Single cell genomics for cellular phenotyping: applications in brain disorder

Neurovascular unit (NVU) is an elaborated multicellular brain-vessel-blood interface supporting a controlled blood-brain communication through the selective-permeable blood-brain barrier (BBB) and an adequate neurovascular and neurometabolic coupling. Impairment of NVU during aging and neurologic disorders is accompanied by microvascular dysfunction, BBB opening, neurovascular uncoupling, and neuroinflammation, with deleterious effects on brain microenvironment and neuronal signaling. After stroke, neurons are usually lost in the infarct core and astrocytes become reactive and proliferative, dysregulating the balance between neuronal and non-neuronal cells of the NVU in the lesioned area. In this review, we present major cytological responses of the NVU to cerebral ischemia with an emphasis on the aged brain. Early responses of the neurovascular unit to chronic hypoxia include neutrophils infiltration, brain edema, blood vessel disintegration, astrocytes and endothelial cells proliferation as well as conversion of resident microglia to phagocytic microglia. Later responses include the confinement of the peri-infarcted region by a scar tissue composed mainly of reactive astrocytes and endothelial cells, and angiogenesis. However, the newly formed capillary network is disorganized and the blood vessels are leaky making a successful regeneration of the damaged area, unlikely.

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Screening for Interacting Proteins with Peptide Biomarker of Blood–Brain Barrier Alteration under Inflammatory Conditions

International Journal of Molecular Sciences ◽

10.3390/ijms22094725 ◽

2021 ◽

Vol 22 (9) ◽

pp. 4725

Author(s):

Karina Vargas-Sanchez ◽

Monica Losada-Barragán ◽

Maria Mogilevskaya ◽

Susana Novoa-Herrán ◽

Yehidi Medina ◽

...

Keyword(s):

Endothelial Cells ◽

Blood Brain Barrier ◽

Neurovascular Unit ◽

Specific Interaction ◽

Brain Barrier ◽

Mass Spectrometry Analysis ◽

Peptide Ligand ◽

Inflammatory Conditions ◽

Blood Brain ◽

Potential Biomarker

Neurodegenerative diseases are characterized by increased permeability of the blood–brain barrier (BBB) due to alterations in cellular and structural components of the neurovascular unit, particularly in association with neuroinflammation. A previous screening study of peptide ligands to identify molecular alterations of the BBB in neuroinflammation by phage-display, revealed that phage clone 88 presented specific binding affinity to endothelial cells under inflammatory conditions in vivo and in vitro. Here, we aimed to identify the possible target receptor of the peptide ligand 88 expressed under inflammatory conditions. A cross-link test between phage-peptide-88 with IL-1β-stimulated human hCMEC cells, followed by mass spectrometry analysis, was used to identify the target of peptide-88. We modeled the epitope–receptor molecular interaction between peptide-88 and its target by using docking simulations. Three proteins were selected as potential target candidates and tested in enzyme-linked immunosorbent assays with peptide-88: fibronectin, laminin subunit α5 and laminin subunit β-1. Among them, only laminin subunit β-1 presented measurable interaction with peptide-88. Peptide-88 showed specific interaction with laminin subunit β-1, highlighting its importance as a potential biomarker of the laminin changes that may occur at the BBB endothelial cells under pathological inflammation conditions.

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Human pluripotent stem cell–derived brain pericyte–like cells induce blood-brain barrier properties

Science Advances ◽

10.1126/sciadv.aau7375 ◽

2019 ◽

Vol 5 (3) ◽

pp. eaau7375 ◽

Cited By ~ 40

Author(s):

Matthew J. Stebbins ◽

Benjamin D. Gastfriend ◽

Scott G. Canfield ◽

Ming-Song Lee ◽

Drew Richards ◽

...

Keyword(s):

Stem Cells ◽

Endothelial Cells ◽

Blood Brain Barrier ◽

Neurovascular Unit ◽

Barrier Properties ◽

Cell Types ◽

Brain Barrier ◽

Human Model ◽

Blood Brain ◽

Brain Pericytes

Brain pericytes play important roles in the formation and maintenance of the neurovascular unit (NVU), and their dysfunction has been implicated in central nervous system disorders. While human pluripotent stem cells (hPSCs) have been used to model other NVU cell types, including brain microvascular endothelial cells (BMECs), astrocytes, and neurons, hPSC-derived brain pericyte–like cells have not been integrated into these models. In this study, we generated neural crest stem cells (NCSCs), the embryonic precursor to forebrain pericytes, from hPSCs and subsequently differentiated NCSCs to brain pericyte–like cells. These cells closely resembled primary human brain pericytes and self-assembled with endothelial cells. The brain pericyte–like cells induced blood-brain barrier properties in BMECs, including barrier enhancement and reduced transcytosis. Last, brain pericyte–like cells were incorporated with iPSC-derived BMECs, astrocytes, and neurons to form an isogenic human model that should prove useful for the study of the NVU.

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Human pluripotent stem cell-derived brain pericyte-like cells induce blood-brain barrier properties

10.1101/387100 ◽

2018 ◽

Author(s):

Matthew J. Stebbins ◽

Benjamin D. Gastfriend ◽

Scott G. Canfield ◽

Ming-Song Lee ◽

Drew Richards ◽

...

Keyword(s):

Stem Cells ◽

Endothelial Cells ◽

Blood Brain Barrier ◽

Pluripotent Stem Cells ◽

Neurovascular Unit ◽

Human Pluripotent Stem Cells ◽

Brain Barrier ◽

Blood Brain ◽

Brain Pericytes ◽

The Brain

ABSTRACTBrain pericytes play an important role in the formation and maintenance of the neurovascular unit (NVU), and their dysfunction has been implicated in central nervous system (CNS) disorders. While human pluripotent stem cells (hPSCs) have been used to model other components of the NVU including brain microvascular endothelial cells (BMECs), astrocytes, and neurons, cells having brain pericyte-like phenotypes have not been described. In this study, we generated neural crest stem cells (NCSCs), the embryonic precursor to forebrain pericytes, from human pluripotent stem cells (hPSCs) and subsequently differentiated NCSCs to brain pericyte-like cells. The brain pericyte-like cells expressed marker profiles that closely resembled primary human brain pericytes, and they self-assembled with endothelial cells to support vascular tube formation. Importantly, the brain pericyte-like cells induced blood-brain barrier (BBB) properties in BMECs, including barrier enhancement and reduction of transcytosis. Finally, brain pericyte-like cells were incorporated with iPSC-derived BMECs, astrocytes, and neurons to form an isogenic human NVU model that should prove useful for the study of the BBB in CNS health, disease, and therapy.

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Blood-Brain Barrier Dysfunction in the Detrimental Brain Function

10.5772/intechopen.94572 ◽

2021 ◽

Author(s):

Alejandro Gonzalez-Candia ◽

Nicole K. Rogers ◽

Rodrigo L. Castillo

Keyword(s):

Endothelial Cells ◽

Blood Brain Barrier ◽

Brain Function ◽

Neurovascular Unit ◽

Cell Types ◽

Brain Barrier ◽

Barrier Dysfunction ◽

Microvascular Endothelium ◽

Blood Brain ◽

And Function

The blood circulation interface and the neural tissue feature unique characteristics encompassed by the term blood -brain barrier (BBB). The barrier’s primary functions are maintenance of brain homeostasis, selective transport, and protection, all of them determined by its specialized multicellular structure. The BBB primarily exists at the level of the brain microvascular endothelium; however, endothelial cells are not intrinsically capable of forming a barrier. Indeed, the development of barrier characteristics in cerebral endothelial cells requires coordinated cell–cell interactions and signaling from glial cells (i.e., astrocytes, microglia), pericytes, neurons, and extracellular matrix. Such an intricate relationship implies the existence of a neurovascular unit (NVU). The NVU concept emphasizes that the dynamic BBB response to stressors requires coordinated interactions between various central nervous system (CNS) cell types and structures. Every cell type makes an indispensable contribution to the BBBs integrity, and any cell’s failure or dysfunction might result in the barrier breakdown, with dramatic consequences, such as neuroinflammation and neurodegeneration. This chapter will focus on the structure and function of the BBB and discuss how BBB breakdown causes detrimental brain function.

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Endothelial Cells and Astrocytes: AConcerto en Duoin Ischemic Pathophysiology

International Journal of Cell Biology ◽

10.1155/2012/176287 ◽

2012 ◽

Vol 2012 ◽

pp. 1-16 ◽

Cited By ~ 19

Author(s):

Vincent Berezowski ◽

Andrew M. Fukuda ◽

Roméo Cecchelli ◽

Jérôme Badaut

Keyword(s):

Endothelial Cells ◽

Blood Brain Barrier ◽

Time Window ◽

Neurovascular Unit ◽

Recombinant Tissue Plasminogen Activator ◽

Brain Barrier ◽

Peroxisome Proliferator ◽

Edema Formation ◽

Blood Brain ◽

Peroxisome Proliferator Activated Receptors

The neurovascular/gliovascular unit has recently gained increased attention in cerebral ischemic research, especially regarding the cellular and molecular changes that occur in astrocytes and endothelial cells. In this paper we summarize the recent knowledge of these changes in association with edema formation, interactions with the basal lamina, and blood-brain barrier dysfunctions. We also review the involvement of astrocytes and endothelial cells with recombinant tissue plasminogen activator, which is the only FDA-approved thrombolytic drug after stroke. However, it has a narrow therapeutic time window and serious clinical side effects. Lastly, we provide alternative therapeutic targets for future ischemia drug developments such as peroxisome proliferator- activated receptors and inhibitors of the c-Jun N-terminal kinase pathway. Targeting the neurovascular unit to protect the blood-brain barrier instead of a classical neuron-centric approach in the development of neuroprotective drugs may result in improved clinical outcomes after stroke.

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Development of blood-brain barrier under the modulation of HIF activity in astroglial and neuronal cells in vitro

Biomeditsinskaya Khimiya ◽

10.18097/pbmc20166206664 ◽

2016 ◽

Vol 62 (6) ◽

pp. 664-669 ◽

Cited By ~ 1

Author(s):

V.A. Ruzaeva ◽

A.V. Morgun ◽

E.D. Khilazheva ◽

N.V. Kuvacheva ◽

E.A. Pozhilenkova ◽

...

Keyword(s):

Endothelial Cells ◽

Blood Brain Barrier ◽

Neurovascular Unit ◽

Brain Barrier ◽

Gamma Secretase ◽

Blood Brain ◽

Maturation In Vitro ◽

Elevated Expression ◽

The One

Barriergenesis is the process of maturation of the primary vascular network of the brain responsible for the establishment of the blood-brain barrier. It represents a combination of factors that, on the one hand, contribute to the process of migration and tubulogenesis of endothelial cells (angiogenesis), on the other hand, contribute to the formation of new connections between endothelial cells and other elements of the neurovascular unit. Astrocytes play a key role in barriergenesis, however, mechanisms of their action are still poorly examined. We have studied the effects of HIF-1 modulators acting on the cells of non-endothelial origin (neurons and astrocytes) on the development of the blood-brain barrier in vitro. Application of FM19G11 regulating expression of HIF-1 activity and GSI-1 suppressing gamma-secretase and/or proteasomal activity resulted in the elevated expression of thrombospondins and matrix metalloproteinases in the developing blood-brain barrier. However, it caused the opposite effect on VEGF expression thus promoting barrier maturation in vitro.

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Triglyceride-rich lipoprotein lipolysis products increase blood-brain barrier transfer coefficient and induce astrocyte lipid droplets and cell stress

AJP Cell Physiology ◽

10.1152/ajpcell.00120.2016 ◽

2017 ◽

Vol 312 (4) ◽

pp. C500-C516 ◽

Cited By ~ 16

Author(s):

Linda L. Lee ◽

Hnin H. Aung ◽

Dennis W. Wilson ◽

Steven E. Anderson ◽

John C. Rutledge ◽

...

Keyword(s):

Endothelial Cells ◽

Transfer Coefficient ◽

Blood Brain Barrier ◽

Lipid Droplet ◽

Neurovascular Unit ◽

Droplet Formation ◽

Cell Stress ◽

Brain Barrier ◽

Lipid Droplet Formation ◽

Blood Brain

Elevation of blood triglycerides, primarily as triglyceride-rich lipoproteins (TGRL), has been linked to cerebrovascular inflammation, vascular dementia, and Alzheimer’s disease (AD). Brain microvascular endothelial cells and astrocytes, two cell components of the neurovascular unit, participate in controlling blood-brain barrier (BBB) permeability and regulating neurovascular unit homeostasis. Our studies showed that infusion of high physiological concentrations of TGRL lipolysis products (TGRL + lipoprotein lipase) activate and injure brain endothelial cells and transiently increase the BBB transfer coefficient ( Ki = permeability × surface area/volume) in vivo. However, little is known about how blood lipids affect astrocyte lipid accumulation and inflammation. To address this, we first demonstrated TGRL lipolysis products increased lipid droplet formation in cultured normal human astrocytes. We then evaluated the transcriptional pathways activated in astrocytes by TGRL lipolysis products and found upregulated stress and inflammatory-related genes including activating transcription factor 3 (ATF3), macrophage inflammatory protein-3α (MIP-3α), growth differentiation factor-15 (GDF15), and prostaglandin-endoperoxide synthase 2 (COX2). TGRL lipolysis products also activated the JNK/cJUN/ATF3 pathway, induced endoplasmic reticulum stress protein C/EBP homologous protein (CHOP), and the NF-κB pathway, while increasing secretion of MIP-3α, GDF15, and IL-8. Thus our results demonstrate TGRL lipolysis products increase the BBB transfer coefficient ( Ki), induce astrocyte lipid droplet formation, activate cell stress pathways, and induce secretion of inflammatory cytokines. Our observations are consistent with evidence for lipid-induced neurovascular injury and inflammation, and we, therefore, speculate that lipid-induced astrocyte injury could play a role in cognitive decline.

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Multicellular 3D Neurovascular Unit Model for Assessing Hypoxia and Neuroinflammation Induced Blood-Brain Barrier Dysfunction

Scientific Reports ◽

10.1038/s41598-020-66487-8 ◽

2020 ◽

Vol 10 (1) ◽

Cited By ~ 3

Author(s):

Goodwell Nzou ◽

Robert T. Wicks ◽

Nicole R. VanOstrand ◽

Gehad A. Mekky ◽

Stephanie A. Seale ◽

...

Keyword(s):

Endothelial Cells ◽

Blood Brain Barrier ◽

Inflammatory Cytokine ◽

Disease Modeling ◽

Neurovascular Unit ◽

Brain Barrier ◽

Dynamic Component ◽

Blood Brain ◽

Anti Inflammatory ◽

The Brain

AbstractThe blood-brain barrier (BBB) is a dynamic component of the brain-vascular interface that maintains brain homeostasis and regulates solute permeability into brain tissue. The expression of tight junction proteins between adjacent endothelial cells and the presence of efflux proteins prevents entry of foreign substances into the brain parenchyma. BBB dysfunction, however, is evident in many neurological disorders including ischemic stroke, trauma, and chronic neurodegenerative diseases. Currently, major contributors to BBB dysfunction are not well understood. Here, we employed a multicellular 3D neurovascular unit organoid containing human brain microvascular endothelial cells, pericytes, astrocytes, microglia, oligodendrocytes and neurons to model the effects of hypoxia and neuroinflammation on BBB function. Organoids were cultured in hypoxic chamber with 0.1% O2 for 24 hours. Organoids cultured under this hypoxic condition showed increased permeability, pro-inflammatory cytokine production, and increased oxidative stress. The anti-inflammatory agents, secoisolariciresinol diglucoside and 2-arachidonoyl glycerol, demonstrated protection by reducing inflammatory cytokine levels in the organoids under hypoxic conditions. Through the assessment of a free radical scavenger and an anti-inflammatory endocannabinoid, we hereby report the utility of the model in drug development for drug candidates that may reduce the effects of ROS and inflammation under disease conditions. This 3D organoid model recapitulates characteristics of BBB dysfunction under hypoxic physiological conditions and when exposed to exogenous neuroinflammatory mediators and hence may have potential in disease modeling and therapeutic development.

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Resveratrol Reduces Matrix Metalloproteinase-2 Activity Induced by Oxygen-Glucose Deprivation and Reoxygenation in Human Cerebral Microvascular Endothelial Cells

International Journal for Vitamin and Nutrition Research ◽

10.1024/0300-9831/a000119 ◽

2012 ◽

Vol 82 (4) ◽

pp. 267-274 ◽

Cited By ~ 5

Author(s):

Zahide Cavdar ◽

Mehtap Y. Egrilmez ◽

Zekiye S. Altun ◽

Nur Arslan ◽

Nilgun Yener ◽

...

Keyword(s):

Endothelial Cells ◽

Neuroprotective Effect ◽

Ischemia Reperfusion ◽

Neurovascular Unit ◽

Glucose Deprivation ◽

Matrix Metalloproteinase 2 ◽

Microvascular Endothelial Cells ◽

Oxygen Glucose Deprivation ◽

Microvascular Endothelial

The main pathophysiology in cerebral ischemia is the structural alteration in the neurovascular unit, coinciding with neurovascular matrix degradation. Among the human matrix metalloproteinases (MMPs), MMP-2 and -9, known as gelatinases, are the key enzymes for degrading type IV collagen, which is the major component of the basal membrane that surrounds the cerebral blood vessel. In the present study, we investigated the effects of resveratrol on cytotoxicity, reactive oxygen species (ROS), and gelatinases (MMP-2 and -9) in human cerebral microvascular endothelial cells exposed to 6 hours of oxygen-glucose deprivation and a subsequent 24 hours of reoxygenation with glucose (OGD/R), to mimic ischemia/reperfusion in vivo. Lactate dehydrogenase increased significantly, in comparison to that in the normoxia group. ROS was markedly increased in the OGD/R group, compared to normoxia. Correspondingly, ROS was significantly reduced with 50 μM of resveratrol. The proMMP-2 activity in the OGD/R group showed a statistically significant increase from the control cells. Resveratrol preconditioning decreased significantly the proMMP-2 in the cells exposed to OGD/R in comparison to that in the OGD/R group. Our results indicate that resveratrol regulates MMP-2 activity induced by OGD/R via its antioxidant effect, implying a possible mechanism related to the neuroprotective effect of resveratrol.

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