CBIO-21INFLUENCES OF BLOOD BRAIN BARRIER (BBB) BASEMENT MEMBRANE ECM MOLECULES ON HUMAN BRAIN VASCULAR PERICYTES

Abstract

BACKGROUND: Pericytes are specialised cells found within the blood brain barrier (BBB) ensheathed by a basement membrane (BM) containing numerous extracellular matrix (ECM) proteins which regulate vessel stabilisation and barrier formation. Knowledge on ECM influence on pericyte behaviour is limited. This study aimed to determine the effects of BM-ECM components on pericyte proliferation, adhesion, motility and cell-to-cell contact. METHODS: Human brain vascular pericytes (HBVP) were used. Cell seeding number was optimised and cells were plated on different human-derived, previously optimised, ECM molecules including Fibronectin (5 µg/ml), Laminin (75 µg/ml), Collagen IV (10 µg/ml), Agrin (1 µg/ml) and Perlecan (10 µg/ml) and assessed for proliferation and both quantitative and qualitative adhesion. Motility assays were performed using live cell (time-lapse) imaging. Resistance values between pericytes were collated using an Electric Cell-Substrate Impedance Sensing (ECIS) system. Trans-Endothelial Electrical Resistance (TEER) changes when pericytes were seeded onto human endothelial cells under the different ECM culture conditions were also analysed by voltometer and ECIS. RESULTS: Cells adhered best when seeded on Perlecan and least well on Agrin (p < 0.05). Motility assays showed that pericytes travelled the longest distance and fastest on Agrin and least on Perlecan (p < 0.05). Higher resistance values were reached when cells were plated on Perlecan (∼400 Ω/cm2) and least on Agrin (∼300 Ω/cm2). A tighter barrier was noted when pericytes were co-cultured with endothelial cells (1200 Ω/cm2) compared to when pericytes were added to endothelial cell monolayers (1000 Ω/cm2). CONCLUSIONS: Results demonstrate that different BBB basement molecules have varied effects on TEER values, one indicator of BBB function. Findings also suggest that Agrin may function to recruit pericytes by migration while Perlecan may function to serve as an anchor. This study highlights the importance of BM-ECM molecules and suggests a concentration dependant role of the perlecan/agrin axis in BBB establishment and function.