Regulation of human brain vascular pericytes and human astrocytes in a blood-brain barrier model using human brain microvascular endothelial cells: Expression of TGF-β1, VEGF, MMP-9 and P-gp

An in vitro human blood-brain barrier (BBB) model was developed using human brain microvascular endothelial cells (HBMECs) co-cultured with human brain vascular pericytes (HBVPs) and human astrocytes (HAs) at various ratios (HBVPs:HAs = 1:1, 1:2, 1:6), associated with pericyte-conditioned medium (PCM) and astrocyte-conditioned medium (ACM). After 7 days of co-culturing of HBMECs with HBVPs and HAs at a ratio of 1:2, and a 1:1 ratio of PCM and ACM, the transendothelial electrical resistance was enhanced to 319 +/- 16.67 Omega x cm(2) (225% increase) and the permeability coefficient of propidium iodide was reduced to 2.09 +/- 0.5 x 10(-6) cm/s (61% decrease). Analysis of three major barrier integrity modulators: transforming growth factor-31 (TGF-beta 1), vascular endothelial growth factor (VEGF), and matrix metalloproteinases (MMPs), revealed a higher TGF-beta 1 expression and lower VEGF and MMP-9 expressions in a co-culture of HBMECs with HBVPs and HAs at a ratio of 1:2. Calcein-AM analysis showed higher P-glycoprotein (P-gp) activity in the model using PCM and ACM with HBVPs:HAs= 1:2 (100%) than that with HBVPs:HAs =1:1 (55%) and HBVPs:HAs= 1:6 (49%). The effect of TGF-beta 1 receptor inhibitor SB431542, VEGF inhibitor asterric acid, and MMP-9 inhibitor CIT (H-Cys-Thr-Thr-His-Trp-Gly-Phe-Thr-Leu-Cys-OH) on the BBB model suggested that TGF-beta 1 up-regulates P-gp activity and VEGF down-regulates P-gp activity. In the presence of PCM and ACM, co-culturing of HBMECs with HBVPs and HAs at a ratio of 1:2 could approach the in vivo BBB in a well representative manner. (C) 2018 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.