Figure 5
S1P 5 regulates several key features of barrier endothelium. (a) ECIS was used to measure the impedance of the brain endothelial monolayer. S1P5 knockdown cells (1.0 ohm.cm2 ± 0.1) have over 40% reduction in TEER compared to mock cells (1.8 ohm.cm2 ± 0.02). Rb values represent the mean ± SEM of four individual experiments. (b) Paracellular permeability was studied by passive leakage of FITC-dextran molecules (70kD). S1P5 knockdown cells (224.1% ± 4.6 at 5 h) were more permeable FITC-dextrans in time compared to control and mock transduced ECs (133.4% ± 6.1 at 5 h). Fluorescence intensity values represent mean ± SEM of four individual experiments with hCMEC/D3. (c) qPCR analysis of tight junction-associated proteins in S1P5 knockdown cells compared to control cells revealed a 57% decrease in claudin-5 expression (non-targeting control: 100.0% ± 19.4; S1P5 knockdown 42. 8% ± 1.8, n = 3) and a 30% decrease in VE-cadherin expression (non-targeting control: 100.0% ± 2.9; S1P5 knockdown 69.8% ± 3.2, n = 3). (d) qPCR analysis of transporters expressed at the BBB revealed a 23% decrease in GLUT-1 expression (non-targeting control: 100.0% ± 5.1; S1P5 knockdown 77.1% ± 4.7, n = 3), 55% decrease in Pgp expression (non-targeting control: 100.0% ± 8.1; S1P5 knockdown 44.5% ± 1.60, n = 3), and 93% decrease in BCRP1 expression (non-targeting control: 100.0% ± 3.5; S1P5 knockdown 7.0% ± 1.4, n = 3). (e) VE-cadherin localization was also studied by immunocytochemistry. Reduced junctional VE-cadherin localization was observed in S1P5 knockdown cells compared to control cells (VE-cadherin, green and F-actin, red). Statistical significance (t-test) is indicated with asterisks: *P < 0.05, **P < 0.002 and ***P < 0.001.