Figure 1.
Fluorescence microscopy after labeling GEnC with WGA-FITC lectin and nuclear staining with DAPI. Left column: represents ‘control’ images (no treatment). Right column: represents images after treatment with100 µM of H2O2. The rows define the time periods: 1 h, 2 h and 5 h. These images show reduction in the binding of WGA-FITC lectin after treatment with H2O2 over time. B Bar chart showing quantitative comparisons of fluorescence intensity of WGA-FITC lectin between GEnC treated with vehicle only and H2O2 (100 µM) for 1 h, 2 h and 5 h. Fluorescence is quantified by using NIH Image J software. The chart shows significant reduction in the expression of WGA-FITC after treatment with H2O2 (n = 10, p = 0.01, ANOVA).
Figure 2.
Expression of HS GAG on GEnC surface after ROS. A
Immunofluorescence microscopy after staining GEnC with anti-HS antibody and nuclear staining with DAPI. Left column: represents ‘control’ images (no treatment). Right column: represents images after treatment with100 µM of H2O2. 1st and 2nd rows define the time periods: 1 h and 5 h. 3rd row: Image in the right column shows GEnC treated with both H2O2 and inhibitors, superoxide dismutase (SOD) and catalase (Cat). These images show reduction in anti-HS staining after treatment with H2O2 over time. This effect can be blocked in the presence of SOD and Cat. B Fluorescence intensity after immunostaining of GEnC with anti-HS antibody over time. Comparisons are shown between controls, H2O2 (100 µM) and H2O2 (100 µM)+SOD and Cat. Y-axis shows ratio of fluorescein emission (from HS) with nuclear staining (DAPI) which is used as a control for cell numbers. The top bar graph shows significant reduction in HS after 1 h and 2 h of H2O2 treatment (n = 11; p<0.001, t-test). Adding SOD and catalase at the same time as H2O2 blocks the effect of H2O2 analyzed at 2 h (n = 11; p<0.01, t-test). The lower bar graph confirms the effects of H2O2 can be reproduced at 5 h (n = 16; p<0.01, t-test).
Figure 3.
Cell survival assay after ROS.
Bar graph showing measurement of absorbance in arbitrary units (Y axis), which estimates amount of formazan produced by GEnC which is used as a surrogate for cell survival. GEnC were treated with control (vehicle), 50, 100 and 200 µM of H2O2. Results show no significant differences (n = 12; p = ns; ANOVA).
Figure 4.
Biosynthesis of GAG chains after ROS. A&B
Charts showing comparison of A, the cumulative 3H3-glucosamine or B, S35 labeled GAG incorporated into the GEnC under control versus H2O2 conditions. Y-axes represent amount of radioactivity in counts per minute (CPM). Results show no significant changes in the rate of biosynthesis of neither non-sulphated (3H3-glucosamine) nor sulphated GAG (S35) after exposing cells to H2O2 (n = 6 for both experiments; p = 0.12 & 0.19 respectively).
Figure 5.
Estimation of GAG in GEnC supernatant. A
Graph showing analysis of highly anionic GAG present in the supernatant following treatment of GEnC with controls versus H2O2. The experiment utilizes Alcian Blue dye that binds to the anionic residues of proteoglycans. Cumulative data from individual experiments shows significant increase in the quantity of alcian blue staining in the supernatant after H2O2. This confirms cleavage of GAG residues from the surface of GEnC after exposure to ROS (n = 3 experiments (individual experiment replicates = 8–12), p<0.05). B Chart showing comparison of 3H3-glucosamine labeled HS GAG fractions isolated from GEnC supernatant under control and post-H2O2 conditions. Results show marked increase in the HS GAG fractions in the supernatant compared to controls after treatment with H2O2 suggesting cleavage of HS GAG (n = 4; p = 0.0019; t test). C Chart showing comparison of 3H3-glucosamine labeled Hyaluronan GAG (non-sulphated) fractions isolated from GEnC supernatant under control and post-H2O2 conditions. Results show a trend but not a significant increase in the non sulphated/Hyaluronan GAG fractions in the supernatant compared to controls. (n = 4; p = 0.053; t test).
Figure 6.
Transendothelial electrical resistance after ROS.
Graph showing real time trans-endothelial electrical resistance (TEER) recordings of GEnC monolayers during exposure to H2O2. H2O2 is added at time point 0 and measurements are taken every 2 min. TEER (Y-axis) shown as a ratio of baseline recording versus time (X-axis). Results show a reduction in TEER after addition of H2O2 within the first 2 min. This effect is significant and peaks at 24 min followed by recovery by 60 min. There is complete resolution of changes in TEER by 90 min.
Figure 7.
Changes in albumin passage after ROS. A
Graph showing cumulative passage of fluorescein labeled albumin (Y-axis) across GEnC monolayers over time (X-axis). Analysis was performed after a delay of 120 min after addition of H2O2 to allow for recovery of TEER of GEnC monolayers. GEnC exposed to H2O2 show a dose dependant increase in passage of albumin compared to controls (n = 6, p<0.001 for effect of treatment, 2-way ANOVA) B Graph showing data from of a separate experiment confirming the significant increase in passage of labeled albumin across GEnC monolayers when exposed to H2O2 at 100 µM. This effect can be partially blocked when GEnC were pre-treated with free radical scavengers, catalase and superoxide dismutase. (n = 11, p<0.05 by 2 way ANOVA; control vs H2O2100 µM, and H2O2100 µM vs H2O2100 µM+block using bonferroni analysis).