Abstract 174: Roles of Pericyte NADPH Oxidase 4 in Acute Brain Ischemia

Pericytes exist abundantly in the brain and compose the neurovascular unit. It has been elucidated that pericytes play a key role in the formation and maintenance of the blood-brain barrier (BBB), thus being considered to play significant roles in brain ischemia. The NADPH oxidase (Nox) family proteins are a major source of reactive oxygen species (ROS). We have reported previously that Nox4 is abundantly expressed in pericytes among the Nox family. Our goal was to elucidate the roles of Nox4 in brain pericytes during acute brain ischemia. We confirmed by quantitative PCR that Nox4 was abundantly expressed in human cultured brain microvascular pericytes (HBMPC) and was significantly upregulated by hypoxia. We produced a mouse middle cerebral artery occlusion (MCAO) stroke model and examined the expression of Nox4 in the brain. Immunofluorescent double labeling demonstrated that Nox4 expression was upregulated in microvessels particularly in peri-infarct areas and was co-stained with PDGFRβ, a pericyte marker. In order to elucidate the role of Nox4 in brain pericyte during brain ischemia, we generated mice with human Nox4 overexpression using a promoter of SM22α, a pericyte/smooth muscle cell marker (Tg-Nox4). We confirmed that SM22α was expressed in mouse brain pericytes by co-immunostaining with PDGFRβ. We isolated microvessels from Tg-Nox4 brain and confirmed that human Nox4 mRNA was highly expressed. In MCAO model, the infarct volume was significantly larger in Tg-Nox4 than in littermate controls. Confocal microscopy demonstrated that IgG leakage in peri-infarct areas was significantly increased in Tg-Nox4, suggesting that Nox4 overexpression in pericytes enhanced BBB breakdown during acute brain ischemia. To elucidate the mechanisms, we induced adenovirus-mediated overexpression of Nox4 in HBMPC. We demonstrated that Nox4 overexpression increased NFκB phosphorylation and MMP9 expression in the cells.In conclusion, Nox4 may be a major source of ROS in brain pericytes and is upregulated directly by hypoxia in peri-infarct areas during acute brain ischemia. Pericyte Nox4 may enhance BBB breakdown through the activation of NFκB-MMP9 signaling during acute brain ischemia.

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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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The Neurovascular Unit: Focus on the Regulation of Arterial Smooth Muscle Cells

Current Neurovascular Research ◽

10.2174/1567202616666191026122642 ◽

2020 ◽

Vol 16 (5) ◽

pp. 502-515 ◽

Cited By ~ 1

Author(s):

Patrícia Quelhas ◽

Graça Baltazar ◽

Elisa Cairrao

Keyword(s):

Blood Flow ◽

Smooth Muscle ◽

Smooth Muscle Cells ◽

Blood Vessels ◽

Neurovascular Unit ◽

Muscle Cells ◽

Cerebral Blood Vessels ◽

Mural Cells ◽

Brain Pericytes ◽

The Brain

The neurovascular unit is a physiological unit present in the brain, which is constituted by elements of the nervous system (neurons and astrocytes) and the vascular system (endothelial and mural cells). This unit is responsible for the homeostasis and regulation of cerebral blood flow. There are two major types of mural cells in the brain, pericytes and smooth muscle cells. At the arterial level, smooth muscle cells are the main components that wrap around the outside of cerebral blood vessels and the major contributors to basal tone maintenance, blood pressure and blood flow distribution. They present several mechanisms by which they regulate both vasodilation and vasoconstriction of cerebral blood vessels and their regulation becomes even more important in situations of injury or pathology. In this review, we discuss the main regulatory mechanisms of brain smooth muscle cells and their contributions to the correct brain homeostasis.

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NADPH oxidase 4 and its role in the cardiovascular system

Vascular Biology ◽

10.1530/vb-19-0014 ◽

2019 ◽

Vol 1 (1) ◽

pp. H59-H66

Author(s):

Stephen P Gray ◽

Ajay M Shah ◽

Ioannis Smyrnias

Keyword(s):

Nadph Oxidase ◽

Cardiovascular System ◽

Contractile Function ◽

Cellular Level ◽

Nadph Oxidases ◽

Nadph Oxidase 4 ◽

Specific Effects ◽

Different Types ◽

Electron Reduction ◽

Nox Family

The heart relies on complex mechanisms that provide adequate myocardial oxygen supply in order to maintain its contractile function. At the cellular level, oxygen undergoes one electron reduction to superoxide through the action of different types of oxidases (e.g. xanthine oxidases, uncoupled nitric oxide synthases, NADPH oxidases or NOX). Locally generated oxygen-derived reactive species (ROS) are involved in various signaling pathways including cardiac adaptation to different types of physiological and pathophysiological stresses (e.g. hypoxia or overload). The specific effects of ROS and their regulation by oxidases are dependent on the amount of ROS generated and their specific subcellular localization. The NOX family of NADPH oxidases is a main source of ROS in the heart. Seven distinct Nox isoforms (NOX1–NOX5 and DUOX1 and 2) have been identified, of which NOX1, 2, 4 and 5 have been characterized in the cardiovascular system. For the purposes of this review, we will focus on the effects of NADPH oxidase 4 (NOX4) in the heart.

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Serum Ferritin in Stroke: A Marker of Increased Body Iron Stores or Stroke Severity?

Journal of Cerebral Blood Flow & Metabolism ◽

10.1038/sj.jcbfm.9600140 ◽

2005 ◽

Vol 25 (10) ◽

pp. 1386-1393 ◽

Cited By ~ 13

Author(s):

Emilie Millerot ◽

Anne S Prigent-Tessier ◽

Nathalie M Bertrand ◽

Philippe J-C Faure ◽

Claude M Mossiat ◽

...

Keyword(s):

Brain Ischemia ◽

Infarct Volume ◽

Stroke Severity ◽

Iron Dextran ◽

Vessel Occlusion ◽

Free Iron ◽

Serum Free ◽

Body Iron ◽

Iron Stores ◽

The Brain

To evaluate the effect of body iron stores on the vulnerability of the brain to ischemia, a focal permanent brain ischemia was induced by photothrombotic occlusion of cortical vessels in rats with or without chronic treatment with iron dextran (25 mg iron/kg, every other day for 20 days, intraperitoneally). Iron dextran induced systemic iron overload as evidenced by high ferritin (Ft) (x 5) and total iron levels (x 3) in serum as well as increased Ft expression in the liver and heart. Conversely, neither serum free iron levels nor Ft expression in the brain were changed by iron dextran. Finally, infarct volume was not modified by iron dextran. In addition, induction of ischemia in rats treated with FeCl3 (560 μg iron/kg, intravenously) as a means of increasing serum free iron levels during the ischemic period did not enlarge infarct volume. We then explored the effect of brain ischemia itself on serum Ft by measuring serum Ft before and after induction of brain ischemic insults with different neurologic outcomes in rats (brain embolization with microspheres, photothrombotic occlusion of cortical vessels, four-vessel occlusion). Serum Ft levels were found higher at day 1 after ischemia than before ischemia only in rats subjected to the most severe insult (brain embolization). In conclusion, our study showed that increased body iron stores do not increase the vulnerability of the brain to ischemia and that brain ischemia, if severe, results in the elevation of serum Ft levels.

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Interactions between Amyloid-Β Proteins and Human Brain Pericytes: Implications for the Pathobiology of Alzheimer’s Disease

Journal of Clinical Medicine ◽

10.3390/jcm9051490 ◽

2020 ◽

Vol 9 (5) ◽

pp. 1490 ◽

Cited By ~ 1

Author(s):

Donald J. Alcendor

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Neurovascular Unit ◽

Amyloid Β ◽

Brain Parenchyma ◽

Blood Retinal Barrier ◽

Endothelin 1 ◽

Brain Pericytes ◽

Aging Populations ◽

The Brain

Alzheimer’s disease (AD) is a progressive neurodegenerative disease that is the most common cause of dementia, especially among aging populations. Despite advances in AD research, the underlying cause and the discovery of disease-modifying treatments have remained elusive. Two key features of AD pathology are the aberrant deposition of amyloid beta (amyloid-β or Aβ) proteins in the brain parenchyma and Aβ toxicity in brain pericytes of the neurovascular unit/blood–brain barrier (NVU/BBB). This toxicity induces oxidative stress in pericytes and leads to capillary constriction. The interaction between pericytes and Aβ proteins results in the release of endothelin-1 in the pericytes. Endothelin-1 interacts with ETA receptors to cause pericyte contraction. This pericyte-mediated constriction of brain capillaries can cause chronic hypoperfusion of the brain microvasculature, subsequently leading to the neurodegeneration and cognitive decline observed in AD patients. The interaction between Aβ proteins and brain pericytes is largely unknown and requires further investigation. This review provides an updated overview of the interaction between Aβ proteins with pericytes, one the most significant and often forgotten cellular components of the BBB and the inner blood–retinal barrier (IBRB). The IBRB has been shown to be a window into the central nervous system (CNS) that could allow the early diagnosis of AD pathology in the brain and the BBB using modern photonic imaging systems such as optical coherence tomography (OCT) and two-photon microscopy. In this review, I explore the regulation of Aβ proteins in the brain parenchyma, their role in AD pathobiology, and their association with pericyte function. This review discusses Aβ proteins and pericytes in the ocular compartment of AD patients as well as strategies to rescue or protect pericytes from the effects of Aβ proteins, or to replace them with healthy cells.

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Abstract 114: Neuroprotective Role of Brain Pericytes through PDGFRβ-Akt Signaling in Ischemic Stroke

Stroke ◽

10.1161/str.43.suppl_1.a114 ◽

2012 ◽

Vol 43 (suppl_1) ◽

Author(s):

Koichi Arimura ◽

Tetsuro Ago ◽

Masahiro Kamouchi ◽

Hiroshi Sugimori ◽

Junya Kuroda ◽

...

Keyword(s):

Cell Death ◽

Ischemic Stroke ◽

Infarct Volume ◽

Neurovascular Unit ◽

Neuronal Cell Death ◽

Neuronal Cell ◽

Akt Signaling ◽

Artery Occlusion ◽

Brain Functions ◽

Brain Pericytes

Brain pericytes are a constituent of the neurovascular unit and play various important roles in brain functions, such as regulation of capillary blood flow, maintenance of blood-brain barrier and angiogenesis. Previous reports have elucidated that PDGF-B prevents neuronal cell death during ischemic insults in adult rodent models; however, the detailed mechanisms by which PDGF-B signaling protects neurons from ischemic damage are not fully understood. In the present study, we investigated whether brain pericytes play neuroprotective roles in brain ischemia, using a permanent middle cerebral artery occlusion stroke model (MCAO) and cultured human brain pericytes. Immunohistochemistry revealed that the expression of PDGF receptorβ(PDGFRβ) was induced predominantly in pericytes in peri-infarct areas. PDGF-B induced marked phosphorylation of Akt in cultured pericytes. Consistently, Akt was markedly phosphorylated in the PDGFRβ-expressing pericytes in peri-infarct areas. PDGF-B upregulated the expression of neurotrophins, such as neuronal growth factor (NGF) and neurotrophin-3 (NT-3), through Akt activation in the cultured pericytes. We subjected PDGFRβheterozygous knockout (PDGFRβ+/-) mice to MCAO. Infarct volume, as assessed by MAP2 immunostaining, was significantly greater in PDGFRβ+/- than wild-type mice ( 48% increase at day 7, p < 0.01 , n=5). The number of TUNEL positive apoptotic cells was significantly greater in PDGFRβ+/- mice (54 % increase at day 4, p < 0.001 , n=6). Production of NGF and NT-3 at mRNA and protein levels in infarct areas was significantly decreased in PDGFRβ+/- mice (NGF: 28% decrease, p<0.05, NT-3: 22% decrease, p<0.05). Since it has been established that neurotrophin receptors are induced in peri-infarct areas, the decreases in neurotrophin production may increase apoptotic neuronal cell death in the PDGFRβ+/- mice. In conclusion, brain pericytes may have a direct neuroprotective role through secreting neurotrophins via PDGFRβ-Akt signaling, thereby decreasing infarct volume in ischemic stroke.

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The Blood–Brain Barrier, an Evolving Concept Based on Technological Advances and Cell–Cell Communications

Cells ◽

10.3390/cells11010133 ◽

2021 ◽

Vol 11 (1) ◽

pp. 133

Author(s):

Camille Menaceur ◽

Fabien Gosselet ◽

Laurence Fenart ◽

Julien Saint-Pol

Keyword(s):

Blood Brain Barrier ◽

Current Knowledge ◽

Neurovascular Unit ◽

Cell Communication ◽

Brain Barrier ◽

Blood Brain ◽

Brain Pericytes ◽

History Of ◽

The Brain ◽

Cell Cell

The construction of the blood–brain barrier (BBB), which is a natural barrier for maintaining brain homeostasis, is the result of a meticulous organisation in space and time of cell–cell communication processes between the endothelial cells that carry the BBB phenotype, the brain pericytes, the glial cells (mainly the astrocytes), and the neurons. The importance of these communications for the establishment, maturation and maintenance of this unique phenotype had already been suggested in the pioneering work to identify and demonstrate the BBB. As for the history of the BBB, the evolution of analytical techniques has allowed knowledge to evolve on the cell–cell communication pathways involved, as well as on the role played by the cells constituting the neurovascular unit in the maintenance of the BBB phenotype, and more particularly the brain pericytes. This review summarises the key points of the history of the BBB, from its origin to the current knowledge of its physiology, as well as the cell–cell communication pathways identified so far during its development, maintenance, and pathophysiological alteration.

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NOX4-dependent neuronal autotoxicity and BBB breakdown explain the superior sensitivity of the brain to ischemic damage

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1705034114 ◽

2017 ◽

Vol 114 (46) ◽

pp. 12315-12320 ◽

Cited By ~ 48

Author(s):

Ana I. Casas ◽

Eva Geuss ◽

Pamela W. M. Kleikers ◽

Stine Mencl ◽

Alexander M. Herrmann ◽

...

Keyword(s):

Nadph Oxidase ◽

Ex Vivo ◽

Common Denominator ◽

Ischemic Damage ◽

Ex Vivo Model ◽

Nadph Oxidase 4 ◽

Model Free ◽

Medical Need ◽

Organ Specific ◽

The Brain

Ischemic injury represents the most frequent cause of death and disability, and it remains unclear why, of all body organs, the brain is most sensitive to hypoxia. In many tissues, type 4 NADPH oxidase is induced upon ischemia or hypoxia, converting oxygen to reactive oxygen species. Here, we show in mouse models of ischemia in the heart, brain, and hindlimb that only in the brain does NADPH oxidase 4 (NOX4) lead to ischemic damage. We explain this distinct cellular distribution pattern through cell-specific knockouts. Endothelial NOX4 breaks down the BBB, while neuronal NOX4 leads to neuronal autotoxicity. Vascular smooth muscle NOX4, the common denominator of ischemia within all ischemic organs, played no apparent role. The direct neuroprotective potential of pharmacological NOX4 inhibition was confirmed in an ex vivo model, free of vascular and BBB components. Our results demonstrate that the heightened sensitivity of the brain to ischemic damage is due to an organ-specific role of NOX4 in blood–brain-barrier endothelial cells and neurons. This mechanism is conserved in at least two rodents and humans, making NOX4 a prime target for a first-in-class mechanism-based, cytoprotective therapy in the unmet high medical need indication of ischemic stroke.

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