Cerebral Pericytes and Endothelial Cells Communicate through Inflammasome-Dependent Signals

By upregulation of cell adhesion molecules and secretion of proinflammatory cytokines, cells of the neurovascular unit, including pericytes and endothelial cells, actively participate in neuroinflammatory reactions. As previously shown, both cell types can activate inflammasomes, cerebral endothelial cells (CECs) through the canonical pathway, while pericytes only through the noncanonical pathway. Using complex in vitro models, we demonstrate here that the noncanonical inflammasome pathway can be induced in CECs as well, leading to a further increase in the secretion of active interleukin-1β over that observed in response to activation of the canonical pathway. In parallel, a more pronounced disruption of tight junctions takes place. We also show that CECs respond to inflammatory stimuli coming from both the apical/blood and the basolateral/brain directions. As a result, CECs can detect factors secreted by pericytes in which the noncanonical inflammasome pathway is activated and respond with inflammatory activation and impairment of the barrier properties. In addition, upon sensing inflammatory signals, CECs release inflammatory factors toward both the blood and the brain sides. Consequently, CECs activate pericytes by upregulating their expression of NLRP3 (NOD-, LRR-, and pyrin domain-containing protein 3), an inflammasome-forming pattern recognition receptor. In conclusion, cerebral pericytes and endothelial cells mutually activate each other in inflammation.

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Characterization of human fetal brain endothelial cells reveals barrier properties suitable for in vitro modeling of the BBB with syngenic co-cultures

Journal of Cerebral Blood Flow & Metabolism ◽

10.1177/0271678x17708690 ◽

2017 ◽

Vol 38 (5) ◽

pp. 888-903 ◽

Cited By ~ 10

Author(s):

Allison M Andrews ◽

Evan M Lutton ◽

Lee A Cannella ◽

Nancy Reichenbach ◽

Roshanak Razmpour ◽

...

Keyword(s):

Endothelial Cells ◽

Neurovascular Unit ◽

Fetal Brain ◽

Barrier Properties ◽

Endothelial Activation ◽

Fetal Tissue ◽

In Vitro Models ◽

Adult Brain ◽

Alternative Source

Endothelial cells (ECs) form the basis of the blood–brain barrier (BBB), a physical barrier that selectively restricts transport into the brain. In vitro models can provide significant insight into BBB physiology, mechanisms of human disease pathology, toxicology, and drug delivery. Given the limited availability of primary human adult brain microvascular ECs ( aBMVECs), human fetal tissue offers a plausible alternative source for multiple donors and the opportunity to build syngenic tri-cultures from the same host. Previous efforts to culture fetal brain microvascular ECs ( fBMVECs) have not been successful in establishing mature barrier properties. Using optimal gestational age for isolation and flow cytometry cell sorting, we show for the first time that fBMVECs demonstrate mature barrier properties. fBMVECs exhibited similar functional phenotypes when compared to aBMVECs for barrier integrity, endothelial activation, and gene/protein expression of tight junction proteins and transporters. Importantly, we show that tissue used to culture fBMVECs can also be used to generate a syngenic co-culture, creating a microfluidic BBB on a chip. The findings presented provide a means to overcome previous challenges that limited successful barrier formation by fBMVECs. Furthermore, the source is advantageous for autologous reconstitution of the neurovascular unit for next generation in vitro BBB modeling.

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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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Abstract TP86: The Ischemic Blood-brain Barrier In A Dish: Use Of Patient-derived Stem Cells To Model The Response Of The Neurovascular Unit To Oxygen-glucose Deprivation Stress

Stroke ◽

10.1161/str.48.suppl_1.tp86 ◽

2017 ◽

Vol 48 (suppl_1) ◽

Author(s):

Shyanne Page ◽

Ronak Patel ◽

Abraham Alahmad

Keyword(s):

Cell Death ◽

Ischemic Stroke ◽

Blood Brain Barrier ◽

Barrier Function ◽

Neurovascular Unit ◽

Barrier Properties ◽

Cell Types ◽

Brain Barrier ◽

Blood Brain

The blood-brain barrier (BBB) constitutes a component of the neurovascular unit formed by specialized brain microvascular endothelial cells (BMECs) surrounded by astrocytes, pericytes and neurons. During ischemic stroke injury, the BBB constitutes the first responding element resulting in the opening of the BBB and eventually neural cell death by excitotoxicity. A better understanding of the cellular mechanisms underlying the opening of the BBB during ischemic stroke is essential to identify targets to restore such barrier function after injury. Current in vitro models of the human BBB, based on primary or immortalized BMECs monocultures, display poor barrier properties but also lack one or two cellular components of the neurovascular unit.In this study, we designed an integrative in vitro model of the BBB by generating BMECs, astrocytes and neurons using patient-derived BMECs from two iPSC lines (IMR90-c4 and CTR66M). We were able to obtain all three cell types from these two cell lines. iPSC-derived BMECs showed barrier properties similar or better barrier function than hCMEC/D3 monolayer (an immortalized adult somatic BMEC). Furthermore, iPSC—derived astrocytes were capable to induce barrier properties in BMECs upon co-cultures. whereas iPSC-derived neurons were capable to form extensive and branched neurites. Upon OGD stress, iPSC-derived BMECs showed a disruption of their barrier function as early as 6 hours of OGD stress and showed a complete disruption by 24 hours. Such disruption was reversed by reoxygenation. Interestingly such barrier disruption occurs through a VEGF-independent mechanism. In the other hand, iPSC-derived neurons showed a significant decrease in cell metabolic activity preceding neurites pruning. Finally, astrocytes showed the most robust phenotype, as we noted no cell death by 24 hours OGD.In this study, we demonstrated the ability to differentiate three cell types from the same patient in two iPSC lines. We also demonstrated the ability of these cells to respond to OGD/reoxygenation stress in agreement with the current literature. We are currently investigating the molecular mechanisms by which OGD/reoxygenation drive the cellular response in these cell types.

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Three-Dimensional Biofabrication Models of Endometriosis and the Endometriotic Microenvironment

10.20944/preprints202010.0228.v1 ◽

2020 ◽

Author(s):

Jillian R. H. Wendel ◽

Xiyin Wang ◽

Lester J. Smith ◽

Shannon M. Hawkins

Keyword(s):

Cell Line ◽

Three Dimensional ◽

Uterine Cavity ◽

Building Blocks ◽

Cell Types ◽

In Vitro Models ◽

Inflammatory Factors ◽

Biologically Relevant ◽

Increased Risk

Endometriosis occurs when endometrial-like tissue grows outside the uterine cavity, leading to pelvic pain, infertility, and increased risk of ovarian cancer. The present study describes the optimization and characterization of cellular spheroids as building blocks for Kenzan scaffold-free method biofabrication and proof-of-concept models of endometriosis and the endometriotic microenvironment. The spheroid building blocks must be a specific diameter (~500 m), compact, round, and smooth to withstand Kenzan biofabrication. Under optimized spheroid conditions for biofabrication, the endometriotic epithelial-like cell line, 12Z, expressed high levels of estrogen-related genes and secreted high amounts of endometriotic inflammatory factors that were independent of TNF stimulation. Heterotypic spheroids, composed of 12Z and T-HESC, an immortalized endometrial stromal cell line, self-assembled into a biologically relevant pattern, consisting of epithelial cells on the outside of the spheroids and stromal cells in the core. 12Z spheroids were biofabricated into large three-dimensional constructs alone, with HEYA8 spheroids, or as heterotypic spheroids with T-HESC. These three-dimensional biofabricated constructs containing multiple monotypic or heterotypic spheroids represent the first scaffold-free biofabricated in vitro models of endometriosis and the endometriotic microenvironment. These efficient and innovative models will allow us to study the complex interactions of multiple cell types within a biologically relevant microenvironment.

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Endothelial cellular response to altered shear stress

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.2001.281.3.l529 ◽

2001 ◽

Vol 281 (3) ◽

pp. L529-L533 ◽

Cited By ~ 229

Author(s):

Aron B. Fisher ◽

Shu Chien ◽

Abdul I. Barakat ◽

Robert M. Nerem

Keyword(s):

Endothelial Cells ◽

Shear Stress ◽

Endothelial Cell ◽

Cellular Response ◽

Membrane Lipids ◽

Cell Types ◽

In Vitro Models ◽

Cell Responses ◽

Flow Adaptation

Endothelial cells are normally exposed constantly to mechanical forces that significantly influence their phenotype. This symposium presented recent information concerning endothelial cell responses to shear stress associated with blood flow. Endothelial cell shear stress mechanosensors that have been proposed include membrane receptor kinases, integrins, G proteins, ion channels, intercellular junction proteins, membrane lipids (e.g., those associated with caveolae), and the cytoskeleton. These sensors are linked to signaling cascades that interact with or result in generation of reactive oxygen species, nitric oxide, and various transcription factors among other responses. Endothelial cells adapt to sustained shear stress, and either an increase or decrease from normal shear leads to signaling events. In vitro models for the study of endothelial cell responses must consider the pattern of shear stress (e.g., steady vs. oscillatory flow), the scaffold for cell growth (e.g., basement membrane or other cell types such as smooth muscle cells), and the extent of flow adaptation. These cellular responses have major relevance for understanding the pathophysiological effects of increased shear stress associated with hypertension or decreased shear stress associated with thrombotic occlusion.

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Review of Design Considerations for Brain-on-a-Chip Models

Micromachines ◽

10.3390/mi12040441 ◽

2021 ◽

Vol 12 (4) ◽

pp. 441

Author(s):

Tiffany Cameron ◽

Tanya Bennet ◽

Elyn Rowe ◽

Mehwish Anwer ◽

Cheryl Wellington ◽

...

Keyword(s):

Brain Research ◽

Neurovascular Unit ◽

Cell Monolayer ◽

Cell Types ◽

In Vitro Models ◽

Research Field ◽

Design Development ◽

Organ On A Chip

In recent years, the need for sophisticated human in vitro models for integrative biology has motivated the development of organ-on-a-chip platforms. Organ-on-a-chip devices are engineered to mimic the mechanical, biochemical and physiological properties of human organs; however, there are many important considerations when selecting or designing an appropriate device for investigating a specific scientific question. Building microfluidic Brain-on-a-Chip (BoC) models from the ground-up will allow for research questions to be answered more thoroughly in the brain research field, but the design of these devices requires several choices to be made throughout the design development phase. These considerations include the cell types, extracellular matrix (ECM) material(s), and perfusion/flow considerations. Choices made early in the design cycle will dictate the limitations of the device and influence the end-point results such as the permeability of the endothelial cell monolayer, and the expression of cell type-specific markers. To better understand why the engineering aspects of a microfluidic BoC need to be influenced by the desired biological environment, recent progress in microfluidic BoC technology is compared. This review focuses on perfusable blood–brain barrier (BBB) and neurovascular unit (NVU) models with discussions about the chip architecture, the ECM used, and how they relate to the in vivo human brain. With increased knowledge on how to make informed choices when selecting or designing BoC models, the scientific community will benefit from shorter development phases and platforms curated for their application.

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Combination of Alanine and Glutathione as Targeting Ligands of Nanoparticles Enhances Cargo Delivery into the Cells of the Neurovascular Unit

Pharmaceutics ◽

10.3390/pharmaceutics12070635 ◽

2020 ◽

Vol 12 (7) ◽

pp. 635

Author(s):

Gergő Porkoláb ◽

Mária Mészáros ◽

András Tóth ◽

Anikó Szecskó ◽

András Harazin ◽

...

Keyword(s):

Endothelial Cells ◽

Cultured Cells ◽

Neurological Diseases ◽

Neurovascular Unit ◽

Cell Types ◽

Target Cells ◽

Brain Endothelial Cells ◽

Multiple Ligands ◽

The Brain

Inefficient drug delivery across the blood–brain barrier (BBB) and into target cells in the brain hinders the treatment of neurological diseases. One strategy to increase the brain penetration of drugs is to use vesicular nanoparticles functionalized with multiple ligands of BBB transporters as vehicles. Once within the brain, however, drugs must also be able to reach their therapeutic targets in the different cell types. It is, therefore, favorable if such nanocarriers are designed that can deliver their cargo not only to brain endothelial cells, but to other cell types as well. Here, we show that alanine-glutathione dual-targeting of niosomes enhances the delivery of a large protein cargo into cultured cells of the neurovascular unit, namely brain endothelial cells, pericytes, astrocytes and neurons. Furthermore, using metabolic and endocytic inhibitors, we show that the cellular uptake of niosomes is energy-dependent and is partially mediated by endocytosis. Finally, we demonstate the ability of our targeted nanovesicles to deliver their cargo into astroglial cells after crossing the BBB in vitro. These data indicate that dual-labeling of nanoparticles with alanine and glutathione can potentially be exploited to deliver drugs, even biopharmacons, across the BBB and into multiple cell types in the brain.

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Three-Dimensional Biofabrication Models of Endometriosis and the Endometriotic Microenvironment

Biomedicines ◽

10.3390/biomedicines8110525 ◽

2020 ◽

Vol 8 (11) ◽

pp. 525

Author(s):

Jillian R. H. Wendel ◽

Xiyin Wang ◽

Lester J. Smith ◽

Shannon M. Hawkins

Keyword(s):

Cell Line ◽

Three Dimensional ◽

Uterine Cavity ◽

Building Blocks ◽

Cell Types ◽

In Vitro Models ◽

Inflammatory Factors ◽

Biologically Relevant ◽

Increased Risk

Endometriosis occurs when endometrial-like tissue grows outside the uterine cavity, leading to pelvic pain, infertility, and increased risk of ovarian cancer. The present study describes the optimization and characterization of cellular spheroids as building blocks for Kenzan scaffold-free method biofabrication and proof-of-concept models of endometriosis and the endometriotic microenvironment. The spheroid building blocks must be of a specific diameter (~500 μm), compact, round, and smooth to withstand Kenzan biofabrication. Under optimized spheroid conditions for biofabrication, the endometriotic epithelial-like cell line, 12Z, expressed high levels of estrogen-related genes and secreted high amounts of endometriotic inflammatory factors that were independent of TNFα stimulation. Heterotypic spheroids, composed of 12Z and T-HESC, an immortalized endometrial stromal cell line, self-assembled into a biologically relevant pattern, consisting of epithelial cells on the outside of the spheroids and stromal cells in the core. 12Z spheroids were biofabricated into large three-dimensional constructs alone, with HEYA8 spheroids, or as heterotypic spheroids with T-HESC. These three-dimensional biofabricated constructs containing multiple monotypic or heterotypic spheroids represent the first scaffold-free biofabricated in vitro models of endometriosis and the endometriotic microenvironment. These efficient and innovative models will allow us to study the complex interactions of multiple cell types within a biologically relevant microenvironment.

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Morphilogy and Ultrastructure of in Vitro Cultures of Aortic Endothelial Cells Interacting with Antibodies

10.1055/s-0039-1684767 ◽

1979 ◽

Author(s):

S. Korach ◽

D. Ngo

Keyword(s):

Endothelial Cells ◽

Cell Surface ◽

Vascular Endothelial Cells ◽

Intercellular Junctions ◽

Cell Types ◽

Endothelial Damage ◽

Antibody Concentration ◽

High Yields ◽

Scanning Em

Adult pig aortas, sectioned longitudinally, were incubated in 0.1% collagenase-PBS (15 mn, 37°C). Gentle scraping of the lumenal surface resulted in high yields (3-4 x 106 cell/aorta) of viable endothelial cells, essentially devoid of other cell types by morphological and immunochemical (F VIII-antigen) criteria. Confluent monolayers were incubated for various times (5 mn to 1 wk) with decomplemented rabbit antisera raised against pig endothelial cells. Changes in cell morphology appeared to depend on antibody concentration rather than on duration of contact with antiserum. High concentrations of antiserum (5 to 20%) led to cytoplasmic shredding, bulging of cells and extensive vacuolization, whereas at lower concentrations, cells appeared almost normal. Transmission EM studies by the indirect immunoperoxydase method showed antibodies reacting with unfixed cells to be distributed all over the upper cell surface, in the outer parts of intercellular junctions, and within numerous pinocytotic vesicles. Much weaker reactions could also be seen at the lower cell surface. When viewed under the Scanning EM, antiserum-treated endothelial cells also disclosed antibody concentration-dependent bulging and release of cells from their substrate. In vitro studies of gradual modifications of vascular endothelial cells acted upon by antibodies should provide a better understanding of the structural and biochemical processes underlying endothelial damage and detachment.

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Radiobiological Studies of Microvascular Damage through In Vitro Models: A Methodological Perspective

Cancers ◽

10.3390/cancers13051182 ◽

2021 ◽

Vol 13 (5) ◽

pp. 1182

Author(s):

Luca Possenti ◽

Laura Mecchi ◽

Andrea Rossoni ◽

Veronica Sangalli ◽

Simone Bersini ◽

...

Keyword(s):

Endothelial Cells ◽

In Vitro Models ◽

Microvascular Endothelial Cells ◽

Microvascular Damage ◽

Essential Information ◽

Reliable Model ◽

Critical Issues ◽

Irradiation Scheme ◽

In Vitro Effects

Ionizing radiation (IR) is used in radiotherapy as a treatment to destroy cancer. Such treatment also affects other tissues, resulting in the so-called normal tissue complications. Endothelial cells (ECs) composing the microvasculature have essential roles in the microenvironment’s homeostasis (ME). Thus, detrimental effects induced by irradiation on ECs can influence both the tumor and healthy tissue. In-vitro models can be advantageous to study these phenomena. In this systematic review, we analyzed in-vitro models of ECs subjected to IR. We highlighted the critical issues involved in the production, irradiation, and analysis of such radiobiological in-vitro models to study microvascular endothelial cells damage. For each step, we analyzed common methodologies and critical points required to obtain a reliable model. We identified the generation of a 3D environment for model production and the inclusion of heterogeneous cell populations for a reliable ME recapitulation. Additionally, we highlighted how essential information on the irradiation scheme, crucial to correlate better observed in vitro effects to the clinical scenario, are often neglected in the analyzed studies, limiting the translation of achieved results.

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