Single-Cell Analysis of Blood-Brain Barrier Response to Pericyte Loss

Rationale: Pericytes are capillary mural cells playing a role in stabilizing newly formed blood vessels during development and tissue repair. Loss of pericytes has been described in several brain disorders, and genetically induced pericyte deficiency in the brain leads to increased macromolecular leakage across the blood-brain barrier (BBB). However, the molecular details of the endothelial response to pericyte deficiency remain elusive. Objective: To map the transcriptional changes in brain endothelial cells resulting from lack of pericyte contact at single-cell level, and to correlate them with regional heterogeneities in BBB function and vascular phenotype. Methods and Results: We reveal transcriptional, morphological and functional consequences of pericyte absence for brain endothelial cells using a combination of methodologies, including single-cell RNA sequencing, tracer analyses and immunofluorescent detection of protein expression in pericyte-deficient adult Pdgfbret/ret mice. We find that endothelial cells without pericyte contact retain a general BBB-specific gene expression profile, however, they acquire a venous-shifted molecular pattern and become transformed regarding the expression of numerous growth factors and regulatory proteins. Adult Pdgfbret/ret brains display ongoing angiogenic sprouting without concomitant cell proliferation providing unique insights into the endothelial tip cell transcriptome. We also reveal heterogeneous modes of pericyte-deficient BBB impairment, where hotspot leakage sites display arteriolar-shifted identity and pinpoint putative BBB regulators. By testing the causal involvement of some of these using reverse genetics, we uncover a reinforcing role for angiopoietin 2 at the BBB. Conclusions: By elucidating the complexity of endothelial response to pericyte deficiency at cellular resolution, our study provides insight into the importance of brain pericytes for endothelial arterio-venous zonation, angiogenic quiescence and a limited set of BBB functions. The BBB-reinforcing role of ANGPT2 is paradoxical given its wider role as TIE2 receptor antagonist and may suggest a unique and context-dependent function of ANGPT2 in the brain

A molecular atlas of cell types and zonation in the brain vasculature

Cerebrovascular disease is the third most common cause of death in developed countries, but our understanding of the cells that compose the cerebral vasculature is limited. Here, using vascular single-cell transcriptomics, we provide molecular definitions for the principal types of blood vascular and vessel-associated cells in the adult mouse brain. We uncover the transcriptional basis of the gradual phenotypic change (zonation) along the arteriovenous axis and reveal unexpected cell type differences: a seamless continuum for endothelial cells versus a punctuated continuum for mural cells. We also provide insight into pericyte organotypicity and define a population of perivascular fibroblast-like cells that are present on all vessel types except capillaries. Our work illustrates the power of single-cell transcriptomics to decode the higher organizational principles of a tissue and may provide the initial chapter in a molecular encyclopaedia of the mammalian vasculature.Peer reviewe

The role of PDGF-B in brain blood vessels

The development of blood vessels is dependent on several molecular cues to form properly. A functional PDGF-B/PDGFR-b signaling is paramount for the investment of mural cells, that provide with support, to the developing vasculature. Mutations in PDGFB and PDGFRB are linked to PFBC, an age-dependent neurodegenerative condition manifested by vessel associated calcifications in the brain. The overall aim of the work presented in here was to investigate PFBC related calcifications and analyze the effects of impaired PDGF-B/PDGFR-b signaling on the formation of brain calcifications in different mouse models. In paper I, we functionally analyzed PFBC-related PDGFB and PDGFRB mutations in vitro. While all PDGFB mutations lead to abolished protein function, PDGFRB mutations have more diverse consequences. We also show that reduced Pdgfb and Pdgfrb levels are insufficient for the formation of brain calcifications in several mouse strains. Moreover, region-specific susceptibility factors seem to reside in PFBC pathogenesis that are distinct from pericyte coverage and BBB deficiency. In paper II, we described the molecular composition and cellular association of calcified nodules that develop in two mouse models of PFBC, Pdgfbret/ret and Slc20a2-/- mice. We show that the nodules are composed of pro- and anti-mineralization proteins and that they are in direct association with astrocytes and microglia In paper III, we analyzed the effects of EC-specific ablation of PDGF-B in adult brain vasculature.  We report a substantial decrease of pericyte coverage and altered VSMC morphology and that this phenotype is inadequate to trigger the formation of calcifications or affect BBB integrity. The aim of paper IV was to molecularly define the adult mouse brain vasculature by taking advantage of the scRNAseq technique. Here, we describe a gradual change in expression profile along the arteriovenous axis: ECs present a continuum along the axis while mural cell expression profile is punctuated.  In summary, this thesis present detailed description of calcifications formed in mouse models of PFBC and address the role of impaired PDGF-B/PDGFR-b signaling for the formation of nodules in mice. Furthermore, the scRNaseq analysis performed on healthy adult brain vasculature has paved the way for future analysis in mouse models of PFBC

The role of PDGF-B in brain blood vessels

The development of blood vessels is dependent on several molecular cues to form properly. A functional PDGF-B/PDGFR-b signaling is paramount for the investment of mural cells, that provide with support, to the developing vasculature. Mutations in PDGFB and PDGFRB are linked to PFBC, an age-dependent neurodegenerative condition manifested by vessel associated calcifications in the brain. The overall aim of the work presented in here was to investigate PFBC related calcifications and analyze the effects of impaired PDGF-B/PDGFR-b signaling on the formation of brain calcifications in different mouse models. In paper I, we functionally analyzed PFBC-related PDGFB and PDGFRB mutations in vitro. While all PDGFB mutations lead to abolished protein function, PDGFRB mutations have more diverse consequences. We also show that reduced Pdgfb and Pdgfrb levels are insufficient for the formation of brain calcifications in several mouse strains. Moreover, region-specific susceptibility factors seem to reside in PFBC pathogenesis that are distinct from pericyte coverage and BBB deficiency. In paper II, we described the molecular composition and cellular association of calcified nodules that develop in two mouse models of PFBC, Pdgfbret/ret and Slc20a2-/- mice. We show that the nodules are composed of pro- and anti-mineralization proteins and that they are in direct association with astrocytes and microglia In paper III, we analyzed the effects of EC-specific ablation of PDGF-B in adult brain vasculature.  We report a substantial decrease of pericyte coverage and altered VSMC morphology and that this phenotype is inadequate to trigger the formation of calcifications or affect BBB integrity. The aim of paper IV was to molecularly define the adult mouse brain vasculature by taking advantage of the scRNAseq technique. Here, we describe a gradual change in expression profile along the arteriovenous axis: ECs present a continuum along the axis while mural cell expression profile is punctuated.  In summary, this thesis present detailed description of calcifications formed in mouse models of PFBC and address the role of impaired PDGF-B/PDGFR-b signaling for the formation of nodules in mice. Furthermore, the scRNaseq analysis performed on healthy adult brain vasculature has paved the way for future analysis in mouse models of PFBC

Adult-induced genetic ablation distinguishes PDGFB roles in blood-brain barrier maintenance and development

Platelet-derived growth factor B (PDGFB) released from endothelial cells is indispensable for pericyte recruitment during angiogenesis in embryonic and postnatal organ growth. Constitutive genetic loss-of-function of PDGFB leads to pericyte hypoplasia and the formation of a sparse, dilated and venous-shifted brain microvasculature with dysfunctional blood-brain barrier (BBB) in mice, as well as the formation of microvascular calcification in both mice and humans. Endothelial PDGFB is also expressed in the adult quiescent microvasculature, but here its importance is unknown. We show that deletion of Pdgfb in endothelial cells in 2-months-old mice causes a slowly progressing pericyte loss leading, at 12-18 months of age, to ≈50% decrease in endothelial:pericyte cell ratio, ≈60% decrease in pericyte longitudinal capillary coverage and >70% decrease in pericyte marker expression. Similar to constitutive loss of Pdgfb, this correlates with increased BBB permeability. However, in contrast to the constitutive loss of Pdgfb, adult-induced loss does not lead to vessel dilation, impaired arterio-venous zonation or the formation of microvascular calcifications. We conclude that PDFGB expression in quiescent adult microvascular brain endothelium is critical for the maintenance of pericyte coverage and normal BBB function, but that microvessel dilation, rarefaction, arterio-venous skewing and calcification reflect developmental roles of PDGFB

Adult-induced genetic ablation distinguishes PDGFB roles in blood-brain barrier maintenance and development

Platelet-derived growth factor B (PDGFB) released from endothelial cells is indispensable for pericyte recruitment during angiogenesis in embryonic and postnatal organ growth. Constitutive genetic loss-of-function of PDGFB leads to pericyte hypoplasia and the formation of a sparse, dilated and venous-shifted brain microvasculature with dysfunctional blood-brain barrier (BBB) in mice, as well as the formation of microvascular calcification in both mice and humans. Endothelial PDGFB is also expressed in the adult quiescent microvasculature, but here its importance is unknown. We show that deletion of Pdgfb in endothelial cells in 2-months-old mice causes a slowly progressing pericyte loss leading, at 12–18 months of age, to ≈50% decrease in endothelial:pericyte cell ratio, ≈60% decrease in pericyte longitudinal capillary coverage and >70% decrease in pericyte marker expression. Similar to constitutive loss of Pdgfb, this correlates with increased BBB permeability. However, in contrast to the constitutive loss of Pdgfb, adult-induced loss does not lead to vessel dilation, impaired arterio-venous zonation or the formation of microvascular calcifications. We conclude that PDFGB expression in quiescent adult microvascular brain endothelium is critical for the maintenance of pericyte coverage and normal BBB function, but that microvessel dilation, rarefaction, arterio-venous skewing and calcification reflect developmental roles of PDGFB

Astrocyte–microglial association and matrix composition are common events in the natural history of primary familial brain calcification

Primary familial brain calcification (PFBC) is an age-dependent and rare neurodegenerative disorder characterized by microvascular calcium phosphate deposits in the deep brain regions. Known genetic causes of PFBC include loss-of-function mutations in genes involved in either of three processes-platelet-derived growth factor (PDGF) signaling, phosphate homeostasis or protein glycosylation-with unclear molecular links. To provide insight into the pathogenesis of PFBC, we analyzed murine models of PFBC for the first two of these processes in Pdgfbret/ret and Slc20a2-/- mice with regard to the structure, molecular composition, development and distribution of perivascular calcified nodules. Analyses by transmission electron microscopy and immunofluorescence revealed that calcified nodules in both of these models have a multilayered ultrastructure and occur in direct contact with reactive astrocytes and microglia. However, whereas nodules in Pdgfbret/ret mice were large, solitary and smooth surfaced, the nodules in Slc20a2-/- mice were multi-lobulated and occurred in clusters. The regional distribution of nodules also differed between the two models. Proteomic analysis and immunofluorescence stainings revealed a common molecular composition of the nodules in the two models, involving proteins implicated in bone homeostasis, but also proteins not previously linked to tissue mineralization. While the brain vasculature of Pdgfbret/ret mice has been reported to display reduced pericyte coverage and abnormal permeability, we found that Slc20a2-/- mice have a normal pericyte coverage and no overtly increased permeability. Thus, lack of pericytes and increase in permeability of the blood-brain barrier are likely not the causal triggers for PFBC pathogenesis. Instead, gene expression and spatial correlations suggest that astrocytes are intimately linked to the calcification process in PFBC. © 2019 The Authors. Brain Pathology published by John Wiley & Sons Ltd on behalf of International Society of Neuropathology

Functional characterization of germline mutations in PDGFB and PDGFRB in primary familial brain calcification

Primary Familial Brain Calcification (PFBC), a neurodegenerative disease characterized by progressive pericapillary calcifications, has recently been linked to heterozygous mutations in PDGFB and PDGFRB genes. Here, we functionally analyzed several of these mutations in vitro. All six analyzed PDGFB mutations led to complete loss of PDGF-B function either through abolished protein synthesis or through defective binding and/or stimulation of PDGF-Rβ. The three analyzed PDGFRB mutations had more diverse consequences. Whereas PDGF-Rβ autophosphorylation was almost totally abolished in the PDGFRB L658P mutation, the two sporadic PDGFRB mutations R987W and E1071V caused reductions in protein levels and specific changes in the intensity and kinetics of PLCγ activation, respectively. Since at least some of the PDGFB mutations were predicted to act through haploinsufficiency, we explored the consequences of reduced Pdgfb or Pdgfrb transcript and protein levels in mice. Heterozygous Pdgfb or Pdgfrb knockouts, as well as double Pdgfb+/-;Pdgfrb+/- mice did not develop brain calcification, nor did Pdgfrbredeye/redeye mice, which show a 90% reduction of PDGFRβ protein levels. In contrast, Pdgfbret/ret mice, which have altered tissue distribution of PDGF-B protein due to loss of a proteoglycan binding motif, developed brain calcifications. We also determined pericyte coverage in calcification-prone and non-calcification-prone brain regions in Pdgfbret/ret mice. Surprisingly and contrary to our hypothesis, we found that the calcification-prone brain regions in Pdgfbret/ret mice model had a higher pericyte coverage and a more intact blood-brain barrier (BBB) compared to non-calcification-prone brain regions. While our findings provide clear evidence that loss-of-function mutations in PDGFB or PDGFRB cause PFBC, they also demonstrate species differences in the threshold levels of PDGF-B/PDGF-Rβ signaling that protect against small-vessel calcification in the brain. They further implicate region-specific susceptibility factor(s) in PFBC pathogenesis that are distinct from pericyte and BBB deficiency

Prolonged systemic hyperglycemia does not cause pericyte loss and permeability at the mouse blood-brain barrier

Diabetes mellitus is associated with cognitive impairment and various central nervous system pathologies such as stroke, vascular dementia, or Alzheimer's disease. The exact pathophysiology of these conditions is poorly understood. Recent reports suggest that hyperglycemia causes cerebral microcirculation pathology and blood-brain barrier (BBB) dysfunction and leakage. The majority of these reports, however, are based on methods including in vitro BBB modeling or streptozotocininduced diabetes in rodents, opening questions regarding the translation of the in vitro findings to the in vivo situation, and possible direct effects of streptozotocin on the brain vasculature. Here we used a genetic mouse model of hyperglycemia (Ins2(AKITA)) to address whether prolonged systemic hyperglycemia induces BBB dysfunction and leakage. We applied a variety of methodologies to carefully evaluate BBB function and cellular integrity in vivo, including the quantification and visualization of specific tracers and evaluation of transcriptional and morphological changes in the BBB and its supporting cellular components. These experiments did neither reveal altered BBB permeability nor morphological changes of the brain vasculature in hyperglycemic mice. We conclude that prolonged hyperglycemia does not lead to BBB dysfunction, and thus the cognitive impairment observed in diabetes may have other causes

Single-cell RNA sequencing of mouse brain and lung vascular and vessel-associated cell types

Vascular diseases are major causes of death, yet our understanding of the cellular constituents of blood vessels, including how differences in their gene expression profiles create diversity in vascular structure and function, is limited. In this paper, we describe a single-cell RNA sequencing (scRNA-seq) dataset that defines vascular and vessel-associated cell types and subtypes in mouse brain and lung. The dataset contains 3,436 single cell transcriptomes from mouse brain, which formed 15 distinct clusters corresponding to cell (sub) types, and another 1,504 single cell transcriptomes from mouse lung, which formed 17 cell clusters. In order to allow user-friendly access to our data, we constructed a searchable database (http://betsholtzlab.org/VascularSingleCells/database.html). Our dataset constitutes a comprehensive molecular atlas of vascular and vessel-associated cell types in the mouse brain and lung, and as such provides a strong foundation for future studies of vascular development and diseases.Peer reviewe