Study Protocol.

<p>Diagram of the study setup displaying the time course of experimental interventions performed on transdifferentiated AC-HUVEC.</p

TUNEL Fluorescence Microscopy.

<p>Representative light microscopy images of TUNEL fluorescence in labeled AC-HUVECs after 24 hours of hypoxia, 2 hours of isoflurane exposure with 0 MAC (control; fig. 4a), 0.5 MAC (b), 1 MAC (c), 2 MAC (d), and a subsequent 24-hour recovery. In addition, positive (e) and negative (f) controls are presented.</p

Bax/Bcl-2 Western Blot Curve Fit Analysis.

<p>Western blot data and curve fit of Bax (drawn as crosses and dashed lines) and Bcl-2 (rings, solid lines) in relation to hypoxia, isoflurane concentration, and delay to analysis subsequent to 2 hours of isoflurane exposure. a) No hypoxia. 4-hour latency period: significant positive linear isoflurane concentration dependency for both Bax (R² = 0.44, P = 0.02) and Bcl-2 (R² = 0.87, P<0.001). b) No hypoxia, 24-hour latency period: no significant concentration dependency. c & d) Hypoxia for 24 hours followed by 2 hours of reoxygenation prior to isoflurane treatment. c) 4-hour latency period: positive quadratic dose dependency for Bax (R² = 0.71, P = 0.004); d) 24-hour latency period: positive quadratic dose dependency for Bax (R² = 0.80, P = 0.001) and negative exponential dependency for Bcl-2 (R² = 0.39, P = 0.03).</p

TUNEL Regression Analysis.

<p>Quantification and quadratic regression analysis of TUNEL fluorescence in relation to hypoxia, isoflurane concentrations, and delay to analysis subsequent to 2 hours of isoflurane exposure. a) No hypoxia. 4-hour latency period: R² = 0.91, P = 0.003; 24-hour latency period: R² = 0.95, P<0.001. b) Hypoxia for 24 hours followed by 2 hours of reoxygenation prior to isoflurane treatment. 4-hour latency period: R² = 0.75, P = 0.031; 24-hour latency period: R² = 0.98, P<0.001.</p

Bax/Bcl-2 Western Blot bar graphs.

<p>Median value and interquartile range of Bax (a) and Bcl-2 (b) protein expression in relation to hypoxic challenge and recovery period (4/24 h). N.s.: not significant, *: statistically significant difference to control/0 MAC in Mann-Whitney test.</p

Summary of results.

<p>Results for markers of apoptosis (Bax and Bcl-2 ratio, TUNEL intensity) in the treatment groups with and without previous hypoxia. Values are means and range. Midazolam groups have not been compared against isoflurane and sevoflurane groups.</p><p>* P < 0.05 vs. control and sevoflurane.</p><p>** P < 0.01 vs. control, isoflurane 0.5 MAC, isoflurane 1 MAC, and sevoflurane 2 MAC.</p><p>Summary of results.</p

Anesthetics and endothelial apoptosis measured by TUNEL.

<p>TUNEL intensity of AC-HUVEC without (a) and with hypoxia (b) 24 h after post-treatment with 300 nM of midazolam, 2 MAC of isoflurane, or 2 MAC of sevoflurane. * P < 0.05 (N = 2, ANOVA).</p

TUNEL fluorescence microscopy.

<p>Representative TUNEL fluorescence labeled light microscopy images of AC-HUVEC. Images were taken after the experimental sequence of 24 h of hypoxia, 2 h of reoxygenation, treatment with 300 nM midazolam (a), 2 MAC isoflurane (b), 2 MAC sevoflurane (c), or no anesthetic treatment (d) for 2 h, and a 24 h recovery period. Part (d) shows the positive control.</p

Influence of anesthetics on pro- and anti-apoptotic proteins.

<p>Bax and Bcl-2 ratio of normoxic (a) and post-hypoxic (b) AC-HUVEC after post-treatment with various concentrations of midazolam, isoflurane, and sevoflurane for 2 h, and recovery of 24 h. N = 3. * P < 0.01 (ANOVA).</p

Bax and Bcl-2 Western blots.

<p>Representative Western blot clippings for Bax and Bcl-2 from normoxic (Fig 2a and 2b) and post-hypoxic (Fig 2c and 2d) AC-HUVEC harvested 24 h after treatment with 300 nM of Midazolam, 2 MAC Isoflurane, 2 MAC Sevoflurane or no anesthetic compound (control). Positive: positive control.</p