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Modeling hyperosmotic blood-brain barrier opening within human tissue-engineered in vitro brain microvessels.

ABSTRACT: As the majority of therapeutic agents do not cross the blood-brain barrier (BBB), transient BBB opening (BBBO) is one strategy to enable delivery into the brain for effective treatment of CNS disease. Intra-arterial infusion of the hyperosmotic agent mannitol reversibly opens the BBB; however, widespread clinical use has been limited due to the variability in outcomes. The current model for mannitol-induced BBBO assumes a transient but homogeneous increase in permeability; however, the details are poorly understood. To elucidate the mechanism of hyperosmotic opening at the cellular level, we developed a tissue-engineered microvessel model using stem cell-derived human brain microvascular endothelial cells (BMECs) perturbed with clinically relevant mannitol doses. This model recapitulates physiological shear stress, barrier function, microvessel geometry, and cell-matrix interactions. Using live-cell imaging, we show that mannitol results in dose-dependent and spatially heterogeneous increases in paracellular permeability through the formation of transient focal leaks. Additionally, we find that the degree of BBB opening and subsequent recovery is modulated by treatment with basic fibroblast growth factor. These results show that tissue-engineered BBB models can provide insight into the mechanisms of BBBO and hence improve the reproducibility of hyperosmotic therapies for treatment of CNS disease.

SUBMITTER: Linville RM

PROVIDER: S-EPMC7308510 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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Modeling hyperosmotic blood-brain barrier opening within human tissue-engineered in vitro brain microvessels.

Linville Raleigh M RM DeStefano Jackson G JG Sklar Matt B MB Chu Chengyan C Walczak Piotr P Searson Peter C PC

Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism 20190808 7

As the majority of therapeutic agents do not cross the blood-brain barrier (BBB), transient BBB opening (BBBO) is one strategy to enable delivery into the brain for effective treatment of CNS disease. Intra-arterial infusion of the hyperosmotic agent mannitol reversibly opens the BBB; however, widespread clinical use has been limited due to the variability in outcomes. The current model for mannitol-induced BBBO assumes a transient but homogeneous increase in permeability; however, the details a ...[more]

PMID: 31394959

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Project description:BackgroundProtein expression analysis of isolated brain microvessels provides valuable insights into the function of the blood-brain barrier (BBB). However, isolation of brain microvessels from human brain tissue, particularly in small quantities, poses significant challenges. This study presents a method for isolating brain microvessels from a small amount of frozen human brain tissue, adapting techniques from an established mouse brain capillary isolation method.MethodsBrain microvessel fractions were obtained from approximately 0.3 g of frozen human brain tissue (frontal cortex) using a bead homogenizer for homogenization, followed by purification with a combination of cell strainers and glass beads. Protein expression in the isolated human microvessel fractions and whole-brain lysates was analyzed by western blot and proteomic analysis.ResultsMicroscopic imaging confirmed the successful isolation of brain microvessels from frozen human brain tissue. Protein quantification assays demonstrated that the microvessel fraction yielded sufficient protein for detailed expression analysis. Western blot analysis revealed an enrichment of BBB-selective proteins including multidrug resistance 1 (MDR1)/ATP-binding cassette sub-family B member 1 (ABCB1), glucose transporter protein type 1 (GLUT1)/solute carrier family 2 member 1 (SLC2A1), and claudin 5 (CLDN5), in the brain microvessel fraction compared to whole-brain lysates. Multiple reaction monitoring quantification of six BBB-selective proteins-MDR1, breast cancer resistance protein (BCRP)/ATP binding cassette subfamily G member 2 (ABCG2), GLUT1, monocarboxylate transporter 1 (MCT1)/solute carrier family 16 member 1 (SLC16A1), transferrin receptor, and CLDN5-revealed expression levels consistent with those observed in larger human brain samples. Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH-MS)-based quantitative proteomics further demonstrated significant enrichment of human microvascular endothelial cells in the isolated fraction, corroborating the findings from mouse models.ConclusionsWe successfully developed a method for isolation of brain microvessels from a small amount of frozen human brain tissue, facilitating detailed study of BBB proteome in aging or pathological conditions. This technique provides valuable insights into BBB dysfunction in central nervous system disorders and holds potential for improving brain-targeted drug delivery strategies.

Dataset Information

Modeling hyperosmotic blood-brain barrier opening within human tissue-engineered in vitro brain microvessels.

Publications

Modeling hyperosmotic blood-brain barrier opening within human tissue-engineered in vitro brain microvessels.

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