HO-1 protects CE-induced injury, permeability increase and apoptosis.

<p>HO-1 WT and HO-1 KO mice were exposed to either 20 μl of CE or held as controls for 24 hours. (A) The lung tissues were stained with H&E and images under a laser scanning microscopy. Data shown are representative of three donors. (B) Lung inflammation was detected by BAL cell counts. (C) Lung permeability was evaluated by BAL protein content. (D) The quantitative results from TUNEL assay were presented as percentage of TUNEL positive cells per field. Data are shown as a mean ± SD from three different experiments. * <i>p</i> < 0.05, ** <i>p</i> < 0.01 vs HO-1 WT (-) CE; ## <i>p</i> < 0.01 vs HO-1 KO (-) CE; && <i>p</i> < 0.01 vs HO-1 WT (+) CE by a one-way ANOVA with HSD test.</p

CE-induced apoptosis is caspase-3 dependent.

<p>(A) CE instigates apoptosis in BEAS-2B cells. Cells were pretreated with or without 10 μM ZnPP for overnight. Following exposure of BEAS-2B cells to 0 ppm, 150 ppm or 300 ppm (data not shown) of CE for 1 or 4 hours, flow cytometry dot plots for the simultaneous binding of Annexin V-FITC and PI uptake were shown. Numbers in the gates represent percentages of Dead (D) cells, as well as early (E), and late (L) apoptotic events. The data are representative of three independent experiments. Percentages of dead cells (PI⁺Annexin V^-) and apoptotic cells (Annexin V⁺) were quantified and data are shown as a mean ± SD. * <i>p</i> < 0.05, ** <i>p</i> < 0.01 vs no CE control in the absence of ZnPP; # <i>p</i> < 0.05, ## <i>p</i> < 0.01 vs no CE treatment in the presence of ZnPP; & <i>p</i> < 0.05, && <i>p</i> < 0.01 vs with CE treatment in the absence of ZnPP by a one-way ANOVA with HSD test. (B) Representative western blots and associated quantification for active caspase-3, normalized by β-actin content. BEAS-2B cells were exposed to 0 to 150 ppm of CE for 4 hours. Antibodies specific cleaved caspase-3 was used and β-actin was used as a loading control. The representative blots from three independent experiments are shown. The densities of protein bands were determined by densitometry and the data represent a one-fold increase from the control density. (C) Caspase-3 activity was measured using a DEVD-pNA calorimetric assay. After treatment with different concentration of CE for 4 hours, cells were lysed and 100 μg of protein was incubated with 200 μM DEVD-pNA for 6 hours at 37°C. Absorbance measurements were taken at a wavelength of 405 nm and the fold induction of caspase-3 activity relative to the control was shown. * <i>p</i> < 0.05 and ** <i>p</i> < 0.01 vs control by a one-way ANOVA with HSD test. (D) Mice tracheal explants were isolated and IHC analysis was performed after exposure 0 ppm or 150 ppm CE for 2 hours. (E) Blue crabs were exposed to 0 ppm or 150 ppm CE for 19 hours. Gill tissues were harvested for IHC analysis using a cleaved caspase-3 polyclonal antibody (1:800 dilution) followed by treatment with biotinylated goat anti-rabbit antibody and streptavidin–alkaline phosphatase (AP), which produced a red coloration for cleaved caspase-3 positive areas.</p

Morphological changes, reduction in cell diameter, permeability increase and disruption of intercellular junctions induced by CE.

<p>(A) Gills of zebrafish were exposed to either CE (150 ppm) or held as controls for 24 hours or 56 hours and stained with H&E. Digital micrographs were obtained at 20 x magnifications. Arrows point to edema and blebbing of gill epithelium. The ratios of gill area/gill length were calculated using Image Software (NIH, Bethesda, MD, USA) and presented as 1-dimensional area measurements. Data is quantified and shown as mean ± SD of three independent experiments. ** <i>p</i> < 0.01 vs control by a one-way ANOVA with HSD test. (B) Cell diameter measurements. BEAS-2B cells were grown to confluence in 65 mm dishes and exposed to 0 to 150 ppm of CE for 2 hours (n = 3). Data are shown as a mean ± SD. * <i>p</i> < 0.05 and ** <i>p</i> < 0.01 vs control by a one-way ANOVA with HSD test. (C) Permeability measurement of the bronchial epithelium of the airway. The sub-acute response to CE exposure was modeled by ECIS. BEAS-2B cells were seeded into the ECIS array. Cells were allowed to cover the gold electrodes in each well of the array prior to exposure to CE (0 ppm to 70 ppm). Real-time measurements of the electric resistance of the bronchial epithelial monolayers were obtained at 64 kHz. Resistance measurements were normalized with respect to the values in each well 1 hour prior to the initiation of the exposure. This time period corresponded to 16 hours after the seeding of the cells and was designated as t = 0 hour in the graph. The data are representative of three independent experiments. (D) BREA-2B cells were culture with or without 100 ppm CE for 1 hour. Protein expression of ZO-1 and actin filaments was detected using immunofluorescence microscopy (original magnification, ×40) with rabbit anti-ZO-1 (green) and phalloidin (red). Representative images captured from BEAS-2B cells are shown.</p

Upregulation of HO-1 and NOX4 in response to CE stimulation.

<p>Zebrafish were exposed to either CE 150 ppm or control for 56 hours and IHC analysis was performed for HO-1 (A) and NOX4 (B) expression. CE exposure is accomplished by holding adult zebrafish in 1 liter borosilicate class beakers (acid washed, followed with neutralizing alkali) to eliminate any potential reaction of the CE with plastic. (C) Cell lysates from BEAS-2B cells were analyzed by western blotting with anti-HO-1 and NOX4 antibodies at different concentrations of CE for 4 hours. β-actin was used as loading control. For quantification of the HO-1 and NOX4 expression, membranes were scanned and the bar graphs illustrated the relative expression of HO-1 and NOX4 by densitometry. The signal intensity for HO-1 or NOX4 at control was set to 1.0. (D) BEAS-2B cells were exposed to either CE or control for 4 hours, and HO activity was measured as pmol of bilirubin formed per mg protein per h. Data presents mean values of three independent experiments. Data are shown as a mean ± SD. ** <i>p</i> < 0.01 vs control by a one-way ANOVA with HSD test.</p

Both HO-1 and CORM-2 protect against CE-induced ROS production.

<p>(A) BREA-2B cells pretreated with 10 μM ZnPP or 10 μM CORM-2 were either unlabeled or labeled with 20 μM DCFDA for 30 minutes and then cultured for an additional 3 hours with or without 150 ppm or 300 ppm CE according to the protocol. Cells were then analyzed on flow cytometry. The mean fluorescence intensity (MFI) of DCFDA expression by no DCFDA (gray-filled histogram), with DCFDA no CE (Dashed line histogram) or with DCFDA and with CE treatment (Solid line histogram) were compared and shown in the histogram. Data shown are representative of three experiments. (B) For comparison, the MFI ratio (MFIR) was calculated by dividing the MFI of (+) CE treatments (solid line) by the MFI of the (-) CE with DCFDA (dashed line), respectively. The MFIR of DCFDA staining is shown as mean ± SD of 3 donors. ** <i>p</i> < 0.01 vs control (+) CE 150 ppm; ## <i>p</i> < 0.01 vs control (+) CE 300; && <i>p</i> < 0.01 vs (+) CE 150 ppm in the presence of ZnPP; §§ <i>p</i> < 0.01 vs (+) CE 150 ppm in the presence of CORM-2 by a one-way ANOVA with HSD test.</p

HO-1 stabilizes the adhesion proteins E-cadherin and FAK.

<p>(A) HO-1 WT and HO-KO mice were exposed to either 20 μl of CE or held as controls for 24 hours. Cell lysates were prepared from lung epithelial cells and analyzed by western blotting with anti-E-cadherin and FAK antibodies. (B) The densities of protein bands were determined by densitometry and the data represent a one-fold increase from the control density. Data are shown as a mean ± SD from three different experiments. * <i>p</i> < 0.05, ** <i>p</i> < 0.01 vs HO-1 WT (-) CE; ## <i>p</i> < 0.01 vs HO-1 KO (-) CE; && <i>p</i> < 0.01 vs HO-1 WT (+) CE by a one-way ANOVA with HSD test.</p

CE exposure results in the cleavages of tight junctional proteins (ZO-1, ZO-2 and occluding) and adherens junctional proteins (E-cadherin and FAK).

<p>Cell lysates from BEAS-2B cells were analyzed by western blotting with anti-ZO-1, ZO-2 and Occludin (A) and E-cadherin and FAK (B) antibodies at different concentration of CE for 4 hours. The densities of protein bands were determined by densitometry and the data represent a fold change from the control density. The data are representative of three independent experiments. Data are shown as a mean ± SD. * <i>p</i> < 0.05, ** <i>p</i> < 0.01 vs control by a one-way ANOVA with HSD test.</p

Proposed schematic: HO-1 contributes to protection of epithelium from CE-induced injury, ROS and apoptosis.

<p>CE exposure triggers oxidative stress, which induced cleavage of ZO-1, ZO-2, occludin, F-actin, E-cadherin, FAK cleavage, permeability increase, and activation of CRP, NOX4 and HO-1. ROS production and subsequent caspase-3 dependent apoptosis attribute to the induction of both CRP and NOX4. HO-1 and/or CORM-2 are in charge of the protection of CE-induced injury, ROS generation and apoptosis.</p