Antibody. To confirm that the observed effects of arsenite on bEnd3 endothelial permeability are a real phenomenon that is relevant to human disease, we carried out similar experiments using human vein umbilical endothelial cells (HUVECs). The permeability of HUVEC monolayer was detected after exposed to 1-2 µM arsenite with or without cotreatments of tempol, the VEGF antibody, or the rabbit IgG control antibody for 6 days. The basal leakage of dextran in HUVEC cells was 394.9 ( 17.4 AFU. The permeability of HUVECs exposed to 2 µM arsenite significantly increased to 129% of control (P < 0.05). Tempol and the VEGF antibody efficiently protected cells from arsenite-induced monolayer leakage. The results are in line with our observation on bEnd3 cells. Significantly, the effects of As on HUVEC were more damaging than on bEnd3. The permeability of HUVEC exposed to 2 µM arsenite for 6 days increased 29% (Figure 5), as compared to a 7% increase with bEnd3 cells (Figure 1). Effects of Arsenite on VE-Cadherin and ZO-1. The integrity of endothelial cell-cell junctions and vascular barrier function is regulated by a series of adhesion molecules that make up AJ, TJ, and gap junctions (35). VE-cadherin and ZO-1 are important components to maintain AJ and TJ (36). To determine whether arsenite affects AJ and TJ of bEnd3 cell through upregulating ROS and VEGF, immunostaining of VE-cadherin and ZO-1 was conducted. As Figure 6A shows, VE-cadherin staining changed from a uniform distribution along the cell membrane under control conditions to a diffuse pattern after a 6 day arsenite exposure at 10 µM. Also, gap formation was observed in the bEnd3 monolayer treated with arsenite, as compared to no gap formation in untreated cells. Both the NAC and the VEGF antibody inhibited gap formation and nonuniform distribution of VE-cadherin induced by arsenite. Furthermore, in arsenite-

Figure 4. NAC, tempol, and VEGF antibody decreased arsenite-induced permeability. bEnd3 cells in culture inserts were grown to monolayer and exposed to arsenite (10 µM) with or without NAC (2 mM) or tempol (500 µM) (A), 0.8 µg/mL VEGF antibody, or 0.8 µg/mL rabbit IgG control andibody (B) for 6 days. The fluorescence of FITC-dextran leaked form insert to plate well was assessed to determine the permeability. Data were normalized by the value of control (0 µM arsenite). Results were presented as means ( SDs, n ) 3-5 independent experiments; *, p < 0.05 vs control (0 µM arsenite; #, p < 0.05 vs As + NAC; Φ, p < 0.05 vs As + tempol; and Ψ, p < 0.05 vs As + VEGF Ab.

treated cells, the ZO-1 staining became discontinuous and diffuse, with stitchlike structures and gaps observed at the cell-cell contacts (Figure 6B). Arsenite caused a significant increase of gaps formation between adjacent cells. From both results calculated from VE-cadherin (Figure 6D) and ZO-1 (Figure 6E) staining, there were about 2-fold increases of gap formation in endothelial cell monolayer treated by arsenite, as compared with the control. These effects were inhibited by NAC and the VEGF antibody. Effects of Arsenite on Cell Viability. To corroborate the fact that the observed effects were not due to cells undergoing apoptosis or death, cells were stained with Hoechst 33342. The nuclei of the cells treated with arsenite did not show any apoptosis or death morphology. The cell morphology also showed no obvious change after arsenite exposure (Figure 6C). Besides the morphology observation, the LDH assay was used to demonstrate that the levels of exposure to arsenite used in the studies were not cytotoxic to bEnd3 cell. As shown in Figure 7, the viability of bEnd3 cell was not significantly changed by the treatments of arsenite at the concentrations tested for the periods up to 6 days. This result indicates that effects of arsenite on permeability were not due to its cytotoxicity.

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Figure 5. Arsenite induced permeability increase in human endothelial cells (HUVEC) that could be inhibited by tempol and VEGF antibody. HUVEC cells in culture inserts were grown to monolayer and exposed to 1-2 µM arsenite with or without tempol (500 µM), 0.8 VEGF antibody (µg/mL), or rabbit IgG control antibody (0.8 µg/mL) for 6 days. The fluorescence of FITC-dextran leaked from insert to plate well was assessed to determine the permeability. Data were normalized by the value of control (0 µM arsenite). Results were presented as means ( SDs, n ) 3-5 independent experiments; *, p < 0.05 vs control (0 µM arsenite); #, p < 0.05 vs As + tempol; and Φ, p < 0.05 vs As + VEGF Ab.

Discussion Vascular permeability is considered an indicator of vascular integrity. Increased vascular permeability, such as those occurring in inflammation and physical trauma, is considered to be an early sign of vascular injury (37). Even a small increase in permeability can be pathologically important. Arsenic has been reported to elevate vascular permeability in rats and mice (12, 38). Pereira et al. have demonstrated that arsenite at 10 µM caused a 19% increase of human aortic endothelial cell permeability in 6 h (11). Using an in vitro model, we found that arsenite increased brain endothelial cell monolayer permeability concentration and time dependently. Similar to the observation of Pereira et al., our results revealed that at the concentration of 10 µM or higher arsenite significantly increased the permeability of the endothelial cells in 24 h, and the longer the exposure time is, the greater the increase in permeability. More significantly, our results demonstrated that at an environmentally relevant concentration or nonlethal concentrations (5 µM), arsenic caused significant increases in permeability when the exposure time was prolonged to 6 days, although there was no significant effect in the first 3 days of exposure. Our current results, together with the previous findings, are evidence that exposure to arsenite, especially for a long period, can seriously change vascular integrity and contribute to the arsenic-related cardiovascular diseases, even at environmentally relevant concentrations. We selected to test the arsenite effect on endothelial permeability in the range of 0-20 µM including the concentration points at 2, 5, and 10 µM and focused on 10 µM in many of the experiments. The rationale for this selection follows. (1) The selection is to be in the range of As concentrations occurring in the environment. Although the EPA standard for arsenic is 10 ppb (0.13 µM), the concentration of arsenite in drinking water varies in the range of 0.01-3.7 mg/L (1.3-49 µM) (39). For example, an investigation on groundwater arsenic contamination in Bangladesh and India reported that 5% of the analyzed tubewells had an arsenic concentration above 700 µg/L (9.3 µM) and 2% of tubewells were highly contaminated with 1000 µg/L

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(13.3 µM) arsenic (40). In Inner Mongolia, China, there are over 1 million of people exposed to arsenic-contaminated water with the highest concentration of 1.9 mg/L (24.8 µM) (41). Moreover, environmental exposure to arsenic continues over many years. It is the chronic exposure to relative high contents of arsenic that caused many diseases. (2) The tested arsenite concentrations in this study are in the concentration range used in many previous publications. As summarized in a previous review (13), a wide range of arsenic concentration (0-300 µM) has been tested. (3) In this study, we have tested the effect of As at the lower end of its concentration range being previously tested in the literature, including 2 and 5 µM, which are considered environmentally relevant or nonlethal concentrations. In addition, the selection of 10 µM for subsequent experiments enabled us to study the effects and mechanisms at a shorter exposure time, as well as allowing for a comparison to the previous report (11). VEGF was originally described as a potent vascular permeability factor that contributes to vascular pathobiology. Although it may provide neuroprotection through angiogenesis or other unidentified mechanisms, expression of VEGF can damage vasculature structure and cause vascular remodeling. Indeed, VEGF causes increases in BBB permeability (42, 43). To corroborate the role of VEGF in arsenic-induced permeability increases, VEGF action was inhibited by a VEGF specific antibody. Normally, endothelial cells secrete VEGF to the intercellular space, which regulates the intracellular signal pathway by combining with its specific receptor (44). The VEGF antibody combines with VEGF protein and blocks the interaction between VEGF and its receptor. Neutralization of VEGF with antibodies has been proven to suppress its functions (18, 45). The VEGF antibody efficiently reduces the increased permeability in cells exposed to arsenite, confirming that VEGF causes permeability increases of the brain endothelial cells and human vein umbilical endothelial cells. Our observed changes in VE-cadherin and ZO-1 protein distribution may result from VEGF up-regulation in endothelial cells exposed to arsenic. VEGF has the ability to alter the tight junction structure and increase the vascular permeability (46). VEGF induces strong increases in tyrosine phosphorylation of the AJ components VE-cadherin and the cell-cell adhesion molecule platelet/endothelial cell adhesion molecule-1 (47). VEGF is also able to modify the actin cytoskeletal architecture, TJ protein phosphorylation, and localization (48). In addition to VE-cadherin and ZO-1, Pereira et al. have reported that arsenite activated PKCR and induced phosphorylation of β-cadherin (11). Our results reveal that arsenic disturbs the distribution of ZO-1 on the junction structures between cells, which decreases the integrity of tight junction and increases the permeability (46, 49). Inhibiting VEGF action by its antibody ameliorates the destruction of the tight junction. This proves the damaging role of VEGF in arsenic-induced paracellular permeability. Specifically, the results demonstrate that the VEGF antibody reversed VE-cadherin and ZO-1 disruption induced by arsenite, and this suggests that up-regulated VEGF may play an important role in arsenite-induced permeability increases. Arsenite might cause phosphorylation of AJ and TJ through up-regulating of VEGF. Other mechanisms such as angiogenesis of endothelial cells may also contribute to arsenite-induced vascular barrier dysfunction. Meng et al. have shown that arsenic promotes endothelial cell migration and angiogenesis via heme oxygenase1-dependent mechanism. VEGF expression and release were found to be heme oxygenase 1 (HO-1) dependently (50). AMP-

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Figure 6. Effects of arsenite on VE-cadherin and ZO-1. After they were treated with arsenite (10 µM) for 6 days with or without NAC (2 mM) or VEGF antibody (0.8 µg/mL), cells were fixed and immunostained for VE-cadherin (A) or ZO-1 (B). The morphology of cell monolayer was also examined (C). Arrows indicate nonuniform distribution of VE-cadherin or ZO-1 and gap formation between adjacent cells. Images are representatives of typical fields seen in three experiments. Gaps formed between adjacent cells were counted based on both VE-cadherin staining (D) and ZO-1 staining (E). Results were presented as means ( SDs, n ) 3 independent experiments; *, p < 0.05 vs control (0 µM arsenite); #, p < 0.05 vs As + NAC; and Φ, p < 0.05 vs As + VEGF Ab.

activated protein kinase (AMPK) is also reported to be a critical regulatory component in metal-induced VEGF expression (51). It is also reported that arsenic decreases angiopoietin-1 secretion

and increases VEGF secretion, which may coordinately regulate vascular dysfunction (52). Rac 1 is an essential component of the arsenic-stimulated NADPH oxidase complex and is a key

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Figure 7. Effects of arsenite on cell viability. Cells were exposed to 0-20 µM arsenite for 1, 3, and 6 days. The cell viability was determined by the release of lactate dehydrogenase. Data were normalized by the value of control (0 µM arsenite) of days 1, 3, and 6, respectively. Results were presented as means ( SDs, n ) 3-5 independent experiments; *, p < 0.05 vs control (0 µM arsenite) of days 1, 3, and 6, respectively.

regulator of endothelial cell migration in angiogenesis (53-55). It is also reported that Rac promotes but is not strictly required for VEGF-induced angiogenesis, whereas it is a necessary part of the signal transduction system mediating the effects of VEGF on formation of endothelial fenestrations and vascular permeability (56). NADPH oxidase is an important intracellular ROS source. It is reported that arsenic (less than 10 µM) is able to stimulate both endothelial and smooth muscle NADPH oxidase activity (15, 54, 57). Increased activity of vascular NADPH oxidase and production of ROS has been implied in arsenic-induced angiogenesis. To reveal if NADPH oxidase plays a role in the elevated ROS levels caused by arsenite treatments, we studied the activity of NADPH oxidase and the effects of a NADPH oxidase inhibitor on arsenite-induced endothelial permeability. Our results show that arsenic did not significantly increase NADPH oxidase activity in bEnd3 until its concentration reached 20 µM (Figure 8A). The NADPH oxidase inhibitor DPI did not block changes in barrier function of bEnd3 cells exposed to 10 µM arsenite for 6 days (Figure 8B). This result suggests that NADPH oxidase might not be an important player in arsenite-induced permeability increase in the bEnd3 monolayer at low concentrations. The source of the ROS induced by arsenite may be very complicated. Besides NADPH oxidase, mitochondria are also well recognized as main sources of ROS induced by As (13). In addition, arsenic can increase ROS generation through intermediary arsine species, oxidation of arsenite to arsenate, and depletion of antioxidants such as glutathione (GSH). The sources of ROS generation induced by As in endothelial cells are an interesting and important subject and need additional investigation in future studies. Arsenic-elevated VEGF expression has been reported in human microvascular endothelial cell and human vein umbilical endothelial cells by other researchers (58, 59). Our results reveal that arsenic-mediated ROS generation increased VEGF expression in both brain microvascular endothelial cells and human vein umbilical endothelial cells. The involvement of ROS in up-regulating VEGF expression was confirmed by NAC and tempol, which completely inhibited arsenite-induced VEGF expression. Although the exact mechanism of how ROS elevates

Figure 8. Involvement of NADPH oxidase in arsenic-induced permeability increase. bEnd3 cells were treated with 0-20 µM arsenite for 1 day. The NADPH oxidase activity was detected by lucigenin chemiluminescence assay in the presence of its substrate NADPH (A). DPI was used as a NADPH oxidase inhibitor to determine whether or not inhibition of NADPH oxidase activity could prevent bEnd3 from arsenic-induced permeability increase (B). bEnd3 cells were treated with arsenite (10 µM) for 6 days with or without DPI (20 µM). Data were normalized by the value of control (0 µM arsenite). Results were presented as means ( SDs, n ) 3-5 independent experiments; *, p < 0.05 vs control (0 µM arsenite); and Ψ, p > 0.05.

expression of VEGF is still not fully understood, there are several possibilities. First, VEGF is a well-defined hypoxia inducible factor-1 (HIF-1) downstream gene. Several previous reports have shown that HIF-1 plays a role in arsenic-induced VEGF protein expression in human prostate cancer cells (21) and VEGF mRNA expression in human ovarian cancer cells (60). ROS may contribute to the stabilization of hypoxiainducible factor-1R (HIF-1R) (46, 61, 62), which enhances transcription of the gene encoding VEGF (61, 63, 64). However, a previous report has shown that arsenic-stimulated HIF-1 was not involved in arsenic-induced VEGF transcript and protein expression in vascular smooth muscles cells (65). Second, HO-1 is able to stimulate VEGF expression in human microvascular endothelial cells. However, it is in a ROS-independent manner (50). Third, protein kinase C δ (PKC δ) may play a role in arsenic-induced VEGF expression, as shown in vascular smooth muscle cells (65). Fourth, activating transcription factor 4 (ATF4), which is expressed in response to oxidative stress, may be another potential mediator responsible for increased VEGF

Arsenic Induces Permeability through a ROS-VEGF Pathway

transcription in response to oxidative stressor (66). On the basis of these previous reports, ROS-activated PKC and ATF4 may play a critical role in arsenic-induced VEGF expression. Further investigation is needed to confirm this concept. Besides cell-cell junction destruction, cell death, which damages the integrity of the cell monolayer, can result in increased permeability. To clarify whether cell death contributes to the increased permeability, we examined cell death in the monolayer culture by cell morphology and LDH release. The viability of bEnd3 cells was not significantly decreased after being treated by 0-20 µM arsenite for 1, 3, and 6 days. The cell monolayer morphology also did not have obvious changes as compared to the control. Cells with VE-cadherin or ZO-1 disruption effects did not show any apoptosis or cell death morphology. These results confirm that cell death or apoptosis is not the main reason responsible for arsenite-induced permeability. In summary, our data provide a potential mechanism by which arsenite impairs permeability of microvascular endothelial cell. We have identified that arsenite-induced ROS generation increases VEGF expression, which is involved in disrupting the AJ and TJ of the endothelial monolayer and, subsequently, increases the cell permeability. It is well established that arsenic induces ROS generation. It is also reported that ROS are involved in arsenic-induced vascular leakage (67). Results from several groups provide evidence that VEGF is a potent factor in causing endothelial permeability increases (48, 62, 63, 68). However, there is no study directly addressing the role of ROSmediated VEGF expression in arsenite-induced endothelial permeability increase. To the best of our knowledge, this is the first report demonstrating that arsenite causes microvascular endothelial cell permeability increase through a ROS-VEGF pathway. Arsenic is associated with various subclinical and clinical outcomes of the cardiovascular system, including carotid atherosclerosis, peripheral vascular disease, ischemic heart disease, and cerebrovascular disease (69). Our results provide a new means for understanding the damaging role of arsenic in vascular permeability. Acknowledgment. This study was supported in part by a startup fund from KUCR. We thank Dr. Prabhu Ramamoorthy for his assistance with fluorescence microscopy.

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