Capillary regression leads to sustained local hypoperfusion by inducing constriction of upstream transitional vessels.

In the brain, a microvascular sensory web coordinates oxygen delivery to regions of neuronal activity. This involves a dense network of capillaries that send conductive signals upstream to feeding arterioles to promote vasodilation and blood flow. Although this process is critical to the metabolic supply of healthy brain tissue, it may also be a point of vulnerability in disease. Deterioration of capillary networks is a feature of many neurological disorders and injuries and how this web is engaged during vascular damage remains unknown. We performed in vivo two-photon microscopy on young adult mural cell reporter mice and induced focal capillary injuries using precise two-photon laser irradiation of single capillaries. We found that ~59% of the injuries resulted in regression of the capillary segment 7 to 14 d following injury, and the remaining repaired to reestablish blood flow within 7 d. Injuries that resulted in capillary regression induced sustained vasoconstriction in the upstream arteriole-capillary transition (ACT) zone at least 21 days postinjury in both awake and anesthetized mice. The degree of vasomotor dynamics was chronically attenuated in the ACT zone consequently reducing blood flow in the ACT zone and in secondary, uninjured downstream capillaries. These findings demonstrate how focal capillary injury and regression can impair the microvascular sensory web and contribute to cerebral hypoperfusion.

Meningeal origins and dynamics of perivascular fibroblast development on the mouse cerebral vasculature.

Perivascular fibroblasts (PVFs) are a fibroblast-like cell type that reside on large-diameter blood vessels in the adult meninges and central nervous system (CNS). PVFs contribute to fibrosis following injury but their homeostatic functions are not defined. PVFs were previously shown to be absent from most brain regions at birth and are only detected postnatally within the cerebral cortex. However, the origin, timing and cellular mechanisms of PVF development are not known. We used Col1a1-GFP and Col1a2-CreERT2 transgenic mice to track PVF development postnatally. Using lineage tracing and in vivo imaging we show that brain PVFs originate from the meninges and are first seen on parenchymal cerebrovasculature at postnatal day (P) 5. After P5, PVF coverage of the cerebrovasculature expands via local cell proliferation and migration from the meninges. Finally, we show that PVFs and perivascular macrophages develop concurrently. These findings provide the first complete timeline for PVF development in the brain, enabling future work into how PVF development is coordinated with cell types and structures in and around the perivascular spaces to support normal CNS vascular function.

Serum profiling identifies ibrutinib as a treatment option for young adults with B-cell acute lymphoblastic leukaemia.

Acute lymphoblastic leukaemia (ALL) is a haematological malignancy that is characterized by the uncontrolled proliferation of immature lymphocytes. 80% of cases occur in children where ALL is well understood and treated. However it has a devastating affects on adults, where multi-agent chemotherapy is the standard of care with allogeneic stem cell transplantation for those who are eligible. New treatments are required to extend remission and prevent relapse to improve patient survival rates. We used serum profiling to compare samples from presentation adult B-ALL patients with age- and sex-matched healthy volunteer (HV) sera and identified 69 differentially recognised antigens (P ≤ 0·02). BMX, DCTPP1 and VGLL4 showed no differences in transcription between patients and healthy donors but were each found to be present at higher levels in B-ALL patient samples than HVs by ICC. BMX plays a crucial role in the Bruton's Tyrosine Kinase (BTK) pathway which is bound by the BTK inhibitor, ibrutinib, suggesting adult B-ALL would also be a worthy target patient group for future clinical trials. We have shown the utility of proto-array analysis of B-ALL patient sera, predominantly from young adults, to help characterise the B-ALL immunome and identified a new target patient population for existing small molecule therapy.

Diverse Functions of Retinoic Acid in Brain Vascular Development.

UNLABELLED: As neural structures grow in size and increase metabolic demand, the CNS vasculature undergoes extensive growth, remodeling, and maturation. Signals from neural tissue act on endothelial cells to stimulate blood vessel ingression, vessel patterning, and acquisition of mature brain vascular traits, most notably the blood-brain barrier. Using mouse genetic and in vitro approaches, we identified retinoic acid (RA) as an important regulator of brain vascular development via non-cell-autonomous and cell-autonomous regulation of endothelial WNT signaling. Our analysis of globally RA-deficient embryos (Rdh10 mutants) points to an important, non-cell-autonomous function for RA in the development of the vasculature in the neocortex. We demonstrate that Rdh10 mutants have severe defects in cerebrovascular development and that this phenotype correlates with near absence of endothelial WNT signaling, specifically in the cerebrovasculature, and substantially elevated expression of WNT inhibitors in the neocortex. We show that RA can suppress the expression of WNT inhibitors in neocortical progenitors. Analysis of vasculature in non-neocortical brain regions suggested that RA may have a separate, cell-autonomous function in brain endothelial cells to inhibit WNT signaling. Using both gain and loss of RA signaling approaches, we show that RA signaling in brain endothelial cells can inhibit WNT-ß-catenin transcriptional activity and that this is required to moderate the expression of WNT target Sox17. From this, a model emerges in which RA acts upstream of the WNT pathway via non-cell-autonomous and cell-autonomous mechanisms to ensure the formation of an adequate and stable brain vascular plexus. SIGNIFICANCE STATEMENT: Work presented here provides novel insight into important yet little understood aspects of brain vascular development, implicating for the first time a factor upstream of endothelial WNT signaling. We show that RA is permissive for cerebrovascular growth via suppression of WNT inhibitor expression in the neocortex. RA also functions cell-autonomously in brain endothelial cells to modulate WNT signaling and its downstream target, Sox17. The significance of this is although endothelial WNT signaling is required for neurovascular development, too much endothelial WNT signaling, as well as overexpression of its target Sox17, are detrimental. Therefore, RA may act as a "brake" on endothelial WNT signaling and Sox17 to ensure normal brain vascular development.

Differential Tissue-Specific Function of Adora2b in Cardioprotection.

The adenosine A2b receptor (Adora2b) has been implicated in cardioprotection from myocardial ischemia. As such, Adora2b was found to be critical in ischemic preconditioning (IP) or ischemia/reperfusion (IR) injury of the heart. Whereas Adora2b is present on various cells types, the tissue-specific role of Adora2b in cardioprotection is still unknown. To study the tissue-specific role of Adora2b signaling on inflammatory cells, endothelia, or myocytes during myocardial ischemia in vivo, we intercrossed floxed Adora2b mice with Lyz2-Cre(+), VE-cadherin-Cre(+), or myosin-Cre(+) transgenic mice, respectively. Mice were exposed to 60 min of myocardial ischemia with or without IP (four times for 5 min) followed by 120 min of reperfusion. Cardioprotection by IP was abolished in Adora2b(f/f)-VE-cadherin-Cre(+) or Adora2b(f/f)-myosin-Cre(+), indicating that Adora2b signaling on endothelia or myocytes mediates IP. In contrast, primarily Adora2b signaling on inflammatory cells was necessary to provide cardioprotection in IR injury, indicated by significantly larger infarcts and higher troponin levels in Adora2b(f/f)-Lyz2-Cre(+) mice only. Cytokine profiling of IR injury in Adora2b(f/f)-Lyz2-Cre(+) mice pointed toward polymorphonuclear neutrophils (PMNs). Analysis of PMNs from Adora2b(f/f)-Lyz2-Cre(+) confirmed PMNs as one source of identified tissue cytokines. Finally, adoptive transfer of Adora2b(-/-) PMNs revealed a critical role of Adora2b on PMNs in cardioprotection from IR injury. Adora2b signaling mediates different types of cardioprotection in a tissue-specific manner. These findings have implications for the use of Adora2b agonists in the treatment or prevention of myocardial injury by ischemia.

Identification of hypoxia-inducible factor HIF-1A as transcriptional regulator of the A2B adenosine receptor during acute lung injury.

Although acute lung injury (ALI) contributes significantly to critical illness, resolution often occurs spontaneously through endogenous pathways. We recently found that mechanical ventilation increases levels of pulmonary adenosine, a signaling molecule known to attenuate lung inflammation. In this study, we hypothesized a contribution of transcriptionally controlled pathways to pulmonary adenosine receptor (ADOR) signaling during ALI. We gained initial insight from microarray analysis of pulmonary epithelia exposed to conditions of cyclic mechanical stretch, a mimic for ventilation-induced lung disease. Surprisingly, these studies revealed a selective induction of the ADORA2B. Using real-time RT-PCR and Western blotting, we confirmed an up to 9-fold induction of the ADORA2B following cyclic mechanical stretch (A549, Calu-3, or human primary alveolar epithelial cells). Studies using ADORA2B promoter constructs identified a prominent region within the ADORA2B promoter conveying stretch responsiveness. This region of the promoter contained a binding site for the transcription factor hypoxia-inducible factor (HIF)-1. Additional studies using site-directed mutagenesis or transcription factor binding assays demonstrated a functional role for HIF-1 in stretch-induced increases of ADORA2B expression. Moreover, studies of ventilator-induced lung injury revealed induction of the ADORA2B during ALI in vivo that was abolished following HIF inhibition or genetic deletion of Hif1a. Together, these studies implicate HIF in the transcriptional control of pulmonary adenosine signaling during ALI.

Impaired drainage through capillary-venous networks contributes to age-related white matter loss.

The gradual loss of cerebral white matter contributes to cognitive decline during aging. However, microvascular networks that support the metabolic demands of white matter remain poorly defined. We used in vivo deep multi-photon imaging to characterize microvascular networks that perfuse cortical layer 6 and corpus callosum, a highly studied region of white matter in the mouse brain. We show that these deep tissues are exclusively drained by sparse and wide-reaching venules, termed principal cortical venules, which mirror vascular architecture at the human cortical-U fiber interface. During aging, capillary networks draining into deep branches of principal cortical venules are selectively constricted, reduced in density, and diminished in pericyte numbers. This causes hypo-perfusion in deep tissues, and correlates with gliosis and demyelination, whereas superficial tissues become relatively hyper-perfused. Thus, age-related impairment of capillary-venular drainage is a key vascular deficit that contributes to the unique vulnerability of cerebral white matter during brain aging.

Mutations in a Sar1 GTPase of COPII vesicles are associated with lipid absorption disorders.

Dietary fat is an important source of nutrition. Here we identify eight mutations in SARA2 that are associated with three severe disorders of fat malabsorption. The Sar1 family of proteins initiates the intracellular transport of proteins in COPII (coat protein)-coated vesicles. Our data suggest that chylomicrons, which vastly exceed the size of typical COPII vesicles, are selectively recruited by the COPII machinery for transport through the secretory pathways of the cell.

The elusive brain perivascular fibroblast: a potential role in vascular stability and homeostasis.

In the brain, perivascular fibroblasts (PVFs) reside within the perivascular spaces (PVSs) of arterioles and large venules, however their physiological and pathophysiological roles remain largely unknown. PVFs express numerous extracellular matrix proteins that are found in the basement membrane and PVS surrounding large diameter vessels. PVFs are sandwiched between the mural cell layer and astrocytic endfeet, where they are poised to interact with mural cells, perivascular macrophages, and astrocytes. We draw connections between the more well-studied PVF pro-fibrotic response in ischemic injury and the less understood thickening of the vascular wall and enlargement of the PVS described in dementia and neurodegenerative diseases. We postulate that PVFs may be responsible for stability and homeostasis of the brain vasculature, and may also contribute to changes within the PVS during disease.

Capillary regression leads to sustained local hypoperfusion by inducing constriction of upstream transitional vessels.

In the brain, a microvascular sensory web coordinates oxygen delivery to regions of neuronal activity. This involves a dense network of capillaries that send conductive signals upstream to feeding arterioles to promote vasodilation and blood flow. Although this process is critical to the metabolic supply of healthy brain tissue, it may also be a point of vulnerability in disease. Deterioration of capillary networks is a hallmark of many neurological disorders and how this web is engaged during vascular damage remains unknown. We performed in vivo two-photon microscopy on young adult mural cell reporter mice and induced focal capillary injuries using precise two-photon laser irradiation of single capillaries. We found that ∼63% of the injuries resulted in regression of the capillary segment 7-14 days following injury, and the remaining repaired to re-establish blood flow within 7 days. Injuries that resulted in capillary regression induced sustained vasoconstriction in the upstream arteriole-capillary transition (ACT) zone at least 21 days post-injury in both awake and anesthetized mice. This abnormal vasoconstriction involved attenuation of vasomotor dynamics and uncoupling from mural cell calcium signaling following capillary regression. Consequently, blood flow was reduced in the ACT zone and in secondary, uninjured downstream capillaries. These findings demonstrate how capillary injury and regression, as often seen in age-related neurological disease, can impair the microvascular sensory web and contribute to cerebral hypoperfusion. SIGNIFICANCE: Deterioration of the capillary network is a characteristic of many neurological diseases and can exacerbate neuronal dysfunction and degeneration due to poor blood perfusion. Here we show that focal capillary injuries can induce vessel regression and elicit sustained vasoconstriction in upstream transitional vessels that branch from cortical penetrating arterioles. This reduces blood flow to broader, uninjured regions of the same microvascular network. These findings suggest that widespread and cumulative damage to brain capillaries in neurological disease may broadly affect blood supply and contribute to hypoperfusion through their remote actions.

Endothelial structure contributes to heterogeneity in brain capillary diameter.

The high metabolic demand of brain tissue is supported by a constant supply of blood flow through dense microvascular networks. Capillaries are the smallest class of vessels in the brain and their lumens vary in diameter between ~2 and 5 µm. This diameter range plays a significant role in optimizing blood flow resistance, blood cell distribution, and oxygen extraction. The control of capillary diameter has largely been ascribed to pericyte contractility, but it remains unclear if the architecture of the endothelial wall also contributes to capillary diameter. Here, we use public, large-scale volume electron microscopy data from mouse cortex (MICrONS Explorer, Cortical mm3) to examine how endothelial cell number, endothelial cell thickness, and pericyte coverage relates to microvascular lumen size. We find that transitional vessels near the penetrating arteriole and ascending venule are composed of two to six interlocked endothelial cells, while the capillaries intervening these zones are composed of either one or two endothelial cells, with roughly equal proportions. The luminal area and diameter are on average slightly larger with capillary segments composed of two interlocked endothelial cells vs one endothelial cell. However, this difference is insufficient to explain the full range of capillary diameters seen in vivo. This suggests that both endothelial structure and other influences, including pericyte tone, contribute to the basal diameter and optimized perfusion of brain capillaries.

Meningeal origins and dynamics of perivascular fibroblast development on the mouse cerebral vasculature.

Perivascular fibroblasts (PVFs) are a fibroblast-like cell type that reside on large-diameter blood vessels in the adult meninges and central nervous system (CNS). PVFs drive fibrosis following injury but their homeostatic functions are not well detailed. In mice, PVFs were previously shown to be absent from most brain regions at birth and are only detected postnatally within the cerebral cortex. However, the origin, timing, and cellular mechanisms of PVF development are not known. We used Col1a1-GFP and Col1a2-CreERT transgenic mice to track PVF developmental timing and progression in postnatal mice. Using a combination of lineage tracing and in vivo imaging we show that brain PVFs originate from the meninges and are first seen on parenchymal cerebrovasculature at postnatal day (P)5. After P5, PVF coverage of the cerebrovasculature rapidly expands via mechanisms of local cell proliferation and migration from the meninges, reaching adult levels at P14. Finally, we show that PVFs and perivascular macrophages (PVMs) develop concurrently along postnatal cerebral blood vessels, where the location and depth of PVMs and PVFs highly correlate. These findings provide the first complete timeline for PVF development in the brain, enabling future work into how PVF development is coordinated with cell types and structures in and around the perivascular spaces to support normal CNS vascular function. Summary: Brain perivascular fibroblasts migrate from their origin in the meninges and proliferate locally to fully cover penetrating vessels during postnatal mouse development.

Endothelial structure contributes to heterogeneity in brain capillary diameter.

The high metabolic demand of brain tissue is supported by a constant supply of blood through dense microvascular networks. Capillaries are the smallest class of vessels and vary in diameter between ∼2 to 5 µm in the brain. This diameter range plays a significant role in the optimization of blood flow resistance, blood cell distribution, and oxygen extraction. The control of capillary diameter has largely been ascribed to pericyte contractility, but it remains unclear if endothelial wall architecture also contributes to capillary diameter heterogeneity. Here, we use public, large-scale volume electron microscopy data from mouse cortex (MICrONS Explorer, Cortical MM^3) to examine how endothelial cell number, endothelial cell thickness, and pericyte coverage relates to microvascular lumen size. We find that transitional vessels near the penetrating arteriole and ascending venule are composed of 2 to 5 interlocked endothelial cells, while the numerous capillary segments intervening these zones are composed of either 1 or 2 endothelial cells, with roughly equal proportions. The luminal area and diameter is on average slightly larger with capillary segments composed of 2 interlocked endothelial cells versus 1 endothelial cell. However, this difference is insufficient to explain the full range of capillary diameters seen in vivo. This suggests that both endothelial structure and other influences, such as pericyte tone, contribute to the basal diameter and optimized perfusion of brain capillaries.

Formation and function of the meningeal arachnoid barrier around the developing mouse brain.

The arachnoid barrier, a component of the blood-cerebrospinal fluid barrier (B-CSFB) in the meninges, is composed of epithelial-like, tight-junction-expressing cells. Unlike other central nervous system (CNS) barriers, its' developmental mechanisms and timing are largely unknown. Here, we show that mouse arachnoid barrier cell specification requires the repression of Wnt-ß-catenin signaling and that constitutively active ß-catenin can prevent its formation. We also show that the arachnoid barrier is functional prenatally and, in its absence, a small molecular weight tracer and the bacterium group B Streptococcus can cross into the CNS following peripheral injection. Acquisition of barrier properties prenatally coincides with the junctional localization of Claudin 11, and increased E-cadherin and maturation continues after birth, where postnatal expansion is marked by proliferation and re-organization of junctional domains. This work identifies fundamental mechanisms that drive arachnoid barrier formation, highlights arachnoid barrier fetal functions, and provides novel tools for future studies on CNS barrier development.

Adora2b signaling on bone marrow derived cells dampens myocardial ischemia-reperfusion injury.

BACKGROUND: Cardiac ischemia-reperfusion (I-R) injury represents a major cause of cardiac tissue injury. Adenosine signaling dampens inflammation during cardiac I-R. The authors investigated the role of the adenosine A2b-receptor (Adora2b) on inflammatory cells during cardiac I-R. METHODS: To study Adora2b signaling on inflammatory cells, the authors transplanted wild-type (WT) bone marrow (BM) into Adora2b(-/-) mice or Adora2b(-/-) BM into WT mice. To study the role of polymorphonuclear leukocytes (PMNs), neutrophil-depleted WT mice were treated with an Adora2b agonist. After treatments, mice were exposed to 60 min of myocardial ischemia and 120 min of reperfusion. Infarct sizes and troponin I concentrations were determined by triphenyltetrazolium chloride staining and enzyme-linked immunosorbent assay, respectively. RESULTS: Transplantation of WT BM into Adora2b(-/-) mice decreased infarct sizes by 19 ± 4% and troponin I by 87.5 ± 25.3 ng/ml (mean ± SD, n = 6). Transplantation of Adora2b(-/-) BM into WT mice increased infarct sizes by 20 ± 3% and troponin I concentrations by 69.7 ± 17.9 ng/ml (mean ± SD, n = 6). Studies on the reperfused myocardium revealed PMNs as the dominant cell type. PMN depletion or Adora2b agonist treatment reduced infarct sizes by 30 ± 11% or 26 ± 13% (mean ± SD, n = 4); however, the combination of both did not produce additional cardioprotection. Cytokine profiling showed significantly higher cardiac tumor necrosis factor α concentrations in Adora2b(-/-) compared with WT mice (39.3 ± 5.3 vs. 7.5 ± 1.0 pg/mg protein, mean ± SD, n = 4). Pharmacologic studies on human-activated PMNs revealed an Adora2b-dependent tumor necrosis factor α release. CONCLUSION: Adora2b signaling on BM-derived cells such as PMNs represents an endogenous cardioprotective mechanism during cardiac I-R. The authors' findings suggest that Adora2b agonist treatment during cardiac I-R reduces tumor necrosis factor α release of PMNs, thereby dampening tissue injury.

Distinct features of brain perivascular fibroblasts and mural cells revealed by in vivo two-photon imaging.

Perivascular fibroblasts (PVFs) are recognized for their pro-fibrotic role in many central nervous system disorders. Like mural cells, PVFs surround blood vessels and express Pdgfrß. However, these shared attributes hinder the ability to distinguish PVFs from mural cells. We used in vivo two-photon imaging and transgenic mice with PVF-targeting promoters (Col1a1 or Col1a2) to compare the structure and distribution of PVFs and mural cells in cerebral cortex of healthy, adult mice. We show that PVFs localize to all cortical penetrating arterioles and their offshoots (arteriole-capillary transition zone), as well as the main trunk of only larger ascending venules. However, the capillary zone is devoid of PVF coverage. PVFs display short-range mobility along the vessel wall and exhibit distinct structural features (flattened somata and thin ruffled processes) not seen with smooth muscle cells or pericytes. These findings clarify that PVFs and mural cells are distinct cell types coexisting in a similar perivascular niche.

Corrigendum: In vivo Single Cell Optical Ablation of Brain Pericytes.

[This corrects the article DOI: 10.3389/fnins.2022.900761.].

Public Volume Electron Microscopy Data: An Essential Resource to Study the Brain Microvasculature.

Electron microscopy is the primary approach to study ultrastructural features of the cerebrovasculature. However, 2D snapshots of a vascular bed capture only a small fraction of its complexity. Recent efforts to synaptically map neuronal circuitry using volume electron microscopy have also sampled the brain microvasculature in 3D. Here, we perform a meta-analysis of 7 data sets spanning different species and brain regions, including two data sets from the MICrONS consortium that have made efforts to segment vasculature in addition to all parenchymal cell types in mouse visual cortex. Exploration of these data have revealed rich information for detailed investigation of the cerebrovasculature. Neurovascular unit cell types (including, but not limited to, endothelial cells, mural cells, perivascular fibroblasts, microglia, and astrocytes) could be discerned across broad microvascular zones. Image contrast was sufficient to identify subcellular details, including endothelial junctions, caveolae, peg-and-socket interactions, mitochondria, Golgi cisternae, microvilli and other cellular protrusions of potential significance to vascular signaling. Additionally, non-cellular structures including the basement membrane and perivascular spaces were visible and could be traced between arterio-venous zones along the vascular wall. These explorations revealed structural features that may be important for vascular functions, such as blood-brain barrier integrity, blood flow control, brain clearance, and bioenergetics. They also identified limitations where accuracy and consistency of segmentation could be further honed by future efforts. The purpose of this article is to introduce these valuable community resources within the framework of cerebrovascular research. We do so by providing an assessment of their vascular contents, identifying features of significance for further study, and discussing next step ideas for refining vascular segmentation and analysis.

Three-dimensional ultrastructure of the brain pericyte-endothelial interface.

Pericytes and endothelial cells share membranous interdigitations called "peg-and-socket" interactions that facilitate their adhesion and biochemical crosstalk during vascular homeostasis. However, the morphology and distribution of these ultrastructures have remained elusive. Using a combination of 3D electron microscopy techniques, we examined peg-and-socket interactions in mouse brain capillaries. We found that pegs extending from pericytes to endothelial cells were morphologically diverse, exhibiting claw-like morphologies at the edge of the cell and bouton-shaped swellings away from the edge. Reciprocal endothelial pegs projecting into pericytes were less abundant and appeared as larger columnar protuberances. A large-scale 3D EM data set revealed enrichment of both pericyte and endothelial pegs around pericyte somata. The ratio of pericyte versus endothelial pegs was conserved among the pericytes examined, but total peg abundance was heterogeneous across cells. These data show considerable investment between pericytes and endothelial cells, and provide morphological evidence for pericyte somata as sites of enriched physical and biochemical interaction.

Single-Cell Transcriptomic Analyses of the Developing Meninges Reveal Meningeal Fibroblast Diversity and Function.

The meninges are a multilayered structure composed of fibroblasts, blood and lymphatic vessels, and immune cells. Meningeal fibroblasts secrete a variety of factors that control CNS development, yet strikingly little is known about their heterogeneity or development. Using single-cell sequencing, we report distinct transcriptional signatures for fibroblasts in the embryonic dura, arachnoid, and pia. We define new markers for meningeal layers and show conservation in human meninges. We find that embryonic meningeal fibroblasts are transcriptionally distinct between brain regions and identify a regionally localized pial subpopulation marked by the expression of µ-crystallin. Developmental analysis reveals a progressive, ventral-to-dorsal maturation of telencephalic meninges. Our studies have generated an unparalleled view of meningeal fibroblasts, providing molecular profiles of embryonic meningeal fibroblasts by layer and yielding insights into the mechanisms of meninges development and function.