VEROCYTOTOXIN-1 (VT-1) INDUCES APOPTOSIS IN HUMAN GLOMERULAR CAPILLARY ENDOTHELIAL CELLS (GCEC) AND MESANGIAL CELLS (MC) IN VITRO. † 2168

Endothelin (ET)-1 is a powerful vasoconstrictor known to be produced and secreted by endothelial cells lining large vessels. Because ET-1 stimulates glomerular mesangial cell contraction, glomerular capillary endothelial cells (GEN), normally situated in close apposition to mesangial cells, were examined for potential ET expression and secretion. Cultured bovine GEN released ET in a time-dependent fashion. ET secretion was significantly stimulated by bradykinin, an agonist known to activate phospholipase C in these cells. Preproendothelin 1 (preproET-1) mRNA levels in GEN rose in a biphasic manner on stimulation with bradykinin. The early increments (at 30 min) were not dependent on new protein synthesis, whereas the late rise (6 h after addition of bradykinin) appeared to be protein synthesis dependent. Neither early or late bradykinin-stimulated preproET-1 mRNA expression in glomerular endothelial cells was due to inhibition of mRNA breakdown. Both phases of preproET-1 mRNA expression were observed with other glomerular endothelial cell calcium-mobilizing agonists, namely thrombin, and were mimicked by the calcium ionophore ionomycin. By contrast, the protein kinase C activator phorbol myristate acetate only enhanced preproET-1 mRNA expression at 30 min and suppressed expression thereafter. It is concluded that GEN have the potential to express and secrete ET-1 in a phospholipase C-regulated fashion. Furthermore, because glomerular mesangial cells respond to this peptide, the findings raise the possibility of paracrine regulation of mesangial cell tone by glomerular endothelial cell-derived ET-1.

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Age influences susceptibility of brain capillary endothelial cells to La Crosse virus infection and cell death

Journal of Neuroinflammation ◽

10.1186/s12974-021-02173-4 ◽

2021 ◽

Vol 18 (1) ◽

Author(s):

Rahul Basu ◽

Vinod Nair ◽

Clayton W. Winkler ◽

Tyson A. Woods ◽

Iain D. C. Fraser ◽

...

Keyword(s):

Endothelial Cells ◽

Cell Death ◽

Virus Infection ◽

La Crosse Virus ◽

Brain Capillary ◽

Adult Mice ◽

Age Related ◽

Capillary Endothelial Cells ◽

The Brain

Abstract Background A key factor in the development of viral encephalitis is a virus crossing the blood-brain barrier (BBB). We have previously shown that age-related susceptibility of mice to the La Crosse virus (LACV), the leading cause of pediatric arbovirus encephalitis in the USA, was associated with the ability of the virus to cross the BBB. LACV infection in weanling mice (aged around 3 weeks) results in vascular leakage in the olfactory bulb/tract (OB/OT) region of the brain, which is not observed in adult mice aged > 6–8 weeks. Thus, we studied age-specific differences in the response of brain capillary endothelial cells (BCECs) to LACV infection. Methods To examine mechanisms of LACV-induced BBB breakdown and infection of the CNS, we analyzed BCECs directly isolated from weanling and adult mice as well as established a model where these cells were infected in vitro and cultured for a short period to determine susceptibility to virus infection and cell death. Additionally, we utilized correlative light electron microscopy (CLEM) to examine whether changes in cell morphology and function were also observed in BCECs in vivo. Results BCECs from weanling, but not adult mice, had detectable infection after several days in culture when taken ex vivo from infected mice suggesting that these cells could be infected in vitro. Further analysis of BCECs from uninfected mice, infected in vitro, showed that weanling BCECs were more susceptible to virus infection than adult BCECs, with higher levels of infected cells, released virus as well as cytopathic effects (CPE) and cell death. Although direct LACV infection is not detected in the weanling BCECs, CLEM analysis of brain tissue from weanling mice indicated that LACV infection induced significant cerebrovascular damage which allowed virus-sized particles to enter the brain parenchyma. Conclusions These findings indicate that BCECs isolated from adult and weanling mice have differential viral load, infectivity, and susceptibility to LACV. These age-related differences in susceptibility may strongly influence LACV-induced BBB leakage and neurovascular damage allowing virus invasion of the CNS and the development of neurological disease.

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Interaction of Neutrophils with Endothelial Cells, Fibroblasts and Their Extracellular Matrices: Microscopic and Computerised Analysis

Alternatives to Laboratory Animals ◽

10.1177/026119298801600111 ◽

1988 ◽

Vol 16 (1) ◽

pp. 48-53

Author(s):

Marina Ziche ◽

Lucia Morbidelli ◽

Annalisa Rubino ◽

Piero Dolara ◽

Stefano Bianchi ◽

...

Keyword(s):

Endothelial Cells ◽

Image Analysis ◽

Vascular Endothelial Cells ◽

Extracellular Matrices ◽

Polymorphonuclear Neutrophil ◽

Initial Event ◽

Computerised Image Analysis ◽

Capillary Endothelial Cells ◽

Vitro Model

Polymorphonuclear neutrophil (PMN) interaction with vascular endothelial cells is the initial event in the migration of neutrophils through blood vessel walls before reaching inflammation sites in tissues. The interaction between fibroblasts and endothelial cells and their extracellular matrices might be modulated by the activation of neutrophils that occurs at inflammatory reaction sites. We have used an in vitro model to study PMN function, measuring the adhesion of human PMNs to capillary endothelial cells and fibroblasts grown in culture and to their extracellular matrices. The interaction was measured in basal conditions and in the presence of the chemotactic effector, formyl-methionyl-leucyl-phenylalanine (FMLP at the concentration of 10 7M). Adhesion was expressed by the number of adherent PMNs/mm2 on a histological specimen. Moreover, we have adapted a program for image analysis to quantify neutrophil adhesion. Three times more PMNs adhered to matrices than to monolayers, and adherence could be increased by the presence of 10-7M FMLP, except in the case of fibroblast monolayers. We found a good correlation between microscopic observation and computerised image analysis measuring PMN adhesiveness to extracellular matrices.

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The role of matrix metalloproteinase activity in the maturation of human capillary endothelial cells in vitro

Journal of Cell Science ◽

10.1242/jcs.112.10.1599 ◽

1999 ◽

Vol 112 (10) ◽

pp. 1599-1609 ◽

Cited By ~ 2

Author(s):

B.M. Kraling ◽

D.G. Wiederschain ◽

T. Boehm ◽

M. Rehn ◽

J.B. Mulliken ◽

...

Keyword(s):

Cell Culture ◽

Cell Proliferation ◽

Endothelial Cells ◽

Endothelial Cell ◽

Endothelial Cell Proliferation ◽

Protein Levels ◽

Matrix Deposition ◽

Capillary Endothelial Cells ◽

Vessel Maturation

Vessel maturation during angiogenesis (the formation of new blood vessels) is characterized by the deposition of new basement membrane and the downregulation of endothelial cell proliferation in the new vessels. Matrix remodeling plays a crucial, but still poorly understood role, in angiogenesis regulation. We present here a novel assay system with which to study the maturation of human capillary endothelial cells in vitro. When human dermal microvascular endothelial cells (HDMEC) were cultured in the presence of dibutyryl cAMP (Bt2) and hydrocortisone (HC), the deposition of a fibrous lattice of matrix molecules consisting of collagens type IV, type XVIII, laminin and thrombospondin was induced. In basal medium (without Bt2 and HC), HDMEC released active matrix metalloproteinases (MMPs) into the culture medium. However, MMP protein levels were significantly reduced by treatment with Bt2 and HC, while protein levels and activity of endogenous tissue inhibitor of MMPs (TIMP) increased. This shift in the proteolytic balance and matrix deposition was inhibited by the specific protein kinase A inhibitors RpcAMP and KT5720 or by substituting analogues without reported glucocorticoid activity for HC. The addition of MMP inhibitors human recombinant TIMP-1 or 1,10-phenanthroline to cultures under basal conditions induced matrix deposition in a dose-dependent manner, which was not observed with the serine protease inhibitor epsilon-amino-n-caproic acid (ACA). The deposited basement membrane-type of matrix reproducibly suppressed HDMEC proliferation and increased HDMEC adhesion to the substratum. These processes of matrix deposition and downregulation of endothelial cell proliferation, hallmarks of differentiating new capillaries in the end of angiogenesis, were recapitulated in our cell culture system by decreasing the matrix-degrading activity. These data suggest that our cell culture assay provides a simple and feasible model system for the study of capillary endothelial cell differentiation and vessel maturation in vitro.

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The Proteome of Brain Capillary Endothelial Cells: Toward a Molecular Characterization of an In Vitro Blood–Brain Barrier Model

Expression Profiling in Neuroscience - Neuromethods ◽

10.1007/978-1-61779-448-3_10 ◽

2011 ◽

pp. 161-179 ◽

Cited By ~ 2

Author(s):

Sophie Duban-Deweer ◽

Christophe Flahaut ◽

Yannis Karamanos

Keyword(s):

Endothelial Cells ◽

Molecular Characterization ◽

Blood Brain Barrier ◽

Brain Barrier ◽

Brain Capillary ◽

Blood Brain Barrier Model ◽

Capillary Endothelial Cells ◽

Barrier Model

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Endothelin stimulates phosphatidylinositol hydrolysis and DNA synthesis in brain capillary endothelial cells

Biochemical Journal ◽

10.1042/bj2660415 ◽

1990 ◽

Vol 266 (2) ◽

pp. 415-420 ◽

Cited By ~ 118

Author(s):

P Vigne ◽

R Marsault ◽

J P Breittmayer ◽

C Frelin

Keyword(s):

Endothelial Cells ◽

Dna Synthesis ◽

Cardiac Cells ◽

Brain Capillary ◽

Aortic Endothelial Cells ◽

Brain Capillary Endothelial Cells ◽

Important Target ◽

Receptor Sites ◽

Capillary Endothelial Cells

Endothelin-1 (ET-1) is a novel vasoconstricting and cardiotonic peptide that is synthesized by the vascular endothelium. Bovine aortic endothelial cells which secrete ET in vitro lack membrane receptor sites for the peptide. Endothelial cells from rat brain microvessels that do not secrete ET in vitro express large amounts of high-affinity receptors for 125I-labelled ET-1 (Kd 0.8 nM). The ET receptor is recognized by sarafotoxin S6b and the different ET peptides with the following order of potency: ET-1 (Kd 0.5 nM) approximately equal to ET-2 (Kd 0.7 nM) greater than sarafotoxin S6b (Kd 27 nM) greater than ET-3 (Kd 450 nM). This structure-activity relationship is different from those found in vascular smooth muscle cells, renal cells and cardiac cells. ET-1 stimulates DNA synthesis in brain capillary endothelial cells. It is more potent than basic fibroblast growth factor. The action of ET on endothelial cells from microvessels involves phosphatidylinositol hydrolysis and intracellular Ca2+ mobilization. These observations suggest that brain endothelial cells might be an important target for ET.

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