title: Three-dimensional culture conditions differentially affect astrocyte modulation of brain endothelial barrier function in response to transforming growth factor β1.
abstract: Blood-brain barrier (BBB) function is regulated by dynamic interactions among cell types within the neurovascular unit, including astrocytes and endothelial cells. Co-culture models of the BBB typically involve astrocytes seeded on two-dimensional (2D) surfaces, which recent studies indicate cause astrocytes to express a phenotype similar to that of reactive astrocytes in situ. We hypothesized that the culture conditions of astrocytes would differentially affect their ability to modulate BBB function in vitro. Brain endothelial cells were grown alone or in co-culture with astrocytes. Astrocytes were grown either as conventional (2D) monolayers, or in a collagen-based gel which allows them to grow in a three-dimensional (3D) construct. Astrocytes were viable in 3D conditions, and displayed a marked reduction in their expression of glial fibrillary acidic protein (GFAP), suggesting reduced activation. Stimulation of astrocytes with transforming growth factor (TGF)β1 decreased transendothelial electrical resistance (TEER) and reduced expression of claudin-5 in co-cultures, whereas treatment of endothelial cells in the absence of astrocytes was without effect. The effect of TGFβ1 on TEER was significantly more pronounced in endothelial cells cultured with 3D astrocytes compared to 2D astrocytes. These results demonstrate that astrocyte culture conditions differentially affect their ability to modulate brain endothelial barrier function, and suggest a direct relationship between reactive gliosis and BBB permeability. Moreover, these studies demonstrate the potential importance of physiologically relevant culture conditions to in vitro modeling of disease processes that affect the neurovascular unit.
authors.affiliation: RTI International, 3040 Cornwallis Road, Research Triangle Park, NC 27709, USA. Electronic address: bhawkins@rti.org.
authors.forename: Brian T
authors.initials: BT
authors.lastname: Hawkins
authors.affiliation: RTI International, 3040 Cornwallis Road, Research Triangle Park, NC 27709, USA. Electronic address: sgrego@rti.org.
authors.forename: Sonia
authors.initials: S
authors.lastname: Grego
authors.affiliation: RTI International, 3040 Cornwallis Road, Research Triangle Park, NC 27709, USA. Electronic address: ksellgren@rti.org.
authors.forename: Katelyn L
authors.initials: KL
authors.lastname: Sellgren
mesh_terms: D000818:Animals; D001253:Astrocytes / Q000187:drug effects* / Q000502:physiology; D001692:Biological Transport / Q000187:drug effects; D001921:Brain / Q000166:cytology*; D002461:Cell Line, Transformed; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000502:physiology; D049109:Cell Proliferation / Q000187:drug effects; D018920:Coculture Techniques; D042783:Endothelial Cells / Q000187:drug effects* / Q000502:physiology; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D021621:Imaging, Three-Dimensional; D051379:Mice; D013997:Time Factors; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology*
chemical_list: D005904:Glial Fibrillary Acidic Protein; D053773:Transforming Growth Factor beta1
keywords: Astrocytes; Blood–brain barrier; Gliosis; In vitro; Three-dimensional cell culture