Fig. 6

Molecular weight-dependent permeability of the human LEC layers to hydrophilic substances and effects of TNF-α or IL-1β on the permeability of the LEC layers to hydrophilic substances. a A schematic diagram of an in vitro cell barrier permeability kit contained a 24-well cell culture plate insert and polyethylene terephthalate filters with a 1-µm pore size. The fluorescent activity of the solution that had filtered into the lower chamber was determined using a fluorescence plate reader 5 min after the addition of 1 mg/ml of the FITC-labeled dextran or albumin into the upper chamber. b The time-dependent changes in the relative fluorescence units (RFUs) into the lower chamber of the permeability kit evaluated using the fluorescent plate reader with the FITC-labeled 4,400, 12,000, and 66,000 Da dextrans. The ordinate shows the RFUs. The abscissa is the time after the addition of 1 mg/mLlof the FITC-labeled dextrans into the upper chamber. **p < 0.01, a significant difference exists between these columns (n = 8). NS not significant (n = 8). c The permeability of human LEC layers to FITC-labeled 4,400, 12,000, and 66,000 Da dextrans and FITC-labeled albumin. The ordinate shows the normalized FITC-labeled substance flux, which is defined as the ratio of the level of fluorescent activity in the solution at the lower chamber, which was assessed 5 min after the addition of the FITC-labeled hydrophilic substance in the upper chamber to that of a solution containing the same FITC-labeled hydrophilic substance that had only been passed through a polyethylene filter (pore size: 1 µm). **p < 0.01, a significant difference exists between these columns (n = 9). NS not significant (n = 9). d The effects of TNF-α [10 (light green) or 100 (green) ng/mL] on the permeability of human LEC layers to FITC-labeled 4- (upper panel), 12- (middle panel), and 66-kDa (lower panel) dextrans. The ordinate shows the relative flux of FITC-labeled dextrans, which is defined as the ratio of the level of fluorescent activity in the solution in the lower chamber at 5 min after the addition of the FITC-labeled hydrophilic substance in the upper chamber to the fluorescent activity of the original test solution. **p < 0.01, significantly different from no treatment with TNF-α (n = 9). NS, not significant (n = 9). e The effect of IL-1β [10 (light orange) or 100 (orange) ng/ml] on the permeability of human LEC layers to FITC-labeled 4- (upper panel), 12- (middle panel), and 66-kDa (lower panel) dextrans. The ordinate is the same item as shown in panel D. **p < 0.01, significantly different from no treatment with IL-1β (n = 9). NS not significant (n = 9). Data from Kawai et al. [82], with permission