scholarly journals Neutrophil-Derived Microvesicle Induced Dysfunction of Brain Microvascular Endothelial Cells In Vitro

2019 ◽  
Vol 20 (20) ◽  
pp. 5227 ◽  
Author(s):  
Anjana Ajikumar ◽  
Merete B. Long ◽  
Paul R. Heath ◽  
Stephen B. Wharton ◽  
Paul G. Ince ◽  
...  
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
The Body ◽  
Confocal Imaging ◽  
Microvascular Endothelial Cells ◽  
Microvascular Endothelial Cell ◽  
Brain Microvascular Endothelial Cells ◽  
Microvascular Endothelial

The blood-brain barrier (BBB), composed of brain microvascular endothelial cells (BMEC) that are tightly linked by tight junction (TJ) proteins, restricts the movement of molecules between the periphery and the central nervous system. Elevated systemic levels of neutrophils have been detected in patients with altered BBB function, but the role of neutrophils in BMEC dysfunction is unknown. Neutrophils are key players of the immune response and, when activated, produce neutrophil-derived microvesicles (NMV). NMV have been shown to impact the integrity of endothelial cells throughout the body and we hypothesize that NMV released from circulating neutrophils interact with BMEC and induce endothelial cell dysfunction. Therefore, the current study investigated the interaction of NMV with human BMEC and determined whether they altered gene expression and function in vitro. Using flow cytometry and confocal imaging, NMV were shown to be internalized by the human cerebral microvascular endothelial cell line hCMEC/D3 via a variety of energy-dependent mechanisms, including endocytosis and macropinocytosis. The internalization of NMV significantly altered the transcriptomic profile of hCMEC/D3, specifically inducing the dysregulation of genes associated with TJ, ubiquitin-mediated proteolysis and vesicular transport. Functional studies confirmed NMV significantly increased permeability and decreased the transendothelial electrical resistance (TEER) of a confluent monolayer of hCMEC/D3. These findings indicate that NMV interact with and affect gene expression of BMEC as well as impacting their integrity. We conclude that NMV may play an important role in modulating the permeability of BBB during an infection.

scholarly journals Retroviral transformation of cerebral microvascular endothelial cells: macrophage-like and microvascular endothelial cell properties

Blood ◽  
1991 ◽  
Vol 77 (2) ◽  
pp. 294-305
Author(s):  
DH Robinson ◽  
MK Warren ◽  
LT Liang ◽  
JJ Oprandy ◽  
TB Nielsen ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Self Organization ◽  
Microvascular Endothelial Cells ◽  
Microvascular Endothelial Cell ◽  
Focus Formation ◽  
Transformed Cell ◽  
L Cell ◽  
Microvascular Endothelial

We report that L-cell-conditioned medium (LCM) transforms porcine cerebral microvascular (PCMV) endothelial cells into cells with macrophage-like properties. LCM is known to contain both cytokine(s) and the L-cell virus, a murine retrovirus found in the L929 cell and LCM. Our evidence suggests that both LCM cytokine(s) and the L-cell virus are involved in this PCMV endothelial cell transformation. Criteria for transformation include focus formation, decreased serum requirements for growth, changes in morphology including nonadherence, propagation in suspension culture, and a decreased growth response to stimulation with a known endothelial cell mitogen. Macrophage-like characteristics of this transformed cell, designated as RVTE, include pinocytosis of low-density lipoprotein, Fc receptor-mediated phagocytosis, phagocytosis of bacteria and zymosan, the expression of macrophage enzyme markers, and constitutive production of colony- stimulating factor 1. However, the transformed cell retains several properties of the nontransformed cell including the expression of FVIII:RAg and in vitro self-organization into capillary-like structures. Cloning of RVTE cells clearly shows that both macrophage- like and cerebral microvascular endothelial cell properties are present in the same cell. During self-organization, nontransformed cells express morphologic and functional characteristics classically associated with the macrophage. These findings suggest that some brain capillary pathophysiologies could involve macrophage-like cerebral microvascular endothelial cells. Furthermore, the “reticuloendothelial” phenotypic repertoire expressed by this transformed cerebral microvascular endothelial cell may show that the cerebral capillary endothelial cell in vivo is derived from a hematopoietic and/or phagocytic precursor.

scholarly journals Pneumolysin Is the Main Inducer of Cytotoxicity to Brain Microvascular Endothelial Cells Caused by Streptococcus pneumoniae

2001 ◽  
Vol 69 (2) ◽  
pp. 845-852 ◽  
Author(s):  
Gregor Zysk ◽  
Barbara Katharina Schneider-Wald ◽  
Jae Hyuk Hwang ◽  
Levente Bejo ◽  
Kwang Sik Kim ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Streptococcus Pneumoniae ◽  
Pneumococcal Meningitis ◽  
Primary Cultures ◽  
Cell Monolayer ◽  
Microvascular Endothelial Cells ◽  
Microvascular Endothelial Cell ◽  
Brain Microvascular Endothelial Cells ◽  
Microvascular Endothelial

ABSTRACT In pneumococcal meningitis it is assumed that bacteria cross the blood-brain barrier (BBB), which consists mainly of cerebral endothelial cells. The effect of Streptococcus pneumoniaeon the BBB was investigated with an in vitro BBB model using a human brain microvascular endothelial cell line (HBMEC) and primary cultures of bovine brain microvascular endothelial cells (BBMEC). Within a few hours of incubation with pneumococci, rounding and detachment of the HBMEC were observed, and the transendothelial electrical resistance of the BBMEC monolayer decreased markedly. An S. pneumoniaemutant deficient in pneumolysin did not affect the integrity of the endothelial cell monolayer. Neither cell wall fragments nor isolated pneumococcal cell walls induced changes of endothelial cell morphology. However, purified pneumolysin caused endothelial cell damage comparable to that caused by the viable pneumococci. The cell detachment was dependent on de novo protein synthesis and required the activities of caspase and tyrosine kinases. The results show that pneumolysin is an important component for damaging the BBB and may contribute to the entry of pneumococci into the cerebral compartment and to the development of brain edema in pneumococcal meningitis.

scholarly journals Retroviral transformation of cerebral microvascular endothelial cells: macrophage-like and microvascular endothelial cell properties

Blood ◽  
1991 ◽  
Vol 77 (2) ◽  
pp. 294-305 ◽  
Author(s):  
DH Robinson ◽  
MK Warren ◽  
LT Liang ◽  
JJ Oprandy ◽  
TB Nielsen ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Self Organization ◽  
Microvascular Endothelial Cells ◽  
Microvascular Endothelial Cell ◽  
Focus Formation ◽  
Transformed Cell ◽  
L Cell ◽  
Microvascular Endothelial

Abstract We report that L-cell-conditioned medium (LCM) transforms porcine cerebral microvascular (PCMV) endothelial cells into cells with macrophage-like properties. LCM is known to contain both cytokine(s) and the L-cell virus, a murine retrovirus found in the L929 cell and LCM. Our evidence suggests that both LCM cytokine(s) and the L-cell virus are involved in this PCMV endothelial cell transformation. Criteria for transformation include focus formation, decreased serum requirements for growth, changes in morphology including nonadherence, propagation in suspension culture, and a decreased growth response to stimulation with a known endothelial cell mitogen. Macrophage-like characteristics of this transformed cell, designated as RVTE, include pinocytosis of low-density lipoprotein, Fc receptor-mediated phagocytosis, phagocytosis of bacteria and zymosan, the expression of macrophage enzyme markers, and constitutive production of colony- stimulating factor 1. However, the transformed cell retains several properties of the nontransformed cell including the expression of FVIII:RAg and in vitro self-organization into capillary-like structures. Cloning of RVTE cells clearly shows that both macrophage- like and cerebral microvascular endothelial cell properties are present in the same cell. During self-organization, nontransformed cells express morphologic and functional characteristics classically associated with the macrophage. These findings suggest that some brain capillary pathophysiologies could involve macrophage-like cerebral microvascular endothelial cells. Furthermore, the “reticuloendothelial” phenotypic repertoire expressed by this transformed cerebral microvascular endothelial cell may show that the cerebral capillary endothelial cell in vivo is derived from a hematopoietic and/or phagocytic precursor.

Abstract W P407: Shear Stress and Co-culture with Astrocytes Determine Brain Microvascular Endothelial Cell Phenotype

Stroke ◽  
2015 ◽  
Vol 46 (suppl_1) ◽  
Author(s):  
Sina Salehi Omran ◽  
Fernando Garcia Polite ◽  
Elazer Edelman ◽  
Mercedes Balcells-Camps
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Endothelial Cell ◽  
Immunocytochemical Staining ◽  
Microvascular Endothelial Cells ◽  
Cell Phenotype ◽  
Brain Microvascular Endothelial Cells ◽  
Glut 1 ◽  
Microvascular Endothelial

Introduction: Dementia has classically been identified to be of either vascular or neural origin. These domains overlap and are complementary, thus we consider dementia a disease of the single cerebrovascular unit. Our objective was to generate a modular platform for co-culture of brain microvascular endothelial cells and astrocytes that also incorporates mechanical stresses, and to then use this model of the cerebrovascular unit microenvironment to study the unit in vitro . Hypothesis: We assessed the hypothesis that endothelial health and blood-brain barrier integrity are modulated by shear stress and co-culture with astrocytes. Methods: We lithographed polydimethylsiloxane substrate on Teflon negative molds with subjacent rectangular channels of 0.45 and 2 mm depth for seeding of human brain microvascular cells and astrocytes, respectively, separated by a polytetrafluoroethylene (0.45 μm pore size) membrane under no flow or physiologic flow (6.2 dynes/cm 2 ) for one week. Immunocytochemical staining for glial fibrillary acidic protein and CD31 was simultaneously visualized by confocal microscopy. Cells from each channel were detached via trypsinization, and expression of transport proteins P-glycoprotein (P-gp) and glucose transporter-1 (GLUT-1), in addition to junction proteins zona occludens-1 (ZO-1) and CD31, was measured by Western Blot. Results: We stably co-cultured brain microvascular endothelial cells and astrocytes with no chamber leakage or mixing. CD31 staining revealed endothelial cell alignment to direction of flow. Expression of ZO-1 by endothelial cells increased in presence of flow and co-culture independently, by 1.6-fold in combined conditions relative to static monoculture (p<0.05). For P-gp, the increase in combined conditions was 5.5-fold (p<0.05). GLUT-1 and CD31 levels did not change significantly with co-culture or flow. Conclusion: Cell biology devoid of microenvironmental cues provides limited insight, especially when considering whole tissues, on the impact of disease. A co-culture system that introduces multiple cells, flow, controlled stress and independent visualization and sampling of each cell domain adds deeper understanding and greater value to in vitro biological models and tissue biology.

Isolation and characterization of human and rat cardiac microvascular endothelial cells

1993 ◽  
Vol 264 (2) ◽  
pp. H639-H652 ◽  
Author(s):  
M. Nishida ◽  
W. W. Carley ◽  
M. E. Gerritsen ◽  
O. Ellingsen ◽  
R. A. Kelly ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Microvascular Endothelial Cells ◽  
Microvascular Endothelium ◽  
Adult Rat ◽  
Isolation And Characterization ◽  
Ventricular Tissue ◽  
Microvascular Endothelial ◽  
Cardiac Microvascular Endothelial Cells

Although reciprocal intercellular signaling may occur between endocardial or microvascular endothelium and cardiac myocytes, suitable in vitro models have not been well characterized. In this report, we describe the isolation and primary culture of cardiac microvascular endothelial cells (CMEC) from both adult rat and human ventricular tissue. Differential uptake of fluorescently labeled acetylated low-density lipoprotein (Ac-LDL) indicated that primary isolates of rat CMEC were quite homogeneous, unlike primary isolates of human ventricular tissue, which required cell sorting based on Ac-LDL uptake to create endothelial cell-enriched primary cultures. The endothelial phenotype of both primary isolates and postsort subcultured CMEC and their microvascular origin were determined by characteristic histochemical staining for a number of endothelial cell-specific markers, by the absence of cells with fibroblast or pericyte-specific cell surface antigens, and by rapid tube formation on purified basement membrane preparations. Importantly, [3H]-thymidine uptake was increased 2.3-fold in subconfluent rat microvascular endothelial cells 3 days after coculture with adult rat ventricular myocytes because of release of an endothelial cell mitogen(s) into the extracellular matrix, resulting in a 68% increase in cell number compared with CMEC in monoculture. Thus biologically relevant cell-to-cell interactions can be modeled with this in vitro system.

Hydroxysafflor yellow A promotes angiogenesis in rat brain microvascular endothelial cells injured by oxygen-glucose deprivation/reoxygenation(OGD/R) through SIRT1-HIF-1α-VEGFA signaling pathway

2021 ◽  
Vol 18 ◽  
Author(s):  
Juxuan Ruan ◽  
Lei Wang ◽  
Jiheng Dai ◽  
Jing Li ◽  
Ning Wang ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Growth Factor ◽  
Signaling Pathway ◽  
Ischemia Reperfusion ◽  
Tube Formation ◽  
Microvascular Endothelial Cells ◽  
Brain Microvascular Endothelial Cells ◽  
Hydroxysafflor Yellow A ◽  
Microvascular Endothelial

Objective: Angiogenesis led by brain microvascular endothelial cells (BMECs) contributes to the remission of brain injury after brain ischemia reperfusion. In this study, we investigated the effects of hydroxysafflor yellow A(HSYA) on angiogenesis of BMECs injured by OGD/R via SIRT1-HIF-1α-VEGFA signaling pathway. Methods: The OGD/R model of BMECs was established in vitro by OGD for 2h and reoxygenation for 24h. At first, the concentrations of vascular endothelial growth factor (VEGF), Angiopoietin (ang) and platelet-derived growth factor (PDGF) in supernatant were detected by ELISA, and the proteins expression of VEGFA, Ang-2 and PDGFB in BMECs were tested by western blot; the proliferation, adhesion, migration (scratch healing and transwell) and tube formation experiment of BMECs; the expression of CD31 and CD34 were tested by immunofluorescence staining. The levels of sirtuin1(SIRT1), hypoxia-inducible factor-1α (HIF-1α), VEGFA mRNA and protein were tested. Results: HSYA up-regulated the levels of VEGF, Ang and PDGF in the supernatant of BMECs under OGD/R, and the protein expression of VEGFA, Ang-2 and PDGFB were increased; HSYA could significantly alleviate the decrease of cell proliferation, adhesion, migration and tube formation ability of BMECs during OGD/R; HSYA enhanced the fluorescence intensity of CD31 and CD34 of BMECs during OGD/R; HSYA remarkably up-regulated the expression of SIRT1, HIF-1α, VEGFA mRNA and protein after OGD/R, and these increase decreased after SIRT1 was inhibited. Conclusion: SIRT1-HIF-1α-VEGFA signaling pathway is involved in HSYA improves angiogenesis of BMECs injured by OGD/R.

Anthropogenic Iron Oxide Nanoparticles Induce Damage to Brain Microvascular Endothelial Cells Forming the Blood-Brain Barrier

2021 ◽  
Author(s):  
Lorena Gárate-Vélez ◽  
Claudia Escudero-Lourdes ◽  
Daniela Salado-Leza ◽  
Armando González-Sánchez ◽  
Ildemar Alvarado-Morales ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Blood Brain Barrier ◽  
Brain Barrier ◽  
Microvascular Endothelial Cells ◽  
Brain Microvascular Endothelial Cells ◽  
Blood Brain ◽  
Fe3o4 Nps ◽  
Microvascular Endothelial ◽  
The Brain

Background: Iron nanoparticles, mainly in magnetite phase (Fe3O4 NPs), are released to the environment in areas with high traffic density and braking frequency. Fe3O4 NPs were found in postmortem human brains and are assumed to get directly into the brain through the olfactory nerve. However, these pollution-derived NPs may also translocate from the lungs to the bloodstream and then, through the blood-brain barrier (BBB), into the brain inducing oxidative and inflammatory responses that contribute to neurodegeneration. Objective: To describe the interaction and toxicity of pollution-derived Fe3O4 NPs on primary rat brain microvascular endothelial cells (rBMECs), main constituents of in vitro BBB models. Methods: Synthetic bare Fe3O4 NPs that mimic the environmental ones (miFe3O4) were synthesized by co-precipitation and characterized using complementary techniques. The rBMECs were cultured in Transwell® plates. The NPs-cell interaction was evaluated through transmission electron microscopy and standard colorimetric in vitro assays. Results: The miFe3O4 NPs, with a mean diameter of 8.45 ± 0.14 nm, presented both magnetite and maghemite phases, and showed super-paramagnetic properties. Results suggest that miFe3O4 NPs are internalized by rBMECs through endocytosis and that they are able to cross the cells monolayer. The lowest miFe3O4 NPs concentration tested induced mid cytotoxicity in terms of 1) membrane integrity (LDH release) and 2) metabolic activity (MTS transformation). Conclusion: Pollution-derived Fe3O4 NPs may interact and cross the microvascular endothelial cells forming the BBB and cause biological damage.

scholarly journals Single Domain Antibodies Targeting Receptor Binding Pockets of NadA Restrain Adhesion of Neisseria meningitidis to Human Brain Microvascular Endothelial Cells

2020 ◽  
Vol 7 ◽  
Author(s):  
Amod Kulkarni ◽  
Evelína Mochnáčová ◽  
Petra Majerova ◽  
Ján Čurlík ◽  
Katarína Bhide ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Phage Display ◽  
Human Brain ◽  
Receptor Binding ◽  
Phage Library ◽  
Microvascular Endothelial Cells ◽  
Brain Microvascular Endothelial Cells ◽  
Binding Pockets ◽  
Microvascular Endothelial

Neisseria adhesin A (NadA), one of the surface adhesins of Neisseria meningitides (NM), interacts with several cell types including human brain microvascular endothelial cells (hBMECs) and play important role in the pathogenesis. Receptor binding pockets of NadA are localized on the globular head domain (A33 to K69) and the first coiled-coil domain (L121 to K158). Here, the phage display was used to develop a variable heavy chain domain (VHH) that can block receptor binding sites of recombinant NadA (rec-NadA). A phage library displaying VHH was panned against synthetic peptides (NadA-gdA33−K69 or NadA-ccL121−K158), gene encoding VHH was amplified from bound phages and re-cloned in the expression vector, and the soluble VHHs containing disulfide bonds were overexpressed in the SHuffle E. coli. From the repertoire of 96 clones, two VHHs (VHHF3–binding NadA-gdA33−K69 and VHHG9–binding NadA-ccL121−K158) were finally selected as they abrogated the interaction between rec-NadA and the cell receptor. Preincubation of NM with VHHF3 and VHHG9 significantly reduced the adhesion of NM on hBMECs in situ and hindered the traversal of NM across the in-vitro BBB model. The work presents a phage display pipeline with a single-round of panning to select receptor blocking VHHs. It also demonstrates the production of soluble and functional VHHs, which blocked the interaction between NadA and its receptor, decreased adhesion of NM on hBMECs, and reduced translocation of NM across BBB in-vitro. The selected NadA blocking VHHs could be promising molecules for therapeutic translation.

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