Xenobiotic-mediated production of superoxide by primary cultures of rat cerebral endothelial cells, astrocytes, and neurones

Previous works of our group demonstrated that xenobiotic metabolism by brain microsomes or cultured cerebral cells may promote the formation of reactive oxygen species. In order to characterise the risk of oxidative stress to both the central nervous system and the blood-brain barrier, we measured in the present work the release of superoxide in the culture medium of rat cerebrovascular endothelial cells during the metabolism of menadione, anthraquinone, diquat or nitrofurazone. Assays were run in the same experimental conditions on primary cultures of rat neurones and astrocytes. Quinone metabolism efficiently produced superoxide, but the production of radicals during the metabolism of diquat or nitrofurazone was very low, as a probable result of their reduced transport inside the cells. In all cell types assayed, superoxide production was time- and concentration-dependent, and cultured astrocytes always produced the highest amounts of radicals. Superoxide formation by microsomes prepared from the cultured cells was decreased by immunoinhibition of NADPH-cytochrome P450 reductase or by its irreversible inhibition by diphenyliodonium chloride, suggesting the involvement of this flavoprotein in radical production. Cerebrovascular endothelial cells cultured on collagen-coated filters produced equivalent amounts of superoxide both at their luminal side and through the artificial basement membrane, suggesting that in vivo, endothelial superoxide production may endanger adjacent astrocytes and neurones.     

Sex steroid induced morphological changes in primary uterine cell cultures

The ultrastructure of primary cultures of endometrial cells after treatment with diethystilbestrol, progesterone, and hydrocortisone was examined using the method of electronmicroscopic stereology. Cells treated with diethylstilbestrol had the appearance of a rapidly dividing culture having large euchromatic nuclei and prominent nucleoli with a cytoplasm containing many free ribosomes. Progesterone appeared to convert the cells to a more secretory type with large amounts of RER and smaller nucleoli. Mitochondrial enlargement was induced by sex hormones but not by hydrocortisone.     

Inflammatory cytokines increase extracellular procathepsin D in permanent and primary endothelial cell cultures

The protease cathepsin D (Cath D) and its proteolytically inactive proform, procathepsin D (ProCath D), turned out to be multifunctional within and outside the cell. Elevated levels of ProCath D occur in malignant tumors and in organs under chronic inflammation. One important source for this increase of ProCath D might be endothelial cells. Here we examined the expression of Cath D in the human endothelial cell line EA.hy 926 and in primary endothelial cells isolated from human umbilical cord veins (HUVEC). After serum-free incubation with or without human interferon-gamma (hIFN-gamma) and/or human tumor necrosis factor-alpha (hTNF-alpha) immature and mature Cath D forms were examined in cell extracts and in cell-conditioned medium concentrates by Western blotting. Lysates of EA.hy 926 cells as well as of HUVEC contained active Cath D as two-chain form, but only negligible amounts of ProCath D and Cath D intermediates. Yet both endothelial cell cultures accumulated ProCath D in their conditioned media in the absence of any stimulus. The treatment with hIFN-gamma and/or hTNF-alpha had little effect on intracellular levels of Cath D, whereas the cytokine stimulation increased the extracellular presence of ProCath D in both endothelial cell cultures. The extracellular increase of ProCath D was not related to induction of apoptosis, as validated by cleaved caspase-3 in cell lysates. Acidification of cytokine-treated media converted ProCath D into Cath D, which was associated with cathepsin-like activity using a fluorogenic substrate-linked assay. We conclude, in vitro, endothelial cells are a cytokine-dependent source for extracellular ProCath D.     

Francisella tularensis selectively induces proinflammatory changes in endothelial cells

Naturally acquired infections with Francisella tularensis, the bacterial agent of tularemia, occur infrequently in humans. However, the high infectivity and lethality of the organism in humans raise concerns that it might be exploited as a weapon of bioterrorism. Despite this potential for illicit use, the pathogenesis of tularemia is not well understood. To examine how F. tularensis interacts with cells of its mammalian hosts, we tested the ability of a live vaccine strain (LVS) to induce proinflammatory changes in cultured HUVEC. Living F. tularensis LVS induced HUVEC to express the adhesion molecules VCAM-1 and ICAM-1, but not E-selectin, and to secrete the chemokine CXCL8, but not CCL2. Stimulation of HUVEC by the living bacteria was partially suppressed by polymyxin B, an inhibitor of LPS, but did not require serum, suggesting that F. tularensis LVS does not stimulate endothelium through the serum-dependent pathway that is typically used by LPS from enteric bacteria. In contrast to the living organisms, suspensions of killed F. tularensis LVS acquired the ability to increase endothelial expression of both E-selectin and CCL2. Up-regulation of E-selectin and CCL2 by the killed bacteria was not inhibited by polymyxin B. Exposure of HUVEC to either live or killed F. tularensis LVS for 24 h promoted the transendothelial migration of subsequently added neutrophils. These data indicate that multiple components of F. tularensis LVS induce proinflammatory changes in endothelial cells in an atypical manner that may contribute to the exceptional infectivity and virulence of this pathogen.     

The phenolic profile of maize primary cell wall changes in cellulose-deficient cell cultures

Cultured maize cells habituated to grow in the presence of the cellulose synthesis inhibitor dichlobenil (DCB) have a modified cell wall in which the amounts of cellulose are reduced and the amounts of arabinoxylan increased. This paper examines the contribution of cell wall-esterified hydroxycinnamates to the mechanism of DCB habituation. For this purpose, differences in the phenolic composition of DCB-habituated and non-habituated cell walls, throughout the cell culture cycle and the habituation process were characterized by HPLC. DCB habituation was accompanied by a net enrichment in cell wall phenolics irrespective of the cell culture phase. The amount of monomeric phenolics was 2-fold higher in habituated cell walls. Moreover, habituated cell walls were notably enriched in p-coumaric acid. Dehydrodimers were 5–6-fold enhanced as a result of DCB habituation and the steep increase in 8,5′-diferulic acid in habituated cell walls would suggest that this dehydrodimer plays a role in DCB habituation. In summary, the results obtained indicate that cell wall phenolics increased as a consequence of DCB habituation, and suggest that they would play a role in maintaining the functionality of a cellulose impoverished cell wall.     

The small G-protein Rac mediates depolarization-induced superoxide formation in human endothelial cells

Superoxide anions impair nitric oxide-mediated responses and are involved in the development of hypertensive vascular hypertrophy. The regulation of their production in the vascular system is, however, poorly understood. We investigated whether changes in membrane potential that occur in hypertensive vessels modulate endothelial superoxide production. In cultured human umbilical vein endothelial cells, changes in membrane potential were induced by high potassium buffer, the non-selective potassium channel blocker tetrabutylammonium chloride (1 mm), and the non-selective cation ionophore gramicidin (1 micrometer). Superoxide formation was significantly elevated to a similar degree by all three treatments (by approximately 60%, n = 23, p < 0.01), whereas hyperpolarization by the K(ATP) channel activator Hoe234 (1 micrometer) significantly decreased superoxide formation. Depolarization also induced an increased tyrosine phosphorylation of several not yet identified proteins (90-110 kDa) and resulted in a significant increase in membrane association of the small G-protein Rac. Accordingly, the Rac inhibitor Clostridium difficile toxin B blocked the effects of depolarization on superoxide formation. The tyrosine kinase inhibitor genistein (30 micrometer, n = 15) abolished depolarization-induced superoxide formation and also prevented depolarization-induced Rac translocation associated with it. It is concluded that depolarization is an important stimulus of endothelial superoxide production, which involves a tyrosine phosphorylation-dependent translocation of the small G-protein Rac.     

Exogenous,basal, and flow-induced nitric oxide production and endothelial cell proliferation

The role of nitric oxide (NO) from endogenous and exogenous sources in regulating large vessel and microvascular endothelial cell proliferation was investigated. Exogenous NO liberated from five different chemical donors inhibited bovine aortic, bovine retinal microvascular, and human umbilical vein endothelial cell proliferation in a dose-dependent manner as determined by ³H-thymidine incorporation. The potency of the donors varied as a function of the donors' half-lives. Donors with half-lives greater than 30 min were more effective than donors with significantly shorter half-lives. Coincubation of endothelial cells with 0.4 mM deoxyadenosine and 0.4 mM deoxyguanosine reduced the percentage of inhibition due to an NO donor. These data are consistent with a ribonucleotide reductase-dependent mechanism of inhibition. Inhibition of basal NO production with four different inhibitors of nitric oxide synthase (NOS) did not modify proliferation. Laminar flow with a wall shear stress of 22 dyn/cm²inhibited the proliferation of subconfluent bovine aortic endothelial cells. The addition of a NOS inhibitor did not abrogate the flow-induced inhibition of proliferation, suggesting that flow-stimulated release of NO from endothelial cells did not account for flow-induced inhibition of proliferation. Taken together, these data suggest that relatively large concentrations of exogenous NO inhibit endothelial cell proliferation, while endogenous levels of NO are inadequate to inhibit proliferation. J. Cell. Physiol. 171:252–258, 1997. © 1997 Wiley-Liss, Inc.     

3D hepatic cultures simultaneously maintain primary hepatocyte and liver sinusoidal endothelial cell phenotypes

Developing in vitro engineered hepatic tissues that exhibit stable phenotype is a major challenge in the field of hepatic tissue engineering. However, the rapid dedifferentiation of hepatic parenchymal (hepatocytes) and non-parenchymal (liver sinusoidal endothelial, LSEC) cell types when removed from their natural environment in vivo remains a major obstacle. The primary goal of this study was to demonstrate that hepatic cells cultured in layered architectures could preserve or potentially enhance liver-specific behavior of both cell types. Primary rat hepatocytes and rat LSECs (rLSECs) were cultured in a layered three-dimensional (3D) configuration. The cell layers were separated by a chitosan-hyaluronic acid polyelectrolyte multilayer (PEM), which served to mimic the Space of Disse. Hepatocytes and rLSECs exhibited several key phenotypic characteristics over a twelve day culture period. Immunostaining for the sinusoidal endothelial 1 antibody (SE-1) demonstrated that rLSECs cultured in the 3D hepatic model maintained this unique feature over twelve days. In contrast, rLSECs cultured in monolayers lost their phenotype within three days. The unique stratified structure of the 3D culture resulted in enhanced heterotypic cell-cell interactions, which led to improvements in hepatocyte functions. Albumin production increased three to six fold in the rLSEC-PEM-Hepatocyte cultures. Only rLSEC-PEM-Hepatocyte cultures exhibited increasing CYP1A1/2 and CYP3A activity. Well-defined bile canaliculi were observed only in the rLSEC-PEM-Hepatocyte cultures. Together, these data suggest that rLSEC-PEM-Hepatocyte cultures are highly suitable models to monitor the transformation of toxins in the liver and their transport out of this organ. In summary, these results indicate that the layered rLSEC-PEM-hepatocyte model, which recapitulates key features of hepatic sinusoids, is a potentially powerful medium for obtaining comprehensive knowledge on liver metabolism, detoxification and signaling pathways in vitro.     

Exogenous and endogenous force regulation of endothelial cell behavior

Endothelial cells live in a dynamic environment where they are constantly exposed to external hemodynamic forces and generate cytoskeletal-based endogenous forces. These exogenous and endogenous forces are critical regulators of endothelial cell health and blood vessel maintenance at all generations of the vascular system, from large arteries to capillary beds. The first part of this review highlights the role of the primary exogenous hemodynamic forces of shear, cyclic strain, and pressure forces in mediating endothelial cell response. We then discuss the emergent role of the mechanical properties of the extracellular matrix and of cellular endogenous force generation on endothelial cell function, implicating substrate stiffness and cellular traction stresses as important mediators of endothelial cell health. The intersection of exogenous and endogenous forces on endothelial cell function is discussed, suggesting some of the many remaining questions in the field of endothelial mechanobiology.     

Exogenous gangliosides may affect methylation mechanisms in neuronal cell cultures

Primary neurons in culture from chick embryo cerebral hemispheres were treated with a mixture of gangliosides added to the growth medium (final concentration: 10^–5M and 10^–8M) from the 3rd to the 6th day in vitro. Under these conditions methylation processes measured with [³H] and [³⁵S] methionine and [³H]ethanolamine as precursors showed an increased methylation of [³H]ethanolamine containing phospholipids, a correspondent increased conversion of these compounds to [³H]choline containing phospholipids, and a general increased methylation of trichloroacetic acid precipitable macromolecules containing labeled methionine. A small increase in protein synthesis was observed after incubation of neurons with [³H]- and [³⁵S]methionine. This was confirmed after electrophoretic separation of a protein extract with increased³H-and³⁵S-labeling in protein bands with moecular weights between 50 and 60 KDaltons. A protein band of about 55 KDaltons appeared to be preferentially labelled when [³H] methionine was the precursor. The treatment with gangliosides increased the incorporation of [methyl-³H] label after incubation of neurons with [³H] methionine, into total DNA and decreased that of total RNA. The treatment of neurons in culture with exogenous gangliosides hence affects differently methylation processes, a finding which may confirm the involvement of gangliosides on the intracellular mediation of neuronal information mechanisms.     

Sulfated mucopolysaccharide synthesis and secretion in endothelial cell cultures

The cells that line the lumen of rabbit aorta have been obtained in a quantity sufficient for mass culture. Autoradiography of the cells incubated with ³⁵S-sulfuric acid and electrophoretic analysis of ³⁵S mucopolysaccharides extracted from the cells and from the medium indicate that these cells synthesize and secrete various species of sulfated mucopolysaccharides. In the steady state, only one major and one minor component are found inside the cell. The major intracellular component is resistant to degradation by chondroitinase ABC. Two major fractions and one minor fraction are found in the culture medium at the end of a 24 h labelling period. Both of the major components extracted from the medium are not present in the cell in an appreciable amount.     

An interaction between BRG1 and histone modifying enzymes mediates lipopolysaccharide-induced proinflammatory cytokines in vascular endothelial cells

Vascular inflammation is the culprit for a host of human diseases. The underlying mechanism, however, is not definitively elucidated. In the present study, we investigated the interplay between different epigenetic factors during lipopolysaccharide (LPS) induced synthesis of proinflammatory cytokines in cultured vascular endothelial cells. We report that in response to LPS treatment, NF-κB was deplored to its target promoters along with the chromatin remodeling protein BRG1. Paralleling these changes trimethylated H3K9 became erased from while trimethylated H3K4 started to accumulate on the NF-κB target promoters. Further analysis revealed that LPS stimulation resulted in sequential recruitment of the H3K9 tri-demethylase JMJD2A and the H3K4 trimethyltransferase SET1A to the NF-κB target promoters. JMJD2A mediated–H3K9 demethylation served as a prerequisite for SET1A to bind to the NF-κB target promoters. Both JMJD2A and SET1A were essential for LPS-induced transactivation of proinflammatory cytokines by sustaining the binding of NF-κB. Of key importance, BRG1 coordinated the sequential recruit of and the interplay between JMJD2A and SET1A. In conclusion, our data unveil a novel epigenetic mechanism that contributes to LPS-induced vascular inflammation.     

Endothelial cell P-selectin mediates a proinflammatory and prothrombogenic phenotype in cerebral venules of sickle cell transgenic mice

Whereas the adhesion of leukocytes and erythrocytes to vascular endothelium has been implicated in the vasooclusive events associated with sickle cell disease, the role of platelet-vessel wall interactions in this process remains undefined. The objectives of this study were to: 1) determine whether the adhesion of platelets and leukocytes in cerebral venules differs between sickle cell transgenic (betaS) mice and their wild-type (WT) counterparts (C57Bl/6) under both resting and posthypoxic conditions, and 2) define the contributions of P-selectin to these adhesion processes. Animals were anesthetized, and platelet and leukocyte interactions with endothelial cells of cerebral postcapillary venules were monitored and quantified using intravital fluorescence microscopy in WT, betaS, and chimeric mice produced by transplanting bone marrow from WT or betaS mice into WT or P-selectin-deficient (P-sel(-/-)) mice. Platelet and leukocyte adhesion to endothelial cells in both unstimulated and posthypoxic betaS mice were significantly elevated over WT levels. Chimeric mice involving bone marrow transfer from betaS mice to P-sel(-/-) mice exhibited a profound attenuation of both platelet and leukocyte adhesion compared with betaS bone marrow transfer to WT mice. These findings indicate that betaS mice assume both an inflammatory and prothrombogenic phenotype, with endothelial cell P-selectin playing a major role in mediating these microvascular responses.     

The VirB type IV secretion system of Bartonella henselae mediates invasion, proinflammatory activation and antiapoptotic protection of endothelial cells

Bartonella henselae is an arthropod-borne zoonotic pathogen causing intraerythrocytic bacteraemia in the feline reservoir host and a broad range of clinical manifestations in incidentally infected humans. Remarkably, B. henselae can specifically colonize the human vascular endothelium, resulting in inflammation and the formation of vasoproliferative lesions known as bacillary angiomatosis and bacillary peliosis. Cultured human endothelial cells provide an in vitro system to study this intimate interaction of B. henselae with the vascular endothelium. However, little is known about the bacterial virulence factors required for this pathogenic process. Recently, we identified the type IV secretion system (T4SS) VirB as an essential pathogenicity factor in Bartonella, required to establish intraerythrocytic infection in the mammalian reservoir. Here, we demonstrate that the VirB T4SS also mediates most of the virulence attributes associated with the interaction of B. henselae during the interaction with human endothelial cells. These include: (i) massive rearrangements of the actin cytoskeleton, resulting in the formation of bacterial aggregates and their internalization by the invasome structure; (ii) nuclear factor kappaB-dependent proinflammatory activation, leading to cell adhesion molecule expression and chemokine secretion, and (iii) inhibition of apoptotic cell death, resulting in enhanced endothelial cell survival. Moreover, we show that the VirB system mediates cytostatic and cytotoxic effects at high bacterial titres, which interfere with a potent VirB-independent mitogenic activity. We conclude that the VirB T4SS is a major virulence determinant of B. henselae, required for targeting multiple endothelial cell functions exploited by this vasculotropic pathogen.     

Exogenous glycosaminoglycans modulate chondrogenesis, cyclic AMP level and cell growth in limb bud mesenchyme cultures

Effects of hyaluronate, heparin and chondroitin-6-sulfate were studied on micromass cultures of chick limb bud mesenchyme (Hamburger and Hamilton stages 23-24). Histochemical, electron microscopical, biochemical and radiochemical investigations of day 4 cultures revealed dose-dependent inhibitory effects of these glycosaminoglycans on chondrogenesis, cyclic AMP level and growth of cells. In addition, hyaluronate with 100 micrograms/ml dose caused a displacement of newly formed proteoglycan from cultures into the medium. It is supposed that exogenous glycosaminoglycans influence ionic equilibrium in the immediate vicinity of cells and disturb the organization of the prechondrogenic extracellular matrix resulting in alterations of cell membrane--cytoskeleton associations. These alterations may provoke a reduction in cyclic AMP level and DNA synthesis. It is suggested that a reduction in cyclic AMP level preceding the expression of cartilage phenotype results in the inhibition of chondrogenesis.     

Intercellular junctions and transfer of small molecules in primary vascular endothelial cultures

The ultrastructure of gap and tight junctions and the cell-to-cell transfer of small molecules were studied in primary cultures and freshly isolated sheets of endothelial cells from calf aortae and umbilical veins. In thin sections and in freeze-fracture replicas, the gap and tight junctions in the freshly isolated cells from both sources appeared similar to those found in the intimal endothelium. Most of the interfaces in replicas had complex arrays of multiple gap junctions either intercalated within tight junction networks or interconnected by linear particle strands. The particle density in the center of most gap junctions was noticeably reduced. In confluent monolayers, after 3-5 days in culture, gap and tight junctions were present, although reduced in complexity and apparent extent. Despite the relative simplicity of the junctions, the cell-to-cell transfer of potential changes, dye (Lucifer Yellow CH), and nucleotides was readily detectable in cultures of both endothelial cell types. The extent and rapidity of dye transfer in culture was only slightly less than that in sheets of freshly isolated cells, perhaps reflecting a reduced gap junctional area combined with an increase in cell size in vitro.     

Notch signaling mediates melanoma–endothelial cell communication and melanoma cell migration

Stromal and cellular components within the tumor microenvironment significantly influence molecular signals mediating tumor growth and progression. We recently performed a screen to evaluate critical mediators of melanoma–endothelial communication and identified several molecular pathways associated with these cellular networks, including Notch3. Here, we evaluate the nature of melanoma–endothelial communication mediated by Notch3 and its functional significance. We find that Notch3 is specifically upregulated in melanoma–endothelial cell cocultures and is functionally associated with increased Notch signaling in melanoma cells. Furthermore, induced Notch3 signaling in melanoma cell lines leads to enhanced tumor cell migration without associated increases in tumor cell growth. Additionally, Notch3 expression is specifically associated with malignant patient samples and is not evident in benign nevi. We conclude that Notch3 mediates melanoma–endothelial cell communication and tumor cell migration and may serve as a meaningful therapeutic target for this aggressive malignancy.     

Cell identification in primary cell cultures from skin

Summary Primary cell cultures can readily be obtained from human skin using the explant method. With special care it is possible to obtain primary cultures consisting of epidermal keratinocytes without fibroblast contamination. By means of differences in their growth patterns and retention of specific differentiative functions in vitro, keratinocytes and fibroblasts can easily be distinguished. The high degree of confidence in establishing cell identity permits meaningful experimental use of this system. The method of enzymatic separation of epidermis from dermis, followed by culture of cells from the dissociated epidermal tissue, provides both epithelial and dendritic cells. The former are probably keratinocytes, whereas the latter are definitely melanocytes. The possibility of eventual fibroblast overgrowth, using this latter method, has not yet been ruled out with certainty. Presented at the Workshop on Primary Cell Culture, November 1–3, 1973, Convenor Dr. Warren I. Schaeffer, University of Vermont, at the W. Alton Jones Cell Science Center, Lake Placid, N. Y. The editorial assistance of Dr. and Mrs. Joseph Leighton in preparing for press the five papers from that workshop is gratefully acknowledged. This work was supported by grants from the National Cancer Institute, 1 PO 1 CA 11536, and the National Institute of Arthritis and Metabolic Diseases, 1 PO 1 AM 15515.