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Supplemental Digital Content is available in the text., Rationale: Endothelial cells (ECs) are highly glycolytic and generate the majority of their energy via the breakdown of glucose to lactate. At the same time, a main role of ECs is to allow the transport of glucose to the surrounding tissues. GLUT1 (glucose transporter isoform 1/Slc2a1) is highly expressed in ECs of the central nervous system (CNS) and is often implicated in blood-brain barrier (BBB) dysfunction, but whether and how GLUT1 controls EC metabolism and function is poorly understood. Objective: We evaluated the role of GLUT1 in endothelial metabolism and function during postnatal CNS development as well as at the adult BBB. Methods and Results: Inhibition of GLUT1 decreases EC glucose uptake and glycolysis, leading to energy depletion and the activation of the cellular energy sensor AMPK (AMP-activated protein kinase), and decreases EC proliferation without affecting migration. Deletion of GLUT1 from the developing postnatal retinal endothelium reduces retinal EC proliferation and lowers vascular outgrowth, without affecting the number of tip cells. In contrast, in the brain, we observed a lower number of tip cells in addition to reduced brain EC proliferation, indicating that within the CNS, organotypic differences in EC metabolism exist. Interestingly, when ECs become quiescent, endothelial glycolysis is repressed, and GLUT1 expression increases in a Notch-dependent fashion. GLUT1 deletion from quiescent adult ECs leads to severe seizures, accompanied by neuronal loss and CNS inflammation. Strikingly, this does not coincide with BBB leakiness, altered expression of genes crucial for BBB barrier functioning nor reduced vascular function. Instead, we found a selective activation of inflammatory and extracellular matrix related gene sets. Conclusions: GLUT1 is the main glucose transporter in ECs and becomes uncoupled from glycolysis during quiescence in a Notch-dependent manner. It is crucial for developmental CNS angiogenesis and adult CNS homeostasis but does not affect BBB barrier function.

Background Genetic variation in a population has an influence on the manifestation of monogenic as well as multifactorial disorders, with the underlying genetic contribution dependent on several interacting variants. Common laboratory mouse strains used for modelling human disease lack the genetic variability of the human population. Therefore, outcomes of rodent studies show limited relevance to human disease. The functionality of brain vasculature is an important modifier of brain diseases. Importantly, the restrictive interface between blood and brain—the blood–brain barrier (BBB) serves as a major obstacle for the drug delivery into the central nervous system (CNS). Using genetically diverse mouse strains, we aimed to investigate the phenotypic and transcriptomic variation of the healthy BBB in different inbred mouse strains. Methods We investigated the heterogeneity of brain vasculature in recently wild-derived mouse strains (CAST/EiJ, WSB/EiJ, PWK/PhJ) and long-inbred mouse strains (129S1/SvImJ, A/J, C57BL/6J, DBA/2J, NOD/ShiLtJ) using different phenotypic arms. We used immunohistochemistry and confocal laser microscopy followed by quantitative image analysis to determine vascular density and pericyte coverage in two brain regions—cortex and hippocampus. Using a low molecular weight fluorescence tracer, sodium fluorescein and spectrophotometry analysis, we assessed BBB permeability in young and aged mice of selected strains. For further phenotypic characterization of endothelial cells in inbred mouse strains, we performed bulk RNA sequencing of sorted endothelial cells isolated from cortex and hippocampus. Results Cortical vessel density and pericyte coverage did not differ among the investigated strains, except in the cortex, where PWK/PhJ showed lower vessel density compared to NOD/ShiLtJ, and a higher pericyte coverage than DBA/2J. The vascular density in the hippocampus differed among analyzed strains but not the pericyte coverage. The staining patterns of endothelial arteriovenous zonation markers were similar in different strains. BBB permeability to a small fluorescent tracer, sodium fluorescein, was also similar in different strains, except in the hippocampus where the CAST/EiJ showed higher permeability than NOD/ShiLtJ. Transcriptomic analysis of endothelial cells revealed that sex of the animal was a major determinant of gene expression differences. In addition, the expression level of several genes implicated in endothelial function and BBB biology differed between wild-derived and long-inbred mouse strains. In aged mice of three investigated strains (DBA/2J, A/J, C57BL/6J) vascular density and pericyte coverage did not change—expect for DBA/2J, whereas vascular permeability to sodium fluorescein increased in all three strains. Conclusions Our analysis shows that although there were no major differences in parenchymal vascular morphology and paracellular BBB permeability for small molecular weight tracer between investigated mouse strains or sexes, transcriptomic differences of brain endothelial cells point to variation in gene expression of the intact BBB. These baseline variances might be confounding factors in pathological conditions that may lead to a differential functional outcome dependent on the sex or genetic polymorphism.

Background Brain tumors, whether primary or secondary, have limited therapeutic options despite advances in understanding driver gene mutations and heterogeneity within tumor cells. The cellular and molecular composition of brain tumor stroma, an important modifier of tumor growth, has been less investigated to date. Only few studies have focused on the vasculature of human brain tumors despite the fact that the blood-brain barrier (BBB) represents the major obstacle for efficient drug delivery. Methods In this study, we employed RNA sequencing to characterize transcriptional alterations of endothelial cells (EC) isolated from primary and secondary human brain tumors. We used an immunoprecipitation approach to enrich for EC from normal brain, glioblastoma (GBM), and lung cancer brain metastasis (BM). Results Analysis of the endothelial transcriptome showed deregulation of genes implicated in cell proliferation, angiogenesis, and deposition of extracellular matrix (ECM) in the vasculature of GBM and BM. Deregulation of genes defining the BBB dysfunction module was found in both tumor types. We identified deregulated expression of genes in vessel-associated fibroblasts in GBM. Conclusion We characterize alterations in BBB genes in GBM and BM vasculature and identify proteins that might be exploited for developing drug delivery platforms. In addition, our analysis on vessel-associated fibroblasts in GBM shows that the cellular composition of brain tumor stroma merits further investigation.

Microglia participate in CNS development and homeostasis and are often implicated in modulating disease processes in the CNS. However, less is known about the role of microglia in the biology of the neurovascular unit (NVU). In particular, data are scant on whether microglia are involved in CNS vascular pathology. In this study, we use a mouse model of primary familial brain calcification (PFBC) –Pdgfbret/retto investigate the role of microglia in calcification of the NVU. We report that microglia enclosing vessel-calcifications, coined calcification-associated microglia (CAM), display a distinct activation signature. Pharmacological ablation of microglia with the CSF1R inhibitor - PLX5622 leads to aggravated vessel calcification. Additionally, depletion of microglia in wild-type andPdgfbret/retmice causes the development of bone protein (osteocalcin, osteopontin) containing axonal spheroids in the white matter. Mechanistically, we show that microglia require functional TREM2 for controlling vessel-associated calcification. In conclusion, our results demonstrate that microglial activity in the setting of pathological vascular calcification is beneficial. In addition, we identify a new, previously unrecognized function of microglia in halting the expansion of ectopic calcification.

Brain endothelium possesses several organ-specific features collectively known as the blood-brain barrier (BBB). In addition, trafficking of immune cells in the healthy central nervous system (CNS) is tightly regulated by CNS vasculature. In CNS autoimmune diseases such as multiple sclerosis (MS), these homeostatic mechanisms are overcome by autoreactive lymphocyte entry into the CNS causing inflammatory demyelinating immunopathology. Previous studies have shown that pericytes regulate the development of organ-specific characteristics of brain vasculature such as the BBB and astrocytic end-feet. Whether pericytes are involved in the control of leukocyte trafficking remains elusive. Using adult, pericyte-deficient mice (Pdgfbret/ret), we show here that brain vasculature devoid of pericytes shows increased expression of VCAM-1 and ICAM-1, which is accompanied by increased leukocyte infiltration of dendritic cells, monocytes and T cells into the brain, but not spinal cord parenchyma. Regional differences enabling leukocyte trafficking into the brain as opposed to the spinal cord inversely correlate with the pericyte coverage of blood vessels. Upon induction of experimental autoimmune encephalitomyelitis (EAE), pericyte-deficient mice succumb to severe neurological impairment. Treatment with first line MS therapy - fingolimod significantly reverses EAE, indicating that the observed phenotype is due to the massive influx of immune cells into the brain. Furthermore, pericyte-deficiency in mice that express myelin oligodendrocyte glycoprotein peptide (MOG35-55) specific T cell receptor (Pdgfbret/ret; 2D2Tg) leads to the development of spontaneous neurological symptoms paralleled by massive influx of leukocytes into the brain, suggesting altered brain vascular immune quiescence as a prime cause of exaggerated neuroinflammation. Thus, we show that pericytes indirectly restrict immune cell transmigration into the CNS under homeostatic conditions and during autoimmune-driven neuroinflammation by inducing immune quiescence of brain endothelial cells.