In vitro culture of coxsackievirus group B, type 3 immune spleen cells on infected endothelial cells and biological activity of the cultured cells in vivo.

Vascular endothelial cells (ECs) derived from the central nervous system (CNS) variably lose their unique barrier properties during in vitro culture, hindering the development of robust assays for blood-brain barrier (BBB) function, including drug permeability and extrusion assays. In previous work (Sabbagh et al., 2018) we characterized transcriptional and accessible chromatin landscapes of acutely isolated mouse CNS ECs. In this report, we compare transcriptional and accessible chromatin landscapes of acutely isolated mouse CNS ECs versus mouse CNS ECs in short-term in vitro culture. We observe that standard culture conditions are associated with a rapid and selective loss of BBB transcripts and chromatin features, as well as a greatly reduced level of beta-catenin signaling. Interestingly, forced expression of a stabilized derivative of beta-catenin, which in vivo leads to a partial conversion of non-BBB CNS ECs to a BBB-like state, has little or no effect on gene expression or chromatin accessibility in vitro.

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Antigen-specific suppressor T-cell activity in genetically restricted immune spleen cells.

Journal of Experimental Medicine ◽

10.1084/jem.148.5.1271 ◽

1978 ◽

Vol 148 (5) ◽

pp. 1271-1281 ◽

Cited By ~ 13

Author(s):

C W Pierce ◽

J A Kapp

Keyword(s):

T Cells ◽

T Cell ◽

Cell Activity ◽

Antibody Responses ◽

Spleen Cells ◽

Suppressor T Cells ◽

Specific Suppressor T Cells ◽

Immune Spleen Cells

Virgin spleen cells develop comparable primary antibody responses in vitro to syngeneic or allogeneic macrophages (Mphi) bearing the terpolymer L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT), whereas immune spleen cells primed with syngeneic or allogeneic GAT-Mphi develop secondary responses preferentially when stimulated with GAT-Mphi syngeneic to the GAT-Mphi used for priming in vivo. These restrictions are mediated by products of the I-A subregion of the H-2 complex and are operative at the level of the GAT-Mphi-immune helper T-cell interactions. To investigate why these immune spleen cells fail to develop a significant antibody response to GAT-Mphi other than those used for in vivo immunization and determine the mechanism by which the restriction is maintained, spleen cells from virgin and syngeneic or allogeneic GAT-Mphi-primed mice were co-cultured in the presence of GAT-Mphi of various haplotypes. Antibody responses to GAT developed only in the presence of GAT-Mphi syngeneic to the Mphi used for in vivo priming; responses in cultures with GAT-Mphi allogeneic to the priming Mphi, whether these Mphi were syngeneic or allogeneic with respect to the responding spleen cells, were suppressed. The suppression was mediated by GAT-specific radiosensitive T cells. Thus, development of GAT-specific suppressor T cells appears to be a natural consequence of the immune response to GAT in responder as well as nonresponder mice. The implications of stimulation of genetically restricted immune helper T cells, and antigen-specific, but unrestricted, suppressor T cells after immunization with GAT-Mphi in vivo are discussed in the context of regulatory mechanisms in antibody responses.

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Abstract 580: The Production of a Bio-Engineered Endothelial Intima From Cultured Cells Using Whole Cardiac Cadaveric Extracellular Matrix.

Circulation ◽

10.1161/circ.116.suppl_16.ii_105-a ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

Stefan Kren ◽

Gabriel Caron ◽

Doris A Taylor

Keyword(s):

Endothelial Cells ◽

Rat Heart ◽

Cultured Cells ◽

Fluorescent Microscopy ◽

Nuclear Staining ◽

Endothelial Lining ◽

Arterial Tree ◽

The Matrix

Background; Bioengineered solutions to failing cardiac tissue have been difficult to achieve due partially to adverse interactions between circulating blood and the engineered surface. The aim of this study was to determine if by using naturally-derived ECM and cultured endothelial cells, a bioengineered whole-heart vascular intima could be generated. The matrix substrate for organ culture was produced by a perfusion-based detergent decelluarization of cadaveric rat heart. This process maintained ECM protein integrity as indicated by a glycosaminoglycan assay, with ~ equivalent amounts present relative to cadaveric rat heart. Its acellular nature was confirmed by loss of > 96% DNA (p = 0.001) compared to normal rat heart. In vitro infusion of aqueous dye or Mercox resin suggested a complete arterial tree, with structural preservation of vascular conduits. In vivo perfusability of the ECM was demonstrated by heterotopic transplantation with anticoagulation (n=4) into RNU rats for 7 days. Recellularization of the vascular tree was attempted by In Vitro Langendorff perfusion of 2 x 107 rat aortal endothelial cells (ECs) followed by a 7d incubation with escalating pulsatile flow in a 3D bioreactor. CellTracker Green assessed EC viability and permitted visualization of engrafted cells by fluorescent microscopy. Vessels of different diameters contained “patches” of confluent endothelium with complete circumferential lining of many of the matrix conduits. ECs lining both chamber walls and trabeculae were also observed. Nuclear staining showed 537.8 +/− 67.6 ECs / mm2 on endocardial surfaces, as well as 311.7 +/− 61.8 ECs / mm2 in vessels. To enhance the delivery of cells into the ventricular walls, a microcanulization of the brachiocephalic artery with sustained aortal perfusion was undertaken. This technique diverted more cells to the vasculature and more broadly distributed the cells in each area resulting in a lower cell density; 199.8 +/− 25.0 ECs / mm2 in vessels vs 125.8 +/− 43.4 ECs / mm2 on endocardial surfaces. In conclusion, these data suggest that by using detergent prepared acellular ECM of a whole organ, generation of a complete endothelial lining of vascular structures may be possible.

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Tropoelastin Promotes the Formation of Dense, Interconnected Endothelial Networks

Biomolecules ◽

10.3390/biom11091318 ◽

2021 ◽

Vol 11 (9) ◽

pp. 1318

Author(s):

Aleen Al Halawani ◽

Lea Abdulkhalek ◽

Suzanne M. Mithieux ◽

Anthony S. Weiss

Keyword(s):

Stem Cells ◽

Wound Healing ◽

Endothelial Cells ◽

Wound Repair ◽

Cultured Cells ◽

Tube Formation ◽

Endothelial Tube Formation ◽

Angiogenic Effect

Tropoelastin, the soluble precursor of elastin, has been used for regenerative and wound healing purposes and noted for its ability to accelerate wound repair by enhancing vascularization at the site of implantation. However, it is not clear whether these effects are directly due to the interaction of tropoelastin with endothelial cells or communicated to endothelial cells following interactions between tropoelastin and neighboring cells, such as mesenchymal stem cells (MSCs). We adapted an endothelial tube formation assay to model in vivo vascularization with the goal of exploring the stimulatory mechanism of tropoelastin. In the presence of tropoelastin, endothelial cells formed less tubes, with reduced spreading into capillary-like networks. In contrast, conditioned media from MSCs that had been cultured on tropoelastin enhanced the formation of more dense, complex, and interconnected endothelial tube networks. This pro-angiogenic effect of tropoelastin is mediated indirectly through the action of tropoelastin on co-cultured cells. We conclude that tropoelastin inhibits endothelial tube formation, and that this effect is reversed by pro-angiogenic crosstalk from tropoelastin-treated MSCs. Furthermore, we find that the known in vivo pro-angiogenic effects of tropoelastin can be modeled in vitro, highlighting the value of tropoelastin as an indirect mediator of angiogenesis.

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Cardiac endothelial cells isolated from mouse heart - a novel model for radiobiology.

Acta Biochimica Polonica ◽

10.18388/abp.2011_2253 ◽

2011 ◽

Vol 58 (3) ◽

Cited By ~ 17

Author(s):

Karol Jelonek ◽

Anna Walaszczyk ◽

Dorota Gabryś ◽

Monika Pietrowska ◽

Chryso Kanthou ◽

...

Keyword(s):

Endothelial Cells ◽

Ionizing Radiation ◽

Cultured Cells ◽

Clinical Problem ◽

High Dose ◽

Mouse Heart ◽

Important Clinical Problem ◽

Induced Apoptosis

Cardiovascular disease is recognized as an important clinical problem in radiotherapy and radiation protection. However, only few radiobiological models relevant for assessment of cardiotoxic effects of ionizing radiation are available. Here we describe the isolation of mouse primary cardiac endothelial cells, a possible target for cardiotoxic effects of radiation. Cells isolated from hearts of juvenile mice were cultured and irradiated in vitro. In addition, cells isolated from hearts of locally irradiated adult animals (up to 6 days after irradiation) were tested. A dose-dependent formation of histone γH2A.X foci was observed after in vitro irradiation of cultured cells. However, such cells were resistant to radiation-induced apoptosis. Increased levels of actin stress fibres were observed in the cytoplasm of cardiac endothelial cells irradiated in vitro or isolated from irradiated animals. A high dose of 16 Gy did not increase permeability to Dextran in monolayers formed by endothelial cells. Up-regulated expression of Vcam1, Sele and Hsp70i genes was detected after irradiation in vitro and in cells isolated few days after irradiation in vivo. The increased level of actin stress fibres and enhanced expression of stress-response genes in irradiated endothelial cells are potentially involved in cardiotoxic effects of ionizing radiation.

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100 TIMING OF BLASTOCOELIC CAVITY RE-EXPANSION REPRESENTS A QUALITY INDEX OF IN VITRO PRODUCED OVINE BLASTOCYSTS

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab100 ◽

2006 ◽

Vol 18 (2) ◽

pp. 158

Author(s):

G. G. Leoni ◽

F. Berlinguer ◽

S. Succu ◽

F. Mossa ◽

M. Galioto ◽

...

Keyword(s):

In Vitro Culture ◽

Embryo Transfer ◽

Embryo Quality ◽

Glucose Transporter ◽

Pregnancy Rates ◽

Simple Method ◽

Group A ◽

Group B

There is general consensus that the quality of in vitro-produced embryos is inferior to that of their in vivo counterparts and that along with other factors this may be responsible for the poorer cryosurvival and the lower pregnancy rates reported for these embryos. The feasibility to accurately select viable embryos would be valuable for improving pregnancy rates and avoiding futile transfer attempts. The aim of our study was to assess whether in vitro-produced ovine embryo quality could be determined by the timing blastocoelic cavity re-expansion after vitrification, thawing, and in vitro culture. Embryos were produced in vitro from ovine ovaries collected at the local slaughterhouse following a standard protocol developed for ovine oocytes, as previously described (Berlinguer et al. 2005 Reprod. Fertil. Dev. 17, 201). Blastocysts were then vitrified/warmed according to a simple method (Leoni et al. 2002 Cryobiology 45, 204-212) and cultured in vitro in TCM-199 supplemented with 10% FCS for 72 h in 5% CO2 in air at 39�C. Vitrified embryos were divided into two groups: (A) expanded within 8 h of in vitro culture after warming; (B) expanded during 8 to 16 h of in vitro culture after warming. Of the 338 vitrified/warmed embryos, 173 (51.1%) showed a re-expanded blastocoelic cavity after 8 h of in vitro culture, whereas 58 (17.1%) re-expanded during 8 to 16 h of in vitro culture. We also analyzed by semiquantitative RT-PCR a panel of genes expressed during pre-implantation embryo development (glucose transporter, HPS-10, e-caderin, poly(A)polimerase, and ubiquitine); our results showed that these genes, apart from ubiquitine which showed no difference in the two groups, were more expressed in Group A blastocysts compared to Group B. Group A blastocysts showed higher hatching rates (67.6%) than Group B blastocysts (43.1%; P < 0.01). The total cell number calculated for the hatched blastocysts after staining with Hoechst 33342 was significantly higher in Group A (140.7 � 8.3, n = 42) than Group B (102.2 � 8.4, n = 27; P < 0.01). Pregnancy rate (detected by ultrasonography 30 days after embryo transfer) after laparoscopic embryo transfer to 12 synchronized recipient Sarda sheep was 40% (6/15) in Group A and 13.4% (2/15) in Group B. The results indicated that timing of blastocoelic cavity re-expansion after vitrification/warming and in vitro culture can be considered as a reliable index of in vitro-produced ovine embryo quality, and in addition, a better prediction for improved survival rate after transfer to synchronized recipients. This work was supported by Cofin 2003.

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Ultrastructural Correlations between Clonal Human Tumor Colonies and in Vivo Neoplasms

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100053711 ◽

1982 ◽

Vol 40 ◽

pp. 210-211

Author(s):

M.J. Murphy ◽

R.R. Price ◽

J.C. Sloman

Keyword(s):

Cultured Cells ◽

Human Tumor ◽

Cell Type ◽

Tumor Mass ◽

Initial Cell ◽

Cloning Assay ◽

Human Tumor Cloning ◽

The Relationship

The in vitro human tumor cloning assay originally described by Salmon and Hamburger has been applied recently to the investigation of differential anti-tumor drug sensitivities over a broad range of human neoplasms. A major problem in the acceptance of this technique has been the question of the relationship between the cultured cells and the original patient tumor, i.e., whether the colonies that develop derive from the neoplasm or from some other cell type within the initial cell population. A study of the ultrastructural morphology of the cultured cells vs. patient tumor has therefore been undertaken to resolve this question. Direct correlation was assured by division of a common tumor mass at surgical resection, one biopsy being fixed for TEM studies, the second being rapidly transported to the laboratory for culture.

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Preparation of cultured astrocytes for x-ray microanalysis

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100156924 ◽

1989 ◽

Vol 47 ◽

pp. 988-989

Author(s):

N.K.R. Smith ◽

K.E. Hunter ◽

P. Mobley ◽

L.P. Felpel

Keyword(s):

Cultured Cells ◽

Elemental Content ◽

Elemental Distribution ◽

Sprague Dawley ◽

X Ray ◽

Cultured Astrocytes ◽

Freeze Dried ◽

Ultimate Objective

Electron probe energy dispersive x-ray microanalysis (XRMA) offers a powerful tool for the determination of intracellular elemental content of biological tissue. However, preparation of the tissue specimen , particularly excitable central nervous system (CNS) tissue , for XRMA is rather difficult, as dissection of a sample from the intact organism frequently results in artefacts in elemental distribution. To circumvent the problems inherent in the in vivo preparation, we turned to an in vitro preparation of astrocytes grown in tissue culture. However, preparations of in vitro samples offer a new and unique set of problems. Generally, cultured cells, growing in monolayer, must be harvested by either mechanical or enzymatic procedures, resulting in variable degrees of damage to the cells and compromised intracel1ular elemental distribution. The ultimate objective is to process and analyze unperturbed cells. With the objective of sparing others from some of the same efforts, we are reporting the considerable difficulties we have encountered in attempting to prepare astrocytes for XRMA.Tissue cultures of astrocytes from newborn C57 mice or Sprague Dawley rats were prepared and cultured by standard techniques, usually in T25 flasks, except as noted differently on Cytodex beads or on gelatin. After different preparative procedures, all samples were frozen on brass pins in liquid propane, stored in liquid nitrogen, cryosectioned (0.1 μm), freeze dried, and microanalyzed as previously reported.

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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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