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. Author manuscript; available in PMC: 2013 Aug 7.
Published in final edited form as: Microvasc Res. 2008 Dec 11;77(2):174–186. doi: 10.1016/j.mvr.2008.11.007

Figure 3. PEG does not act simply as a physical barrier.

Figure 3

EC cultured on gold microelectrodes were pretreated with vehicle EGM2 or PEG (8%, 24 hrs) and then stimulated with the A) calcium ionophore, ionomycin (10 μM). In the control, the influx of extracellular calcium causes a potent endothelial barrier disruption, but PEG pretreatment delays the barrier disruption. The ability of ionomycin to gradually bring the resistance down to baseline suggests that even in the presence of cells, PEG does not act as a physical barrier. B) To validate the TER data, confluent cells grown in co-culture inserts were pretreated with either EGM2 or 8% PEG for 2hr and then challenged with 10 μM ionomycin. FITC-dextran tracking dyes were added to the media. Inserts were removed to stop the experiment after 4hr and 24hr post-PEG challenge and media from the lower well were sample for fluorescence with a luminometer. Normalized data to the unstimulated time points demonstrate that PEG significantly decreased FITC-dextran clearance at 4hr and 24hr post-challenge which correlates with the TER data. Using a physiological relevant agonist, similar experiment was performed measuring TER in which ECs were pretreated with vehicle EGM2 or PEG (8%, 1hr) and then stimulated with C) 1 U/ml thrombin. In the control, thrombin causes a transient drop in TER. PEG pretreatment did not block this transient drop in TER, suggesting thrombin was able to access cell surface PAR receptors further supporting the absence of a physical barrier produced by PEG. Note, however, that the maximum drop in thrombin-induced TER in the presence of PEG is still significantly higher than baseline resistance and thereby offering endothelial barrier protection by enhancing overall barrier resistance. D) We confirm that the elevated baseline resistance provided protection by performing in vitro permeability assay measuring FITC-dextran clearance. EC were pretreated with 8% PEG for 2hr, followed by addition of either thrombin (1U/ml, 2hr) or LPS (1μg/ml, 4hr) with FITC-dextran tracking dye. Pretreatment with PEG significantly attenuated both thrombin and LPS-induced increase in FITC-dextran clearance.