(M1330-04-25) Endothelial Cell-Pericyte Interactions Regulate Brain Glucose Transport

Purpose: Capillary pericytes play a crucial role in maintaining the blood-brain barrier (BBB) integrity and function. Inadequate pericyte coverage in the cerebral microvasculature leads to BBB breakdown and death [1] and is shown to trigger several neurovascular pathologies associated with Alzheimer's disease (AD). The BBB endothelial cells heavily depend on glucose transporters to facilitate glucose uptake and generate energy. Inhibition of the metabolic activity of endothelial cells can potentially cause pericyte loss in the brain vasculature, leading to BBB dysfunction [1]. Despite their abundance and close association with endothelial cells, the exact functions of pericytes and their roles in regulating brain metabolism remain largely unknown. Therefore, the present study seeks to delve into the molecular-level interactions between endothelial cells and pericytes, with the ultimate goal of identifying the mechanisms underlying pericyte degeneration and its potential impact on glucose transport and metabolism at the BBB.

Methods: To investigate the effect of pericytes on the expression of glucose transporters in BBB endothelial cells (ECs), differential expression and pathway enrichment analysis was applied to publicly available RNA-Seq data from the gene expression omnibus database (GEO ID GSE144474). In this study, hematopoietic stem cell‑derived endothelial cells were cultured alone or co-cultured with immortalized human pericytes. The gene expression of endothelial cells in solo cultures and co-cultures with pericytes was evaluated over 96 hours. To investigate the effect of endothelial cell-pericyte interactions on brain glucose transport in vitro, we first differentiated human induced pluripotent stem cells (hiPSCs) into brain-specific microvascular endothelial cells (iBMECs) and then co-cultured them with or without human brain vascular pericytes (hBVPs) on 6-well Transwells^Ò (as shown in Figure 1) for 24, 48, 96 hours, and 7 days. We also cultured hBVPs alone in the same plate for comparison. We evaluated the protein and RNA levels of glucose transporters (GLUT1 and GLUT3) as well as the glucose regulator TXNIP using western blot and RT-PCR, respectively, in iBMECs and hBVPs. Additionally, we measured glucose uptake and ATP production rates in iBMECs with or without hBVPs co-culture using a fluorescence-labeled glucose analog, 2-NBDG, and a Seahorse analyzer, respectively.

Results: According to our results, compared to the solo culture of BBB endothelial cells, the RNA expression of GLUT1 (SLC2A1) and GLUT3 (SLC2A3), and TXNIP [2] was upregulated in endothelial cells co-cultured with pericytes (Figure 2). Furthermore, after 7 days of co-culture, the protein level of GLUT3 was upregulated in iBMEC cells compared to the solo culture, although we did not observe protein level changes in TXNIP and GLUT1 (Figure 3 A&C). In contrast, GLUT1 was upregulated in pericytes co-cultured with iBMECs (Figure 3 B&D). Interestingly, expression of TXNIP, a negative regulator of GLUT1, was significantly downregulated in hBVPs co-cultured with iBMECs, which can potentially increase GLUT1 expression in hBVPs and maintain pericyte metabolism, preventing loss of coverage in brain vasculatures. Furthermore, we observed a significant reduction in the number of 2-NBDG-positive iBMECs in solo cultures compared with co-cultures, indicating the role of pericytes in endothelial glucose uptake. Additionally, we found that both total ATP and glycoATP production was decreased in solo-cultured iBMECs compared to co-cultured iBMECs, suggesting a potential involvement of pericytes in endothelial glucose metabolism.

Conclusion: The results of the study suggest that pericytes play an important role in maintaining normal brain glucose levels by upregulating glucose transporter expression in endothelial cells. Furthermore, TXNIP may play a crucial role in endothelial-pericyte communication and maintenance of brain energy homeostasis.

References: 1. Nikolakopoulou AM, Montagne A, Kisler K, Dai Z, Wang Y, Huuskonen MT, Sagare AP, Lazic D, Sweeney MD, Kong P, Wang M, Owens NC, Lawson EJ, Xie X, Zhao Z, Zlokovic BV. Pericyte loss leads to circulatory failure and pleiotrophin depletion causing neuron loss. Nat Neurosci. 2019 Jul;22(7):1089-1098.
2. Wang L, Curran GL, Min PH, Li L, Lowe VJ, Kandimalla KK. Amyloid Beta Peptides Inhibit Glucose Transport at the Blood-brain Barrier by Disrupting Insulin-Akt Pathway in Alzheimer’s Disease. bioRxiv 2022.11.21.517280.

Acknowledgements:This work was supported by the Minnesota Partnership for Biotechnology and Medical Genomics (MNP#15.31), National Institutes of Health/National Institute of Neurological Disorders and Stroke R01NS125437 and National Institute on Aging RF1 AG058081.

Schematic plot of experiment design. The human induced pluripotent stem cells (hiPSCs) were differentiated into brain-specific microvascular endothelial cells (iBMECs). We then co-cultured iBMEC cells with or without human brain vascular pericytes (hBVPs) on 6-well Transwells for 24, 48, 96 hours, and 7 days and then RNA and protein levels of iBMEC and hBVPs were evaluated.

Log2 fold changes of gene expression of TXNIP, SLC2A1, and SLC2A3 in endothelial cells co-cultured with pericytes at 24, 48, and 96 hours compared to endothelial cell solo-cultures.

Representative immunoblot of TXNIP, GLUTs in (A) iBMECs and (B) hBVPs under solo-culture or co-culture. Corresponding quantification of TXNIP, GLUTs expression in (C) iBMECs and (D) hBVPs.