scholarly journals Molecular atlas of the human brain vasculature at the single-cell level

2021 ◽  
Author(s):  
Thomas Wälchli ◽  
Moheb Ghobrial ◽  
Marc Schwab ◽  
Shigeki Takada ◽  
Hang Zhong ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Human Brain ◽  
Single Cell ◽  
Mhc Class Ii ◽  
Neurovascular Unit ◽  
Brain Atlas ◽  
Molecular Heterogeneity ◽  
Molecular Architecture ◽  
Adult Control ◽  
Brain Vasculature

A broad range of brain pathologies critically relies on the vasculature, and cerebrovascular disease is a leading cause of death worldwide. However, the cellular and molecular architecture of the human brain vasculature remains poorly understood. Here, we performed single-cell RNA sequencing of 599,215 freshly isolated endothelial, perivascular and other tissue-derived cells from 47 fetuses and adult patients to construct a molecular atlas of the developing fetal, adult control and diseased human brain vasculature. We uncover extensive molecular heterogeneity of healthy fetal and adult human brains and across eight vascular-dependent CNS pathologies including brain tumors and brain vascular malformations. We identify alteration of arteriovenous differentiation and reactivated fetal as well as conserved dysregulated pathways in the diseased vasculature. Pathological endothelial cells display a loss of CNS-specific properties and reveal an upregulation of MHC class II molecules, indicating atypical features of CNS endothelial cells. Cell-cell interaction analyses predict numerous endothelial-to-perivascular cell ligand-receptor crosstalk including immune-related and angiogenic pathways, thereby unraveling a central role for the endothelium within brain neurovascular unit signaling networks. Our single-cell brain atlas provides insight into the molecular architecture and heterogeneity of the developing, adult/control and diseased human brain vasculature and serves as a powerful reference for future studies.

Abstract MP17: Upregulating E2F1 Mitigates Senescence-Associated Phenotypes in Cultured Primary Endothelial Cells

Stroke ◽  
2021 ◽  
Vol 52 (Suppl_1) ◽  
Author(s):  
Michael E Maniskas ◽  
Yun-ju Lai ◽  
Sean P Marrelli ◽  
Louise D McCullough ◽  
Jose F Moruno-manchon
Keyword(s):  
Endothelial Cells ◽  
Vascular Endothelial Cells ◽  
Neurovascular Unit ◽  
Glucose Deprivation ◽  
Cerebral Hypoperfusion ◽  
Aged Mouse ◽  
Aged Mice ◽  
Brain Vasculature ◽  
Bilateral Carotid ◽  
The Brain

Vascular contributions to cognitive impairment and dementia (VCID) includes multiple disorders that are identified by cognitive deficits secondary to cerebrovascular pathology. The risk of VCID is higher in people after the age of 70, and, currently, there is no effective treatment. Vascular endothelial cells (VEC) are critical components of the brain vasculature and neurovascular unit and their health is vital to the capacity of the brain vasculature to respond to stressors. However, aged VEC may enter an irreversible replicative-arrest state (senescence), which has been associated with dementia. E2F transcription factor 1 (E2F1) regulates cell cycle progression and DNA damage repair. Importantly, E2F1 deficiency is associated with cell senescence. We hypothesized that E2F1 downregulation contributes to senescence in the cerebral endothelium during aging. We used cultured primary VEC from young (4-months old, mo) and aged (18-mo) male and female mice for RNA sequencing, plasmid-based gene delivery, high-resolution microscopy, and (4-, 12-, and 18-mo) mice of the bilateral carotid artery stenosis (BCAS) model, which produces chronic cerebral hypoperfusion and recapitulates some of the features seen in patients with VCID. We found that overexpression of E2F1 reduced the levels of senescence-associated phenotypes in cultured VEC from young mice that were exposed to oxygen and glucose deprivation (p<0.001), which induces endothelial senescence. Our RNA seq data showed that the expression of E2f1 was reduced (~40%) in cultured primary VEC from aged mouse brains compared with young cells (p<0.001). E2F1 levels were reduced in the brains of aged mice. Interestingly, we found sex differences in E2F1 levels, with less protein levels (~30%) in males vs females (p<0.05), independently of age. Also, aged BCAS mice (1 month after surgery) had more severe senescence phenotypes, reduced cerebral blood flow, and worse memory deficits compared with control mice (p<0.05). The effect of BCAS was more prominent in aged mice compared with younger (4- and 12-mo) mice. In conclusion , our study identifies E2F1 as a potential regulator of endothelial senescence in mice and highlights the contribution of aging as an important factor in losing endothelial resilience.

scholarly journals Estradiol Inhibits Human Brain Vascular Pericyte Migration Activity: A Functional and Transcriptomic Analysis

Cells ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 2314
Author(s):  
Lisa Kurmann ◽  
Michal Okoniewski ◽  
Raghvendra K. Dubey
Keyword(s):  
Endothelial Cells ◽  
Cell Migration ◽  
Human Brain ◽  
Neurovascular Unit ◽  
Active Role ◽  
Migration Activity ◽  
Akt Phosphorylation ◽  
And Function ◽  
The Impact

Stroke is the third leading cause of mortality in women and it kills twice as many women as breast cancer. A key role in the pathophysiology of stroke plays the disruption of the blood–brain barrier (BBB) within the neurovascular unit. While estrogen induces vascular protective actions, its influence on stroke remains unclear. Moreover, experiments assessing its impact on endothelial cells to induce barrier integrity are non-conclusive. Since pericytes play an active role in regulating BBB integrity and function, we hypothesize that estradiol may influence BBB by regulating their activity. In this study using human brain vascular pericytes (HBVPs) we investigated the impact of estradiol on key pericyte functions known to influence BBB integrity. HBVPs expressed estrogen receptors (ER-α, ER-β and GPER) and treatment with estradiol (10 nM) inhibited basal cell migration but not proliferation. Since pericyte migration is a hallmark for BBB disruption following injury, infection and inflammation, we investigated the effects of estradiol on TNFα-induced PC migration. Importantly, estradiol prevented TNFα-induced pericyte migration and this effect was mimicked by PPT (ER-α agonist) and DPN (ER-β agonist), but not by G1 (GPR30 agonist). The modulatory effects of estradiol were abrogated by MPP and PHTPP, selective ER-α and ER-β antagonists, respectively, confirming the role of ER-α and ER-β in mediating the anti-migratory actions of estrogen. To delineate the intracellular mechanisms mediating the inhibitory actions of estradiol on PC migration, we investigated the role of AKT and MAPK activation. While estradiol consistently reduced the TNFα-induced MAPK and Akt phosphorylation, only the inhibition of MAPK, but not Akt, significantly abrogated the migratory actions of TNFα. In transendothelial electrical resistance measurements, estradiol induced barrier function (TEER) in human brain microvascular endothelial cells co-cultured with pericytes, but not in HBMECs cultured alone. Importantly, transcriptomics analysis of genes modulated by estradiol in pericytes showed downregulation of genes known to increase cell migration and upregulation of genes known to inhibit cell migration. Taken together, our findings provide the first evidence that estradiol modulates pericyte activity and thereby improves endothelial integrity.

scholarly journals Dysfunction of brain pericytes in chronic neuroinflammation

2015 ◽  
Vol 36 (4) ◽  
pp. 794-807 ◽  
Author(s):  
Yuri Persidsky ◽  
Jeremy Hill ◽  
Ming Zhang ◽  
Holly Dykstra ◽  
Malika Winfield ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Growth Factor ◽  
Human Brain ◽  
Transforming Growth Factor ◽  
Smooth Muscle Actin ◽  
Neurovascular Unit ◽  
Brain Endothelial Cells ◽  
Monocyte Migration ◽  
Brain Pericytes ◽  
Hiv 1

Brain pericytes are uniquely positioned within the neurovascular unit to provide support to blood brain barrier (BBB) maintenance. Neurologic conditions, such as HIV-1-associated neurocognitive disorder, are associated with BBB compromise due to chronic inflammation. Little is known about pericyte dysfunction during HIV-1 infection. We found decreased expression of pericyte markers in human brains from HIV-1-infected patients (even those on antiretroviral therapy). Using primary human brain pericytes, we assessed expression of pericyte markers (α1-integrin, α-smooth muscle actin, platelet-derived growth factor-B receptor β, CX-43) and found their downregulation after treatment with tumor necrosis factor-α (TNFα) or interleukin-1 β (IL-1β). Pericyte exposure to virus or cytokines resulted in decreased secretion of factors promoting BBB formation (angiopoietin-1, transforming growth factor-β1) and mRNA for basement membrane components. TNFα and IL-1β enhanced expression of adhesion molecules in pericytes paralleling increased monocyte adhesion to pericytes. Monocyte migration across BBB models composed of human brain endothelial cells and pericytes demonstrated a diminished rate in baseline migration compared to constructs composed only of brain endothelial cells. However, exposure to the relevant chemokine, CCL2, enhanced the magnitude of monocyte migration when compared to BBB models composed of brain endothelial cells only. These data suggest an important role of pericytes in BBB regulation in neuroinflammation.

scholarly journals Spatial cell type composition in normal and Alzheimers human brains is revealed using integrated mouse and human single cell RNA sequencing

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Travis S. Johnson ◽  
Shunian Xiang ◽  
Bryan R. Helm ◽  
Zachary B. Abrams ◽  
Peter Neidecker ◽  
...  
Keyword(s):  
Human Brain ◽  
Single Cell ◽  
Rna Sequencing ◽  
Brain Tissue ◽  
Cell Types ◽  
Brain Atlas ◽  
Cell Type ◽  
Cell Type Composition ◽  
Type Composition ◽  
Single Cell Rna Sequencing

Abstract Single-cell RNA sequencing (scRNA-seq) resolves heterogenous cell populations in tissues and helps to reveal single-cell level function and dynamics. In neuroscience, the rarity of brain tissue is the bottleneck for such study. Evidence shows that, mouse and human share similar cell type gene markers. We hypothesized that the scRNA-seq data of mouse brain tissue can be used to complete human data to infer cell type composition in human samples. Here, we supplement cell type information of human scRNA-seq data, with mouse. The resulted data were used to infer the spatial cellular composition of 3702 human brain samples from Allen Human Brain Atlas. We then mapped the cell types back to corresponding brain regions. Most cell types were localized to the correct regions. We also compare the mapping results to those derived from neuronal nuclei locations. They were consistent after accounting for changes in neural connectivity between regions. Furthermore, we applied this approach on Alzheimer’s brain data and successfully captured cell pattern changes in AD brains. We believe this integrative approach can solve the sample rarity issue in the neuroscience.

scholarly journals Foe or friend? Janus-faces of the neurovascular unit in the formation of brain metastases

2017 ◽  
Vol 38 (4) ◽  
pp. 563-587 ◽  
Author(s):  
Imola Wilhelm ◽  
Csilla Fazakas ◽  
Kinga Molnár ◽  
Attila G Végh ◽  
János Haskó ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Endothelial Cells ◽  
Neurovascular Unit ◽  
Malignant Cells ◽  
Metastasis Formation ◽  
Primary Tumors ◽  
Brain Vasculature ◽  
Metastatic Cells ◽  
The Central Nervous System ◽  
The Brain

Despite the potential obstacle represented by the blood–brain barrier for extravasating malignant cells, metastases are more frequent than primary tumors in the central nervous system. Not only tightly interconnected endothelial cells can hinder metastasis formation, other cells of the brain microenvironment (like astrocytes and microglia) can also be very hostile, destroying the large majority of metastatic cells. However, malignant cells that are able to overcome these harmful mechanisms may benefit from the shielding and even support provided by cerebral endothelial cells, astrocytes and microglia, rendering the brain a sanctuary site against anti-tumor strategies. Thus, cells of the neurovascular unit have a Janus-faced attitude towards brain metastatic cells, being both destructive and protective. In this review, we present the main mechanisms of brain metastasis formation, including those involved in extravasation through the brain vasculature and survival in the cerebral environment.

scholarly journals Comparative analysis of human brain organoids of brainstem and midbrain at single-cell resolution

2020 ◽  
Author(s):  
Kaoru Kinugawa ◽  
Joachim Luginbühl ◽  
Takeshi K. Matsui ◽  
Nobuyuki Eura ◽  
Yoshihiko M. Sakaguchi ◽  
...  
Keyword(s):  
Human Brain ◽  
Single Cell ◽  
Neurological Diseases ◽  
Cell Types ◽  
Molecular Heterogeneity ◽  
The Difference ◽  
Brain Organoid ◽  
Cell Transcriptome ◽  
Unique Cell ◽  
Single Cell Transcriptome

ABSTRACTHuman brain organoids provide us the means to investigate human brain development and neurological diseases, and single-cell RNA-sequencing (scRNA-seq) technologies allow us to identify homologous cell types and the molecular heterogeneity between individual cells. Previously established human brain organoids of brainstem (hBSOs) and midbrain (hMBOs) were analyzed by scRNA-seq, but the difference in cellular composition between these organoids remains unclear. Here, we integrated and compared the single-cell transcriptome of hBSOs and hMBOs. Our analysis demonstrated that the hBSOs and hMBOs contain some unique cell types, including inflammatory and mesenchymal cells. Further comparison of the hBSOs and hMBOs with publicly available scRNA-seq dataset of human fetal midbrain (hMB) showed high similarity in their neuronal components. These results provide new insights into human brain organoid technologies.

scholarly journals Integration of Mouse and Human Single-cell RNA Sequencing Infers Spatial Cell-type Composition in Human Brains

2019 ◽  
Author(s):  
Travis S Johnson ◽  
Zachary B Abrams ◽  
Bryan R Helm ◽  
Peter Neidecker ◽  
Raghu Machiraju ◽  
...  
Keyword(s):  
Human Brain ◽  
Single Cell ◽  
Brain Tissue ◽  
Cellular Localization ◽  
Cell Types ◽  
Brain Atlas ◽  
P Value ◽  
Cell Type ◽  
Human Brain Tissue ◽  
Human Data

Technical advances have enabled the identification of high-resolution cell types within tissues based on single-cell transcriptomics. However, such analyses are restricted in human brain tissue due to the limited number of brain donors. In this study, we integrate mouse and human data to predict cell-type proportions in human brain tissue, and spatially map the resulting cellular composition. By applying feature selection and linear modeling, combinations of human and mouse brain single-cell transcriptomics profiles can be integrated to "fill in" missing information. These combined "in silico chimeric" datasets are used to model the composition of nine cell types in 3,702 human brain samples in six Allen Human Brain Atlas (AHBA) donors. Cell types were spatially consistent regardless of the scRNA-Seq dataset (91% significantly correlated) or AHBA donor (p-value = 4.43E-20 by t-test) used in the model. Importantly, neuron nuclei location and neuron mRNA location were correlated only after accounting for neural connectivity (p-value = 1.26E-10), which supports the notion that gene expression is a better indicator than nuclei location of cellular localization for cells with large and irregularly shaped cell bodies, such as neurons. These results advocate for the integration of mouse and human data in models of brain tissue heterogeneity.

scholarly journals Argonaute-2 protects the neurovascular unit from damage caused by systemic inflammation

2022 ◽  
Vol 19 (1) ◽  
Author(s):  
Marta Machado-Pereira ◽  
Cláudia Saraiva ◽  
Liliana Bernardino ◽  
Ana C. Cristóvão ◽  
Raquel Ferreira
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Primary Cultures ◽  
Neurovascular Unit ◽  
Brain Endothelial Cells ◽  
Neuronal Function ◽  
Nitric Oxide Release ◽  
Argonaute 2 ◽  
Brain Vasculature ◽  
The Brain

Abstract Background The brain vasculature plays a pivotal role in the inflammatory process by modulating the interaction between blood cells and the neurovascular unit. Argonaute-2 (Ago2) has been suggested as essential for endothelial survival but its role in the brain vasculature or in the endothelial–glial crosstalk has not been addressed. Thus, our aim was to clarify the significance of Ago2 in the inflammatory responses elicited by these cell types. Methods Mouse primary cultures of brain endothelial cells, astrocytes and microglia were used to evaluate cellular responses to the modulation of Ago2. Exposure of microglia to endothelial cell-conditioned media was used to assess the potential for in vivo studies. Adult mice were injected intraperitoneally with lipopolysaccharide (LPS) (2 mg/kg) followed by three daily intraperitoneal injections of Ago2 (0.4 nM) to assess markers of endothelial disruption, glial reactivity and neuronal function. Results Herein, we demonstrated that LPS activation disturbed the integrity of adherens junctions and downregulated Ago2 in primary brain endothelial cells. Exogenous treatment recovered intracellular Ago2 above control levels and recuperated vascular endothelial-cadherin expression, while downregulating LPS-induced nitric oxide release. Primary astrocytes did not show a significant change in Ago2 levels or response to the modulation of the Ago2 system, although endogenous Ago2 was shown to be critical in the maintenance of tumor necrosis factor-α basal levels. LPS-activated primary microglia overexpressed Ago2, and Ago2 silencing contained the inflammatory response to some extent, preventing interleukin-6 and nitric oxide release. Moreover, the secretome of Ago2-modulated brain endothelial cells had a protective effect over microglia. The intraperitoneal injection of LPS impaired blood–brain barrier and neuronal function, while triggering inflammation, and the subsequent systemic administration of Ago2 reduced or normalized endothelial, glial and neuronal markers of LPS damage. This outcome likely resulted from the direct action of Ago2 over the brain endothelium, which reestablished glial and neuronal function. Conclusions Ago2 could be regarded as a putative therapeutic agent, or target, in the recuperation of the neurovascular unit in inflammatory conditions.

scholarly journals A method for rapid flow-cytometric isolation of endothelial nuclei and RNA from archived frozen brain tissue

2021 ◽  
Author(s):  
Amy L. Kimble ◽  
Jordan Silva ◽  
Omar M. Omar ◽  
Melissa Murphy ◽  
Jessica A. Hensel ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Human Brain ◽  
Single Cell ◽  
Single Cell Analysis ◽  
Specific Marker ◽  
Bulk Analysis ◽  
Fresh Tissue ◽  
Rna Transcripts ◽  
Tissue Digestion ◽  
Brain Tissues

AbstractEndothelial cells are important contributors to brain development, physiology, and disease. Although RNA sequencing has contributed to the understanding of brain endothelial cell diversity, bulk analysis and single-cell approaches have relied on fresh tissue digestion protocols for the isolation of single endothelial cells and flow cytometry-based sorting on surface markers or transgene expression. These approaches are limited in the analysis of the endothelium in human brain tissues, where fresh samples are difficult to obtain. Here, we developed an approach to examine endothelial RNA expression by using an endothelial-specific marker to isolate nuclei from abundant archived frozen brain tissues. We show that this approach rapidly and reliably extracts endothelial nuclei from frozen mouse brain samples, and importantly, from archived frozen human brain tissues. Furthermore, isolated RNA transcript levels are closely correlated with expression in whole cells from tissue digestion protocols and are enriched in endothelial markers and depleted of markers of other brain cell types. As high-quality RNA transcripts could be obtained from as few as 100 nuclei in archived frozen human brain tissues, we predict that this approach should be useful for both bulk analysis of endothelial RNA transcripts in human brain tissues as well as single-cell analysis of endothelial sub-populations.

scholarly journals Optimization of Claudin-5 and ICAM-1 protein detection by using capillary-based immunoassay method in human brain endothelial cells

2021 ◽  
Vol 5 (10) ◽  
Author(s):  
Nurul Farhana Jufri ◽  
Tharsini Salyam ◽  
Farah Wahida Ibrahim ◽  
Mazlyzam Abdul Latif ◽  
Asmah Hamid
Keyword(s):  
Endothelial Cells ◽  
Human Brain ◽  
Standard Procedure ◽  
Protein Detection ◽  
Brain Endothelial Cells ◽  
Protein Loading ◽  
Brain Vasculature ◽  
Reducing Conditions ◽  
Specific Proteins ◽  
Optimized Conditions

Background: Human brain endothelial cells (HBECs) are part of the blood-brain barrier (BBB). BBB acts as a barrier to control the passage of molecules or materials from the blood into the brain.  Identification of specific proteins changes in their expressions that are related to disease state is important in order to understand the disease mechanism involving brain vasculature. To achieve that, the techniques involve in identifying the proteins of interest must be optimized prior to further investigation. Methodology: In this study, identification of Claudin-5 in HBEC lysates was tested using different sample preparation techniques such as; 1) reducing with Dithiothreitol (DTT) and non-reducing conditions; 2) denaturing by heating at 95°C for 5 minutes or 70°C for 20 minutes and 3) protein loading at 3 and 4 µg. The samples were then subjected to an automated capillary-based immunoassay, Jess. Results and Discussion: The results showed that HBEC samples loaded at 4 µg and heated for 5 minutes at 95°C with DTT produced clearer and intense bands for Claudin-5 identification compared to the other set ups. As reducing condition and denaturing by heated at 95°C for 5 minutes conditions demonstrated good results, the conditions were used to identify ICAM-1 expression at different protein loading (3 and 4 µg). The result demonstrated that HBEC samples heated for 5 minutes at 95°C with DTT and loaded at 4 µg produced a good detection for ICAM-1. Conclusion: These optimized conditions could be served as a standard procedure for further identification of Claudin-5 and ICAM-1 proteins in HBEC using a capillary immunoassay instrument.

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