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

Abstract

Abstract 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 Ω × cm2 (225% increase) and the permeability coefficient of propidium iodide was reduced to 2.09 ± 0.5 × 10−6 cm/s (61% decrease). Analysis of three major barrier integrity modulators: transforming growth factor-β1 (TGF-β1), vascular endothelial growth factor (VEGF), and matrix metalloproteinases (MMPs), revealed a higher TGF-β1 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-β1 receptor inhibitor SB431542, VEGF inhibitor asterric acid, and MMP-9 inhibitor CTT (H-Cys-Thr-Thr-His-Trp-Gly-Phe-Thr-Leu-Cys-OH) on the BBB model suggested that TGF-β1 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.