Inorganic phosphate exporter heterozygosity in mice leads to brain vascular calcification, microangiopathy, and microgliosis.

Calcification of the cerebral microvessels in the basal ganglia in the absence of systemic calcium and phosphate imbalance is a hallmark of primary familial brain calcification (PFBC), a rare neurodegenerative disorder. Mutation in genes encoding for sodium-dependent phosphate transporter 2 (SLC20A2), xenotropic and polytropic retrovirus receptor 1 (XPR1), platelet-derived growth factor B (PDGFB), platelet-derived growth factor receptor beta (PDGFRB), myogenesis regulating glycosidase (MYORG), and junctional adhesion molecule 2 (JAM2) are known to cause PFBC. Loss-of-function mutations in XPR1, the only known inorganic phosphate exporter in metazoans, causing dominantly inherited PFBC was first reported in 2015 but until now no studies in the brain have addressed whether loss of one functional allele leads to pathological alterations in mice, a commonly used organism to model human diseases. Here we show that mice heterozygous for Xpr1 (Xpr1WT/lacZ ) present with reduced inorganic phosphate levels in the cerebrospinal fluid and age- and sex-dependent growth of vascular calcifications in the thalamus. Vascular calcifications are surrounded by vascular basement membrane and are located at arterioles in the smooth muscle layer. Similar to previously characterized PFBC mouse models, vascular calcifications in Xpr1WT/lacZ mice contain bone matrix proteins and are surrounded by reactive astrocytes and microglia. However, microglial activation is not confined to calcified vessels but shows a widespread presence. In addition to vascular calcifications, we observed vessel tortuosity and transmission electron microscopy analysis revealed microangiopathy-endothelial swelling, phenotypic alterations in vascular smooth muscle cells, and thickening of the basement membrane.

New Insights in the Complexity and Functionality of the Neurovascular Unit.

The neurovascular unit (NVU) encompasses all brain cells and underlines that neurons, glia and brain vasculature are in intimate physical and functional association. Brain function is dependent on blood flow and local increases in blood flow in response to neural activity - functional hyperaemia takes place at the NVU. Although this is a vital function of the NVU, many studies have demonstrated that the NVU also performs other tasks. Blood vessels in the brain, which are composed of multiple cell types, are essential for correct brain development. They constitute the niche for brain stem cells, sense the environment and communicate changes to neural tissue, and control the immune quiescence of the CNS. In this brief chapter we will discuss new insights into the biology of NVU, which have further revealed the heterogeneity and complexity of the vascular tree and its neurovascular associations.

Characterization of the blood-brain barrier in genetically diverse laboratory mouse strains.

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.

Pericytes regulate vascular immune homeostasis in the CNS.

Pericytes regulate the development of organ-specific characteristics of the brain vasculature such as the blood-brain barrier (BBB) and astrocytic end-feet. Whether pericytes are involved in the control of leukocyte trafficking in the adult central nervous system (CNS), a process tightly regulated by CNS vasculature, remains elusive. Using adult pericyte-deficient mice (Pdgfbret/ret ), we show that pericytes limit leukocyte infiltration into the CNS during homeostasis and autoimmune neuroinflammation. The permissiveness of the vasculature toward leukocyte trafficking in Pdgfbret/ret mice inversely correlates with vessel pericyte coverage. Upon induction of experimental autoimmune encephalomyelitis (EAE), pericyte-deficient mice die of severe atypical EAE, which can be reversed with fingolimod, indicating that the mortality is due to the massive influx of immune cells into the brain. Additionally, administration of anti-VCAM-1 and anti-ICAM-1 antibodies reduces leukocyte infiltration and diminishes the severity of atypical EAE symptoms of Pdgfbret/ret mice, indicating that the proinflammatory endothelium due to absence of pericytes facilitates exaggerated neuroinflammation. Furthermore, we show that the presence of myelin peptide-specific peripheral T cells in Pdgfbret/ret ;2D2tg mice leads to the development of spontaneous neurological symptoms paralleled by the massive influx of leukocytes into the brain. These findings indicate that intrinsic changes within brain vasculature can promote the development of a neuroinflammatory disorder.

Microglia control small vessel calcification via TREM2.

Microglia participate in central nervous system (CNS) development and homeostasis and are often implicated in modulating disease processes. 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, Pdgfbret/ret , to investigate the role of microglia in calcification of the NVU. We report that microglia enclosing vessel calcifications, coined calcification-associated microglia, display a distinct activation phenotype. Pharmacological ablation of microglia with the CSF1R inhibitor PLX5622 leads to aggravated vessel calcification. Mechanistically, we show that microglia require functional TREM2 for controlling vascular calcification. Our results demonstrate that microglial activity in the setting of pathological vascular calcification is beneficial. In addition, we identify a previously unrecognized function of microglia in halting the expansion of vascular calcification.

Blood-brain barrier alterations in human brain tumors revealed by genome-wide transcriptomic profiling.

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.

Role of the GLUT1 Glucose Transporter in Postnatal CNS Angiogenesis and Blood-Brain Barrier Integrity.

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.