Bovine Endothelial Cell Monolayers Research Articles

Bluetongue virus infection alters the impedance of monolayers of bovine endothelial cells as a result of cell death

Bluetongue virus (BTV) is the cause of bluetongue, an emerging, arthropod-transmitted disease of ungulates. Bluetongue is characterized by vascular injury with hemorrhage, tissue infarction and widespread edema, lesions that are consistent with those of the so-called viral hemorrhagic fevers. To further investigate the pathogenesis of vascular injury in bluetongue, we utilized an electrical impedance assay and immunofluorescence staining to compare the effects of BTV infection on cultured bovine endothelial cells (bPAEC) with those of inducers of cell death (Triton X-100) and interendothelial gap formation (tissue necrosis factor [TNF]). The data confirm that the adherens junctions of BTV-infected bPAECs remained intact until 24 h post-infection, and that loss of monolayer impedance precisely coincided with onset of virus-induced cell death. In contrast, recombinant bovine TNF-α caused rapid loss of bPAEC monolayer impedance that was associated with interendothelial gap formation and redistribution of VE-cadherin, but without early cell death. The data from these in vitro studies are consistent with a pathogenesis of bluetongue that involves virus-induced vascular injury leading to thrombosis, hemorrhage and tissue necrosis. However, the contribution of cytokine-induced interendothelial gap formation with subsequent edema and hypovolemic shock contributes to the pathogenesis of bluetongue remains to be fully characterized.

Theileria-infected cell line from an African buffalo (Syncerus caffer)

Mononuclear cells were isolated from the peripheral blood of a buffalo infected with a Theileria sp. using density gradient centrifugation, and the cells were put into culture flasks covered by a monolayer of bovine endothelial cells. Twenty days after culture initiation, cells containing macroschizonts were detected in Giemsa-stained smears. The first subculture was carried out on day 45 of culture propagation. Subsequently, infected cells were subcultured twice a week, and each time 1 to 2 x 10(6) per milliliter cells were harvested. DNA was extracted from culture material and a partial polymerase chain reaction amplification of the 18S ribosomal RNA (rRNA) gene was carried out using Theileria genus-specific primers. Sequence data and phylogenetic analysis using the 18S rRNA gene indicated a close relationship to Theileria sp. buffalo, previously described in literature. Here, the first successful attempt to establish a macroschizont-infected lymphoblastoid cell line of Theileria sp. (buffalo) from an African buffalo is described.

Eimeria bovis infection enhances adhesion of peripheral blood mononuclear cells to and their transmigration through an infected bovine endothelial cell monolayer in vitro

The first schizogony of Eimeria bovis takes place in lymphatic endothelial cells of the ileum, resulting in the formation of macroschizonts within 2-3 weeks. In this study, we analyse early cellular immune responses to infected host cells on the basis of peripheral blood mononuclear cell (PBMC) adhesion on and transmigration through infected bovine umbilical vein endothelial cell (BUVEC) monolayers. Adhesion of PBMC was upregulated by an E. bovis infection. Most marked effects were observed 1 day p.i.; thereafter, PBMC adhesion declined reaching control levels from day 8 p.i. onward. CD8(+) T cells adhered more frequently to infected BUVEC (42%) than CD4(+) T cells (25%). About one third of attached PBMC were represented by gammadelta-TCR(+) T cells. Adhesion of T cells was not restricted to parasitised host cells, but occurred almost equally on non-infected BUVEC within the same monolayer. Furthermore, we found moderately enhanced levels of PBMC transmigration through infected BUVEC monolayers, in particular on day 2 p.i. The data presented here suggest that E. bovis infection of BUVEC induces endothelial cell-derived proinflammatory reactions, which appear suitable for the initiation of both adaptive and innate immune responses.

Neutrophil Adhesion and Transmigration through Bovine Endothelial Cells in vitro by Protein H and LPS of Pasteurella Multocida

This study describes an in vitro investigation on the role of Pasteurella multocida cells and its isolated protein Hand LPS on neutrophil adhesion and migration through bovine endothelial cell monolayers. Pmulticoda cells, protein Hand LPS increased the adhesion and transmigration of neutrophils through BAECThe bacteria/cell ratio of 100 for Pmultocida, protein H concentration 0.05–0.2 μM and LPS concentration 0.5–1.0 μLM respectively, induced the maximum adhesion and transmigration of neutrophils through BAECThe optimal time of incubation with bacteria or bacterial products was 4–6 hOur results confirm the role of Gram-negative bacteria and of components of the outer membrane such as protein H or LPS in activating the neutrophils and in promoting the adhesion and cells transmigration from the vessels to the site of inflammation.

Permeability characteristics of hemoglobin derivatives across cultured endothelial cell monolayers

To better understand the vascular activity of hemoglobin-based (Hb-based) oxygen carriers, the endothelial permeability characteristics of Hb derivatives having various molecular masses were defined by using monolayers of bovine endothelial cells cultured on microporous membranes. The endothelial permeability of unmodified bovine Hb was almost twice that of bovine serum albumin. Intramolecularly cross-linked human Hb showed slightly but significantly reduced permeability as compared with unmodified bovine Hb. Polyethyleneglycol modification or haptoglobin binding to Hb further reduced the permeability. These properties were intensified in conditions in which the endothelial barrier function was reduced by pretreatment with either interleukin-6 (100 ng/mL, 21 hours) or lipopolysaccharide (1 μg/mL, 10 hours). In contrast, there was little permeability of liposomeencapsulated Hb, and it was almost unaffected by the pretreatments. These data provide the first information that Hb derivatives with smaller molecular masses show larger transendothelial flux. Because Hb is a potent scavenger of endotheliumderived relaxing factor (EDRF), our observations support the idea that smaller Hbbased acellular oxygen carriers are potent vasoconstrictors as a result of abluminal EDRF scavenging.

Regulation of endothelial cell myosin light chain phosphorylation and permeability by vanadate.

The involvement of tyrosine protein phosphorylation in the regulation of endothelial cell (EC) contraction and barrier function is poorly understood. We have previously shown that myosin light chain (MLC) phosphorylation catalyzed by a novel 214 kDa EC myosin light chain kinase (MLCK) isoform is a key event in EC contraction and barrier dysfunction [Garcia et al. (1995): J Cell Physiol 163:510-522; Garcia et al. (1997): Am J Respir Cell Mol Biol 16:487-491]. In this study, we tested the hypothesis that tyrosine phosphatases participate in the regulation of EC contraction and barrier function via modulation of MLCK activity. The tyrosine phosphatase inhibitor, sodium orthovanadate (vanadate), significantly decreased electrical resistance across bovine EC monolayers and increased albumin permeability consistent with EC barrier impairment. Vanadate significantly increased EC MLC phosphorylation in a time-dependent manner (maximal increase observed at 10 min) and augmented both the MLC phosphorylation and permeability responses produced by thrombin, an agonist which rapidly increases tyrosine kinase activities. The vanadate-mediated increase in MLC phosphorylation was not associated with alterations in either phosphorylase A Ser/Thr phosphatase activities or in cytosolic [Ca2+] but was strongly associated with significant increases in EC MLCK phosphotyrosine content. These data suggest that tyrosine phosphatase activities may participate in EC contractile and barrier responses via the regulation of the tyrosine phosphorylation status of EC MLCK.

Mathematical simulation of unidirectional tissue formation: in vitro transanastomotic endothelialization model

In vitro transanastomotic endothelialization was studied using a mathematical model with the Fisher equation. The Fisher equation is a nonlinear parabolic equation which has a cell migration term and a population growth term. The mathematical model was used to simulate recent experiments performed to investigate quantitatively the unidirectional formation of a bovine endothelial cell monolayer in vitro. The two parameters included in the equation were estimated using a trial and error method in which the calculated solutions of the various values of the two parameters are compared with the experimental data and the best fit pair is adopted: one parameter, D, which represents the unidirectional cell migration rate and the other, k, which represents the population growth rate. The calculated solutions fit the experimental data well. We also simulated the healing of a mechanically disrupted endothelial monolayer sheet. The significance of cellular biomechanics in tissue formation and design of tissue-engineered devices is discussed.

Enhanced endothelial cell adhesion of human cerebrospinal fluid lymphocytes

Lymphocyte migration into a tissue depends on properties of both the lymphocyte and the tissue's vascular endothelium. The central nervous system (CNS) possesses a specialized microvasculature and lymphocytes appear to enter the CNS less readily than peripheral tissues. We investigated whether those lymphocytes that interact with the CNS, as represented by cerebrospinal fluid (CSF)-derived lymphocytes, express adhesive properties distinct from peripheral blood lymphocytes (PBLs). Adhesion of human lymphocytes to bovine endothelial cell monolayers was quantitated microscopically. A greater number of PBLs adhered to aortic than to retinal endothelial cell cultures (e.g., 10.9 +/- 0.6 and 4.5 +/- 0.2, respectively; p = 0.0023). Preincubation of either endothelial cell type with tumor necrosis factor-alpha (TNF-alpha) enhanced lymphocyte adhesion. Activation of PBLs with concanavalin A or phytohemagglutinin increased endothelial cell adhesion and the effect was additive with that of TNF-alpha. The number of CSF lymphocytes adhering to endothelial cell cultures (retinal, 67.5 +/- 9.0; aortic, 83.7 +/- 10.6) was more than 10 times the number of PBLs (retinal, 5.4 +/- 0.8; aortic, 8.0 +/- 1.3; p < 0.0001). CSF lymphocytes did not, however, adhere preferentially to CNS-derived endothelial cell cultures. These results suggest that CSF may be enriched, compared with peripheral blood, in its content of surveillance lymphocytes, but that these cells might enter target tissues nonspecifically.

Suramin inhibits C6 glioma‐induced angiogenesis in vitro

Aspects of tumor-induced angiogenesis in vitro were examined using an assay involving collagen gel invasion by a surface monolayer of bovine endothelial cells under the influence of serum free conditioned medium produced by C6 cells, an experimentally derived rat glial tumor cell line. The effects of the polyanionic compound suramin, known to interfere with growth factor/cell signaling on this process were evaluated. Collagen gel invasion was quantified by adding C6 conditioned medium with or without various doses of suramin to monolayers of bovine aortic endothelial cells grown on type I collagen gels in transwell inserts. Cultures were monitored with phase-contrast microscopy. After various periods of incubation collagen gels were fixed, embedded in epoxy resin, and 1-micron thick sections were stained with toluidine blue. Additional cultures were used to evaluate the effects of C6 conditioned medium and suramin on endothelial cell proliferation, and on chemotaxis through 8-microns pores. C6 glioma cell conditioned medium induced large vessel endothelial cells to sprout into the underlying collagen matrix and subsequently form networks of capillary like tubes. Conditioned medium was also chemotactic and mitogenic for these cells. The addition of suramin to C6 glioma conditioned medium prevents tube formation in collagen gels, and inhibits both endothelial cell proliferation and chemotaxis in a dose dependent manner. These results suggest that glial tumor cell conditioned medium induces angiogenesis in large vessel endothelial cells in vitro via mechanisms which are disrupted by suramin, most likely involving tumor-derived growth factor release and/or endothelium-mediated matrix proteolysis.

Calcium entry-dependent oscillations of cytoplasmic calcium concentration in cultured endothelial cell monolayers.

Bovine endothelial cell monolayers grown to confluence and stimulated with bradykinin responded with periodic fluctuations in intracellular Ca2+ concentration ([Ca2+]i) when exposed to K(+)-free Hepes-buffered saline. The fluctuations in [Ca2+]i measured with fura-2 were synchronized among the population of cells observed and were sensitive to extracellular Ca2+ concentration ([Ca2+]o). Thapsigargin, which inhibits the endoplasmic reticular Ca2(+)-ATPase, did not inhibit the [Ca2+]i oscillations. Removal of extracellular Ca2+ or inhibition of Ca2+ entry by using La3+ or 1-(beta- [3-(4-methoxyphenyl)proproxy]-4-methoxyphenethyl)-1H-imidazole hydrochloride (SKF 96365) abolished the [Ca2+]i oscillations in endothelial cell monolayers. The fluctuations in [Ca2+]i were therefore dependent on Ca2+ influx rather than Ca2+ mobilization from intracellular stores. Simultaneous measurements of membrane potential (Em) using the potential-sensitive bisoxonol dye bis(1,3-dibutylbarbituric acid)trimethine oxonol [Di-BAC4(3)] and [Ca2+]i using fura-2 showed that Em oscillated at the same frequency as the fluctuations in [Ca2+]i. The peak depolarization signal coincided with the maximum rate of increase in the [Ca2+]i signal. Oscillations in the Em signal were inhibited by removal of Ca2+ or by addition of 1 mM Ni2+ to the external solution. Taken together, these observations suggest that the change in Em is the consequence of oscillatory changes in a membrane conductance that also allows Ca2+ to enter the cell. Oscillations in the DiBAC4(3) signal may reflect a rhythmic entry of Ca2+ through nonselective cation channels.

Cytotoxic effects of ingested Staphylococcus aureus on bovine endothelial cells: Role of S. aureus α-hemolysin

Previously we observed that Staphylococcus aureus phagocytized by cultured bovine endothelial cells do not proliferate intracellularly, but are cytotoxic to bovine endothelial cells. To investigate S. aureus virulence factors which may be produced intracellularly and cause lysis of endothelial cells, we tested S. aureus mutants defective in production of one or more potential virulence factors and corresponding parent strains for cytotoxicity to endothelial cell monolayers subsequent to being ingested. Following incubation of endothelial cell monolayers with S. aureus for 3.5 h, cultures were supplemented with lysostaphin to destroy extracellular but not intracellular S. aureus. At subsequent times, viability of endothelial cells was assayed by retention of 3H-adenine and the number of intracellular S. aureus was measured. The cytotoxic activity of S. aureus culture supernatants was also characterized. The results indicate that S. aureus α-hemolysin is cytotoxic to bovine endothelial cells and plays an important role in the damage suffered by bovine endothelial cell monolayers following ingestion of S. aureus. Ingestion of α-hemolysin-producing S. aureus by endothelial cells in vivo might be expected to result in destruction of endothelium followed by development of platelet-fibrin vegetations. This possible sequence of events is compatible with the frequently fulminant course of S. aureus endocarditis.

ENDOGENOUS NITRIC OXIDE INHIBITS HUMAN PLATELET ADHESION TO VASCULAR ENDOTHELIUM

The adhesion of human platelets to monolayers of bovine endothelial cells in culture was studied to determine the role of endothelium-derived nitric oxide in the regulation of platelet adhesion. The adhesion of unstimulated and thrombin-stimulated platelets, washed and labelled with indium-111, was lower in the presence than in the absence of bradykinin or exogenous nitric oxide. The inhibitory action of both bradykinin and nitric oxide was abolished by haemoglobin, but not by aspirin, and was potentiated by superoxide dismutase to a similar degree. It is suggested that the effect of bradykinin is mediated by the release of nitric oxide from the endothelial cells, and that nitric oxide release contributes to the non-adhesive properties of vascular endothelium.

Interference With Tumor Cell-Induced Degradation of Endothelial Matrix on the Antimetastatic Action of Nafazatrom2

The antithrombotic compound nafazatrom was evaluated in several in vivo and in vitro assays to elucidate the mechanism of its antimetastatic activity. C57BL/6 mice bearing B16 amelanotic subcutaneous tumors treated with 100 mg nafazatrom/kg/day exhibited a sixfold reduction in metastatic pulmonary lesions compared to lesion numbers in controls. The reduction in metastatic lesions was not accompanied by changes in primary tumor growth, and up to 1 microgram nafazatrom/ml did not inhibit tumor cell proliferation in vitro. Treatment of C57BL/6 mice with nafazatrom prior to iv inoculation of tumor cells failed to inhibit lung colony formation. In vitro exposure of exponentially growing B16 amelanotic cells to nafazatrom (1 microgram/ml for 72 hr) in culture did not change their ability to adhere to endothelial cell monolayers. B16 amelanotic cells degraded the matrix material of bovine endothelial cell monolayers; a heparin sulfate proteoglycan appeared to be the predominant matrix component released by these tumor cells, as judged by resistance to chondroitin ABC lyase and sensitivity to heparitinase and pronase degradation. Nafazatrom (1 microgram/ml for 72 hr) inhibited the solubilization of matrix components by approximately 60%. Tumor cell degradation of matrix components is an important event in the pathogenesis of metastasis. Thus the interference with this process appears to provide an explanation for the inhibition of malignant cell dissemination in vivo by nafazatrom.

944 DDAVP INHIBITS PROSTACYCLIN FORMATION: A POTENTIAL MECHANISM FOR BLEEDING TIME (BT) CORRECTION IN HEMOSTATIC DISORDERS

A recent study has shown that DDAVP normalizes the B.T. in plt. functional defects. In vitro evidence also suggests that DDAVP enhances plt-vascular adherence. We assessed the effects of DDAVP on prostacyclin as a possible mediator of these effects. Initial studies on human vessels revealed that incubation of segments with DDAVP ↓ 6KPGF1α formation. Values of 3.4±.0.8 and 2.5±0.7 pmol/mg were obtained in the presence of 0.1 and 1.0 μg/ml DDAVP, with paired control values of 4.1±1.0 and 4.0±1.1 (p<0.01). No differences in 6KPGF1α were observed when segments were incubated with DDAVP vehicle alone (0.1μg/ml), although at 1μg/ml a slight ↓ in 6KPGF1α was seen. Direct assessment of the effects of DDAVP on ionophore (10μM) stimulated release of 6KPGF1α from bovine endothelial cell monolayers revealed that at 1μg/ml both DDAVP and its vehicle caused a ↓ in 6KPGF1α release (33+18 and 31±16 pmol per 106 cells versus control values of 121±46 pmol;p<0.01). Finally, changes in plasma 6KPGF1α are being assessed in patients (n=5) with plt. functional defects in whom DDAVP has been used as a therapeutic modality to correct their prolonged BTs. A ↓ in plasma 6KPGF1α has been observed with mean levels of 0.32 and 0.25 pmol per ml prior to and 90′ post DDAVP respectively. These studies, demonstrating an in vitro and in vivo effect of DDAVP on endothelial cell prostacyclin production, suggest a potential, hitherto unrecognized, mechanism for BT correction in hemostatic disorders.

Role of hydrogen peroxide in the neutrophil-mediated release of prostacyclin from cultured endothelial cells.

We have examined the effect of activated neutrophils on the release of prostacyclin (PGI2) from cultured endothelial cells by radioimmunoassay and thin layer chromatography of its stable metabolite, 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha). Phorbol myristate acetate-activated neutrophils induced a time- and dose-dependent release of 6-keto-PGF1 alpha from human and bovine endothelial cell monolayers, whereas phorbol myristate acetate alone and neutrophils alone did not. Pretreatment of the endothelial cells with aspirin prevented neutrophil-mediated 6-keto-PGF1 alpha release, indicating that it did not depend upon neutrophil-generated endoperoxides. Phorbol myristate acetate-activated neutrophils from a patient with chronic granulomatous disease failed to induce endothelial 6-keto-PGF1 alpha release. Addition of catalase but not of superoxide dismutase significantly reduced human and bovine endothelial 6-keto-PGF1 alpha release by phorbol myristate acetate-activated neutrophils. Catalase-inhibitable endothelial 6-keto-PGF1 alpha release was also observed after the addition of the hydrogen peroxide-generating system, glucose-glucose oxidase, to bovine and human endothelial cell monolayers. Bovine endothelial 6-keto-PGF1 alpha release induced by exogenously generated hydrogen peroxide was attenuated by the phospholipase inhibitor mepacrine, suggesting that hydrogen peroxide may act by triggering endothelial membrane phospholipase activation. The release of 6-keto-PGF1 alpha by enzymatically or neutrophil-generated hydrogen peroxide was not associated with endothelial cell lysis as assessed by 51Cr release. We conclude that exogenously generated hydrogen peroxide or a hydrogen peroxide-derived product mediates rapid nonlytic release of PGI2 from cultured endothelial cells.

Sulfated proteoglycans synthesized by vascular endothelial cells in culture.

Metabolically labeled proteoglycans were isolated both from the culture medium and from the cell layer of cultured bovine aortic endothelial cells. Proteoglycans were fractionated by sequential gel filtration on Sepharose CL-4B, DEAE-cellulose column chromatography, and CsCl density gradient centrifugation. The culture medium contained two distinct proteoglycans: a large proteoglycan (Kav = 0.07) of low buoyant density containing heparan sulfate side chains (Mr congruent to 36,000) and a smaller proteoglycan (Kav = 0.45) of high density containing chondroitin sulfate chains (Mr congruent to 20,000). The chondroitin sulfate proteoglycan fraction contained a small amount (less than 10%) of dermatan sulfate. A very similar low density heparan sulfate proteoglycan was extracted from the cell layer with 2% sodium dodecyl sulfate in the presence of enzyme inhibitors. In addition, there was a high density proteoglycan of small size (Kav = 0.43) containing heparan sulfate side chains (Mr congruent to 20,000) in the cell layer. Analyses of proteoglycans synthesized by cultured human umbilical vein endothelial cells gave similar results, except that these cells produced more dermatan sulfate and unidentified oversulfated chondroitin sulfate chains. Morphologically atypical endothelial cells contained reduced levels of the large heparan sulfate proteoglycan. Both indirect immunofluorescence and direct immunoelectron microscopy revealed that the basement membrane-like matrix under monolayers of bovine endothelial cells reacted with antibodies against the basement membrane proteoglycan isolated from a basement membrane-producing tumor. By electron microscopy, this material was shown to consist of a fine filamentous meshwork containing discrete 10-20-nm diameter ruthenium red positive granules resembling those present in basement membranes of intact arteries.

A comparative study of thromboxane and prostacyclin release from ex vivo cultured bovine vascular endothelium

Thromboxane B 2, 6-keto-Prostaglandin F 1α, and Prostaglandin E 2 release have been quantitated from cultured adult by bovine endothelial cell monolayers and from ex Vivo vascular segments employing specific radioimmunoassay and thin layer chromatography. Release of all three prostaglandins was demonstrable from both endothelial cell systems under basal conditions and following exposure to the ionophore A23187 and arachidonic acid. In culture, the quantity of 6-keto-PGF 1α released was diminished compared to amounts released from the vessel segments while thromboxane B 2 and prostaglandin E 2 release were similar in the two endothelial model systems. However, the amount of thromboxane B 2 assayed was small and the quantity of thromboxane A 2 it represents is probably of little in Vivo significance to prostacyclin.

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The effects of treatment with colchicine, cytochalasin B, and low temperature (4 degrees C) on the metastatic behavior of the B16-F10 melanoma cell line were examined. The growth of metastases and the distribution of radiolabeled tumor cells were monitored in inbred C57/BL6 mice given iv injections of B16-F10 cells. Cells treated previously with both drugs, but not with low temperature, produced fewer lung nodules than did control cells and displayed alterations in tumor dissemination patterns. In vitro studies revealed that both drugs reduced the rate of adhesion of the tumor cells to bovine endothelial cell monolayers, the rate of migration from agarose droplets, the formation of homotypic aggregates, and agglutination by wheat germ agglutinin. The drugs also induced morphologic alterations of the cells grown in monolayer culture but had little effect on cell volume.