VEROCYTOTOXIN-1 (VT-1) INDUCTION OF APOPTOSIS IN HUMAN GLOMERULAR CAPILLARY ENDOTHELIAL CELLS (GCEC) AND MESANGIAL CELLS (MC) IN VITRO AND IN VIVO.† 1671

Capillary endothelial cells of rat epididymal fat pad were isolated and cultured in media conditioned by bovine aortic endothelial cells and substrata consisting of interstitial or basement membrane collagens. When these cells were grown on interstitial collagens they underwent proliferation, formed a continuous cell layer and, if cultured for long periods of time, formed occasional tubelike structures. In contrast, when these cells were grown on basement membrane collagens, they did not proliferate but did aggregate and form tubelike structures at early culture times. In addition, cells grown on basement membrane substrata expressed more basement membrane constituents as compared with cells grown on interstitial matrices when assayed by immunoperoxidase methods and quantitated by enzyme-linked immunosorbent inhibition assays. Furthermore, when cells were grown on either side of washed, acellular amnionic membranes their phenotypes were markedly different. On the basement membrane surface they adhered, spread, and formed tubelike structures but did not migrate through the basement membrane. In contrast, when seeded on the stromal surface, these cells were observed to proliferate and migrate into the stromal aspect of the amnion and ultimately formed tubelike structures at high cell densities at longer culture periods (21 d). Thus, connective tissue components play important roles in regulating the phenotypic expression of capillary endothelial cells in vitro, and similar roles of the collagenous components of the extracellular matrix may exist in vivo following injury and during angiogenesis. Furthermore, the culture systems outlined here may be of use in the further study of differentiated, organized capillary endothelial cells in culture.

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Endothelial diaphragmed fenestrae: in vitro modulation by phorbol myristate acetate.

The Journal of Cell Biology ◽

10.1083/jcb.102.5.1965 ◽

1986 ◽

Vol 102 (5) ◽

pp. 1965-1970 ◽

Cited By ~ 20

Author(s):

T Lombardi ◽

R Montesano ◽

M B Furie ◽

S C Silverstein ◽

L Orci

Keyword(s):

Endothelial Cells ◽

Cell Surface ◽

Phorbol Myristate Acetate ◽

Freeze Fracture ◽

Tumor Promoter ◽

Microvascular Endothelial Cells ◽

Myristate Acetate ◽

Capillary Endothelial Cells

Cultured microvascular endothelial cells isolated from fenestrated capillaries have been shown to express many properties of their in vivo differentiated phenotype, yet they contain very few diaphragmed fenestrae. We show here that treatment of capillary endothelial cells with the tumor promoter, 4 beta-phorbol 12-myristate 13-acetate, induces more than a fivefold increase in the frequency of fenestrae per micron 2 of cell surface, as determined from a quantitative evaluation on freeze-fracture replicas. In quick-frozen, deep-etched preparations, the endothelial fenestrae appeared to be bridged by a diaphragm composed of radial fibers interweaving in a central mesh, as previously observed in vivo. These results indicate that diaphragmed fenestrae are inducible structures, and provide an opportunity to study them in vitro.

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In vitro rapid organization of endothelial cells into capillary-like networks is promoted by collagen matrices.

The Journal of Cell Biology ◽

10.1083/jcb.97.5.1648 ◽

1983 ◽

Vol 97 (5) ◽

pp. 1648-1652 ◽

Cited By ~ 428

Author(s):

R Montesano ◽

L Orci ◽

P Vassalli

Keyword(s):

Endothelial Cells ◽

Three Dimensional ◽

Collagen Matrix ◽

Collagen Gels ◽

Collagen Matrices ◽

Narrow Lumen ◽

Capillary Endothelial Cells ◽

Vitro Model

We have studied the behavior of cloned capillary endothelial cells grown inside a three dimensional collagen matrix. Cell monolayers established on the surface of collagen gels were covered with a second layer of collagen. This induced the monolayers of endothelial cells to reorganize into a network of branching and anastomosing capillary-like tubes. As seen by electron microscopy, the tubes were formed by at least two cells (in transverse sections) delimiting a narrow lumen. In addition, distinct basal lamina material was present between the abluminal face of the endothelial cells and the collagen matrix. These results showed that capillary endothelial cells have the capacity to form vessel-like structures with well-oriented cell polarity in vitro. They also suggest that an appropriate topological relationship of endothelial cells with collagen matrices, similar to that occurring in vivo, has an inducive role on the expression of this potential. This culture system provides a simple in vitro model for studying the factors involved in the formation of new blood vessels (angiogenesis).

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Mesangial Cells and Glomerular Endothelial Cells Crosstalk in Rat Anti-Thy-1 Nephritis through VEGFA/VEGFAR2 and Angpt2/Tie2 pathway

10.21203/rs.3.rs-96698/v1 ◽

2020 ◽

Author(s):

yinghua zhao ◽

Bo Fu ◽

Pu Chen ◽

Qinggang Li ◽

Qing Ouyang ◽

...

Keyword(s):

Endothelial Cells ◽

Signaling Pathway ◽

Mesangial Cells ◽

Proliferative Glomerulonephritis ◽

Mesangial Proliferative Glomerulonephritis ◽

Surface Receptor ◽

Mimic Peptide ◽

Specific Signaling Pathway

Abstract BACKGROUNDMesangial proliferative glomerulonephritis is characterized by the proliferation of mesangial cells (MCs). Endothelial cells (ECs) are affected by signals from MCs, resulting in capillary proliferation, but the specific signaling pathway associated with this activity remains unclear.RESULTSIn this study, the expression of PCNA, RECA-1 and CD34 in the glomeruli increased on the 7th day after anti-Thy-1 nephritis establishment, indicating the occurrence of ECs proliferation. After coculturing MCs and ECs in vitro, we observed that activated MCs could promote ECs proliferation, migration and α-SMA expression. Moreover, activated ECs had the same effects on MCs. RT-qPCR showed that activated MCs could increasingly secrete VEGFA, and Angpt2 expression in VEGFA-activated ECs was enhanced. Considering that Angpt2-mediated inhibition of ECs surface receptor Tie2 phosphorylation causes ECs proliferation, we hypothesized that VEGFA/VEGFR2 and Angpt2/Tie2 signaling is involved in the interaction between MCs and ECs. Our results showed that blocking VEGFA or adding the Angpt2 antagonist Angpt1 to the coculture system decreased the number of EdU-positive cells，Angpt2，p-VEGFR2 and p-MAPK expression, but increased p-Tie2 in ECs. To determine whether Angpt1 could effectively alleviate the pathological changes of anti-Thy-1 nephritis, we performed Vasculotide (Angpt1 mimic peptide) treatment assays in vivo. The results confirmed that the addition of Vasculotide could effectively reduce PCNA, RECA-1 and α-SMA expression and promote p-Tie2.CONCLUSIONIn summary, the study showed that the VEGFA/VEGFR2 and Angpt2/Tie2 signaling pathway mediate interactions between MCs and ECs, providing an important theoretical basis for the treatment of mesangial proliferative glomerulonephritis.

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Intracellular diaphragmed fenestrae in cultured capillary endothelial cells

Journal of Cell Science ◽

10.1242/jcs.89.3.441 ◽

1988 ◽

Vol 89 (3) ◽

pp. 441-447 ◽

Cited By ~ 1

Author(s):

R. Montesano ◽

L. Orci

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Three Dimensional ◽

Collagen Matrix ◽

Cell Cytoplasm ◽

Experimental Conditions ◽

Intracellular Location ◽

Capillary Endothelial Cells

The endothelium of visceral capillaries is characterized by the occurrence of numerous fenestrae, which are usually bridged by a thin, single-layered diaphragm. Both in vivo and in vitro, diaphragmed fenestrae perforate the endothelial cell cytoplasm in the most attenuated regions of the cell. We report here that in capillary endothelial cells grown under experimental conditions promoting the development of intracellular lumina (for example, suspension within a three-dimensional collagen matrix), diaphragmed fenestrae can form in a unique, previously undescribed intracellular location - that is, within thin cytoplasmic septa separating contiguous luminal compartments.

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Regulated expression of endothelin 1 in glomerular capillary endothelial cells

AJP Renal Physiology ◽

10.1152/ajprenal.1991.261.1.f117 ◽

1991 ◽

Vol 261 (1) ◽

pp. F117-F125 ◽

Cited By ~ 9

Author(s):

P. A. Marsden ◽

D. M. Dorfman ◽

T. Collins ◽

B. M. Brenner ◽

S. H. Orkin ◽

...

Keyword(s):

Endothelial Cells ◽

Protein Synthesis ◽

Endothelial Cell ◽

Mrna Expression ◽

Phospholipase C ◽

Mesangial Cell ◽

Mesangial Cells ◽

Glomerular Endothelial Cell ◽

Glomerular Capillary ◽

Capillary Endothelial Cells

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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A tissue factor-cascade-targeted nanoparticle forsite-directed inducing thrombosis

Journal of Biomaterials Applications ◽

10.1177/0885328217722740 ◽

2017 ◽

Vol 32 (3) ◽

pp. 342-348

Author(s):

Weiwei Jin ◽

Yanxue Yin ◽

Bo Zhang ◽

Heng Mei ◽

Huafang Wang ◽

...

Keyword(s):

Endothelial Cells ◽

Tissue Factor ◽

Three Dimensional ◽

In Vitro Study ◽

Fluorescent Imaging ◽

Brain Capillary ◽

Capillary Endothelial Cells ◽

Expected Position

Tissue factor is an upstream component of the cascade and a high-expressing factor under phathological conditions. In this study, a tissue factor cascade-targeted strategy for inducing local thrombosis was developed by combining ENP-HMME and photochemistry. In vitro study showed that protein EGFP-EGF1 conjugation to the nanoparticles could significantly contribute to the uptake of nanoparticles by tissue factor over-expressed brain capillary endothelial cells. Three-dimensional imaging and specklegram of brains in vivo showed that tissue factor cascade-targeted strategy successfully induced thrombosis of expected position. As shown by the in vivo multispectral fluorescent imaging, when ENP-HMME was combined with photochemistry, higher accumulation in the infarction hemisphere was observed, which might suggest that the photochemistry inducing tissue factor cascade recruited more ENP-HMME than HMME-loaded nanoparticles (NP-HMME). The data indicated the tissue factor cascade-targeted strategy has potential to induce local thrombosis, and might be applied in the treatment of a variety of hypervascular diseases.

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Desmopressin (DDAVP) Enhances Platelet Adhesion to the Extracellular Matrix of Cultured Human Endothelial Cells through Increased Expression of Tissue Factor

Thrombosis and Haemostasis ◽

10.1055/s-0038-1656088 ◽

1997 ◽

Vol 77 (05) ◽

pp. 0975-0980 ◽

Cited By ~ 23

Author(s):

Angel Gálvez ◽

Goretti Gómez-Ortiz ◽

Maribel Díaz-Ricart ◽

Ginés Escolar ◽

Rogelio González-Sarmiento ◽

...

Keyword(s):

Extracellular Matrix ◽

Endothelial Cells ◽

Tissue Factor ◽

Platelet Adhesion ◽

Umbilical Vein ◽

Procoagulant Activity ◽

Experimental Conditions ◽

Chromogenic Assay

SummaryThe effect of desmopressin (DDAVP) on thrombogenicity, expression of tissue factor and procoagulant activity (PCA) of extracellular matrix (ECM) generated by human umbilical vein endothelial cells cultures (HUVEC), was studied under different experimental conditions. HUVEC were incubated with DDAVP (1, 5 and 30 ng/ml) and then detached from their ECM. The reactivity towards platelets of this ECM was tested in a perfusion system. Coverslips covered with DD A VP-treated ECMs were inserted in a parallel-plate chamber and exposed to normal blood anticoagulated with low molecular weight heparin (Fragmin®, 20 U/ml). Perfusions were run for 5 min at a shear rate of 800 s1. Deposition of platelets on ECMs was significantly increased with respect to control ECMs when DDAVP was used at 5 and 30 ng/ml (p <0.05 and p <0.01 respectively). The increase in platelet deposition was prevented by incubation of ECMs with an antibody against human tissue factor prior to perfusion. Immunofluorescence studies positively detected tissue factor antigen on DDAVP derived ECMs. A chromogenic assay performed under standardized conditions revealed a statistically significant increase in the procoagulant activity of the ECMs produced by ECs incubated with 30 ng/ml DDAVP (p <0.01 vs. control samples). Northern blot analysis revealed increased levels of tissue factor mRNA in extracts from ECs exposed to DDAVP. Our data indicate that DDAVP in vitro enhances platelet adhesion to the ECMs through increased expression of tissue factor. A similar increase in the expression of tissue factor might contribute to the in vivo hemostatic effect of DDAVP.

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Epinephrine Induces von Willebrand Factor Release from Cultured Endothelial Cells: Involvement of Cyclic AMP-dependent Signalling in Exocytosis

Thrombosis and Haemostasis ◽

10.1055/s-0038-1656135 ◽

1997 ◽

Vol 77 (06) ◽

pp. 1182-1188 ◽

Cited By ~ 74

Author(s):

Ulrich M Vischer ◽

Claes B Wollheinn

Keyword(s):

Endothelial Cells ◽

Adenylate Cyclase ◽

Von Willebrand Factor ◽

Umbilical Vein ◽

Free Calcium ◽

Camp Content ◽

Von Willebrand ◽

Willebrand Factor

Summaryvon Willebrand factor (vWf) is released from endothelial cell storage granules after stimulation with thrombin, histamine and several other agents that induce an increase in cytosolic free calcium ([Ca2+]i). In vivo, epinephrine and the vasopressin analog DDAVP increase vWf plasma levels, although they are thought not to induce vWf release from endothelial cells in vitro. Since these agents act via a cAMP-dependent pathway in responsive cells, we examined the role of cAMP in vWf secretion from cultured human umbilical vein endothelial cells. vWf release increased by 50% in response to forskolin, which activates adenylate cyclase. The response to forskolin was much stronger when cAMP degradation was blocked with IBMX, an inhibitor of phosphodiesterases (+200%), whereas IBMX alone had no effect. vWf release could also be induced by the cAMP analogs dibutyryl-cAMP (+40%) and 8-bromo-cAMP (+25%); although their effect was weak, they clearly potentiated the response to thrombin. Epinephrine (together with IBMX) caused a small, dose-dependent increase in vWf release, maximal at 10-6 M (+50%), and also potentiated the response to thrombin. This effect is mediated by adenylate cyclase-coupled β-adrenergic receptors, since it is inhibited by propranolol and mimicked by isoproterenol. In contrast to thrombin, neither forskolin nor epinephrine caused an increase in [Ca2+]j as measured by fura-2 fluorescence. In addition, the effects of forskolin and thrombin were additive, suggesting that they act through distinct signaling pathways. We found a close correlation between cellular cAMP content and vWf release after stimulation with epinephrine and forskolin. These results demonstrate that cAMP-dependent signaling events are involved in the control of exocytosis from endothelial cells (an effect not mediated by an increase in [Ca2+]i) and provide an explanation for epinephrine-induced vWf release.

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