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Abstract Cholesterol is a vital component of the cell membrane and plays an essential role in mediating integral membrane protein function. Altered cholesterol regulation has been implicated in neurological diseases that are associated with blood–brain barrier breakdown. However, the role of brain barrier function in inherited disorders of cholesterol metabolism, such as Niemann-Pick disease C1 (NP-C1), remains unclear. In this study, we determined how cholesterol depletion with U18666A, a chemical inhibitor of NPC1 protein, as well as with the cholesterol-depleting agent methyl-β cyclodextrin (MβCD), impacted brain endothelial cell barrier function. We hypothesized that cholesterol depletion would decrease barrier integrity by disrupting tight junction protein continuity. To test this hypothesis, we differentiated human-induced pluripotent stem cells into brain microvascular endothelial cells (hiBMECs). We then assessed barrier integrity by quantifying trans-endothelial electrical resistance (TEER), small fluorescent molecule permeability, and tight junction continuity and protein levels. We now show that U18666A-treated hiBMECs demonstrated a 75% decrease in TEER and 9-fold increase in sodium fluorescein permeability. Similar trends were observed for hiBMECs treated with MβCD, which showed significantly lowered TEER (93% decrease) and increased sodium fluorescein permeability (20-fold higher). We also observed decreased continuity of the tight junction proteins occludin (13% lower) and claudin-5 (8% lower) as well as a 53% decrease in claudin-5 protein with U18666A treatment. Co-treating U18666A-treated hiBMECs with hydroxypropyl-β cyclodextrin (HPβCD), which releases lysosomal cholesterol, prevented these changes. Together, our results demonstrate that cholesterol is vital for hiBMEC barrier function and tight junction continuity. This study highlights the potential of therapeutics targeted to brain endothelium in NP-C1 and other cholesterol metabolism disorders. 

The blood-brain barrier (BBB) is a dynamic interface that regulates the molecular exchanges between the brain and peripheral blood. The permeability of the BBB is primarily regulated by the junction proteins on the brain endothelial cells. In vitro BBB models have shown great potential for the investigation of the mechanisms of physiological function, pathologies, and drug delivery in the brain. However, few studies have demonstrated the ability to monitor and evaluate the barrier integrity by quantitatively analyzing the junction presentation in 3D microvessels. This study aimed to fabricate a simple vessel-on-chip, which allows for a rigorous quantitative investigation of junction presentation in 3D microvessels. To this end, we developed a rapid protocol that creates 3D microvessels with polydimethylsiloxane and microneedles. We established a simple vessel-on-chip model lined with human iPSC-derived brain microvascular endothelial-like cells (iBMEC-like cells). The 3D image of the vessel structure can then be “unwrapped” and converted to 2D images for quantitative analysis of cell–cell junction phenotypes. Our findings revealed that 3D cylindrical structures altered the phenotype of tight junction proteins, along with the morphology of cells. Additionally, the cell–cell junction integrity in our 3D models was disrupted by the tumor necrosis factor α. This work presents a “quick and easy” 3D vessel-on-chip model and analysis pipeline, together allowing for the capability of screening and evaluating the cell–cell junction integrity of endothelial cells under various microenvironment conditions and treatments. 

Cells depend on precisely regulating barrier function within the vasculature to maintain physiological stability and facilitate essential substance transport. Endothelial cells achieve this through specialized adherens and tight junction protein complexes, which govern paracellular permeability across vascular beds. Adherens junctions, anchored by vascular endothelial (VE)-cadherin and associated catenins to the actin cytoskeleton, mediate homophilic adhesion crucial for barrier integrity. In contrast, tight junctions composed of occludin, claudin, and junctional adhesion molecule A interact with Zonula Occludens proteins, reinforcing intercellular connections essential for barrier selectivity. Endothelial cell-cell junctions exhibit dynamic conformations during development, maturation, and remodeling, regulated by local biochemical and mechanical cues. These structural adaptations play pivotal roles in disease contexts such as chronic inflammation, where junctional remodeling contributes to increased vascular permeability observed in conditions from cancer to cardiovascular diseases. Conversely, the brain microvasculature’s specialized junctional arrangements pose challenges for therapeutic drug delivery due to their unique molecular compositions and tight organization. This commentary explores the molecular mechanisms underlying endothelial cell-cell junction conformations and their implications for vascular permeability. By highlighting recent advances in quantifying junctional changes and understanding mechanotransduction pathways, we elucidate how physical forces from cellular contacts and hemodynamic flow influence junctional dynamics.