Brain Endothelial Cell Bioenergetics via Extracellular Flux Analysis

Abstract

The neurovascular unit (NVU) is an important functional component in the central nervous system (CNS). The NVU consists of multiple cell types (endothelial cells, pericytes, astrocytes, others) working together as a restrictive blood‐tissue interface and functions to maintain homeostasis within nervous tissue in the CNS. A dysfunctional NVU is associated with many disease states, including Alzheimer's Disease, strokes, and tumors. The endothelial cell is an important barrier‐forming cell of the NVU because of its direct contact with the blood, its intercellular tight junctions, biotransforming enzymes, and asymmetric distribution of active and carrier‐mediated transporters. These properties are important for maintaining the neuro‐microenvironment and require an abundant supply of energy (ATP). Although energy metabolism has been studied in various tissues and cells, little is known about the energy producing pathways of brain endothelial cells. Therefore, using extracellular flux analysis, we characterized the bioenergetics of human brain microvascular endothelial cells (BMECs) in vitro. BMECs are primarily glycolytic and D‐glucose, pyruvate, and glutamine are preferred metabolic substrates. Inhibitors of monocarboxylate transporter (MCT1), mitochondrial pyruvate carrier, and glycolysis significantly alter ATP production, cellular respiration, and/or glycolytic rates. These findings contribute to our understanding of endothelial cell metabolic plasticity in normal and diseased brain as well as active angiogenesis during development or tumorigenesis. Understanding endothelial cell bioenergetics will be useful for future studies regarding development of therapies, that target endothelial cell energetics, for neurological disorders.Support or Funding InformationWhiteside Foundation and the Department of DefenseThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Oxygen gradients dictate angiogenesis but not barriergenesis in a 3D brain microvascular model

“…Once the monolayer was formed, culture medium supplemented with 10‐µl of phosphorescent oxygen probe was added and oxygen consumption was measured over a time span of 30 min. These relative rates were then compared to previously published values to provide absolute quantities (McDonald et al, 2019; Sheikh et al, 2020).…”

“…To determine whether the difference in cell density also affected glucose levels, medium removed from the vessels was measured with a glucose sensor and indicated there was no significant differences between high and low cell density conditions (Supporting Information: Figure S3a). An OCR was then conducted on the cell density representing low density (112,704 cells/well) and high density (175,296 cells/well) as well as a density used in a previous study of HCMEC/D3 oxygen consumption that served as a reference value (McDonald et al, 2019;Sheikh et al, 2020). The consumption rates were measured to be 6.4 × 10 −11 mol/min and 9.6 × 10 −11 mol/min for the low and high densities respectively (Supporting Information: Figure S3b,c).…”

“…There has been substantial innovation in systems to directly measure cellular oxygen consumption including microfluidic‐based approaches (Azimzadeh et al, 2021). Accurate measurements of oxygen consumption are vital to informing computational models of oxygen diffusion within in vitro systems, and previous studies have provided measurements of oxygen consumption by cerebral endothelial cells in culture (McDonald et al, 2019; Rellick et al, 2016; Sheikh et al, 2020). Overall, combining in vitro platforms with computational models informed by cellular oxygen consumption rates can provide new insight into processes affecting angiogenesis and barriergenesis in cerebral vasculature.…”