Transcriptomic analysis of a 3D blood–brain barrier model exposed to disturbed fluid flow

Abstract

Cerebral aneurysms are more likely to form at bifurcations in the vasculature, where disturbed fluid is prevalent due to flow separation at sufficiently high Reynolds numbers. While previous studies have demonstrated that altered shear stress exerted by disturbed flow disrupts endothelial tight junctions, less is known about how these flow regimes alter gene expression in endothelial cells lining the blood–brain barrier. Specifically, the effect of disturbed flow on expression of genes associated with cell–cell and cell–matrix interaction, which likely mediate aneurysm formation, remains unclear. RNA sequencing of immortalized cerebral endothelial cells isolated from the lumen of a 3D blood–brain barrier model reveals distinct transcriptional changes in vessels exposed to fully developed and disturbed flow profiles applied by both steady and physiological waveforms. Differential gene expression, validated by qRT-PCR and western blotting, reveals that lumican, a small leucine-rich proteoglycan, is the most significantly downregulated gene in endothelial cells exposed to steady, disturbed flow. Knocking down lumican expression reduces barrier function in the presence of steady, fully developed flow. Moreover, adding purified lumican into the hydrogel of the 3D blood–brain barrier model recovers barrier function in the region exposed to fully developed flow. Overall, these findings emphasize the importance of flow regimes exhibiting spatial and temporal heterogeneous shear stress profiles on cell–matrix interaction in endothelial cells lining the blood–brain barrier, while also identifying lumican as a contributor to the formation and maintenance of an intact barrier.

Lumican, a Multifunctional Cell Instructive Biomarker Proteoglycan Has Novel Roles as a Marker of the Hypercoagulative State of Long Covid Disease

“…Knocking down LUM expression reduces barrier function in this model; however, adding purified LUM into the hydrogel of this 3D BBB model recovered barrier function under fully developed flow conditions. These findings show shear stress profiles effect cell–matrix interaction in BBB endothelial cells, and identify LUM in the maintenance of barrier function and is required to maintain endothelial cell and BBB barrier integrity [ 61 ]. Cross-sectional staining of endothelial cells shows substantial peripheral staining of LUM, suggesting interactions with endothelial cell surface receptors.…”

“…Alterations in cerebrovascular blood flow in trauma or disturbance of the BBB in stroke results in a disturbance in the endothelial cell tight junctions and a significant downregulation in LUM gene expression [ 61 ] modulating the maintenance of the BBB. Elevated LUM levels have been observed in protein deposits in the brain has been noted in schizophrenia [ 63 ].…”