Endothelial cell confluence regulates cyclooxygenase-2 and prostaglandin E2 production that modulate motility

Endothelial cells line the vasculature and, after mechanical denudation during invasive procedures or cellular loss from natural causes, migrate to reestablish a confluent monolayer. We find confluent monolayers of human umbilical vein endothelial cells were quiescent and expressed low levels of cyclooxygenase-2, but expressed cyclooxygenase-2 at levels comparable with cytokine-stimulated cells when present in a subconfluent culture. Mechanically wounding endothelial cell monolayers stimulated rapid cyclooxygenase-2 expression that increased with the level of wounding. Cyclooxygenase-2 re-expression occurred throughout the culture, suggesting signaling from cells proximal to the wound to distal cells. Media from wounded monolayers stimulated cyclooxygenase-2 expression in confluent monolayers, which correlated with the level of wounding of the donor monolayer. Wounded monolayers and cells in subconfluent cultures secreted enhanced levels of prostaglandin (PG) E(2) that depended on cyclooxygenase-2 activity, and PGE(2) stimulated cyclooxygenase-2 expression in confluent endothelial cell monolayers. Cells from subconfluent monolayers migrated through filters more readily than those from confluent monolayers, and the cyclooxygenase-2-selective inhibitor NS-398 suppressed migration. Adding PGE(2) to NS-398-treated cells augmented migration. Endothelial cells also migrated into mechanically denuded areas of confluent monolayers, and this too was suppressed by NS-398. We conclude that endothelial cells not in contact with neighboring cells express cyclooxygenase-2 that results in enhanced release of PGE(2), and that this autocrine and paracrine loop enhances endothelial cell migration to cover denuded areas of the endothelium.     

Serum, trypsin, and cell shape but not cell-to-cell contact influence the X-ray sensitivity of Chinese hamster V79 cells in monolayers and in spheroids

Nutrient concentration in the growth medium and trypsin affect cellular radiosensitivity in a manner that is related to cell shape (Reddy, Stevenson, and Lange, Int. J. Radiat. Biol. 55, 105-117 (1989); Reddy and Lange, Radiat. Res. 119, 338-347 (1989]. Hence we hypothesized that the concentration of serum in the medium could influence the X-ray sensitivity of cells and that the spread cells in monolayers and round cells in spheroids may differ in their response to the radiosensitizing effect of trypsin. We compared the X-ray sensitivity of monolayer and spheroid cells grown for 19 +/- 1 h in MEM supplemented with 5 or 15% serum. Cells were trypsinized and plated either immediately before, or 2.5 +/- 0.5 h after, irradiation and incubation for repair in situ. Survival of cells in monolayers and in spheroids was higher in MEM with 5% serum than with 15% serum. Trypsin treatment affected the shape and radiosensitivity of cells in monolayers but not in spheroids. When all cells were grown in the same serum concentration and a 2.5-h postirradiation incubation was allowed prior to trypsinization, the X-ray sensitivity of cells in spheroids was greater than that of cells in monolayers. The survival of cells in spheroids became equal to that of monolayer cells when cells in spheroids were converted to monolayers by placing them in 25-cm2 flasks and allowing them 3 h to attach and spread. Cell cycle distributions were nearly the same in monolayers and spheroids cultured in MEM with 5 or 15% serum. We conclude that: (1) serum concentration in the growth medium and trypsin do appear to contribute to the differences in the radiosensitivity of spheroids and monolayer V79 cells; (2) these differences are associated with changes in cell morphology.     

An ultra performance liquid chromatographic method for determining phytosterol uptake by Caco-2 cells

A simple method for the determination of cellular uptake of phytosterols by Caco-2 cells has been developed by ultra performance liquid chromatography with ultraviolet detection (UPLC-UV). UPLC-UV was established using an ODS column, acetonitrile/H(2)O (9:1, v/v) as a mobile phase, and a detection wavelength at 210 nm. As analytes, β-sitosterol, campesterol, stigmasterol, and brassicasterol were selected based on the abundance in foods and the similarity of their structures. A linear relation was observed between the peak area and the amount of sterol injected from 50 to 2000 pmol (r>0.999) with a relative standard deviation (RSD) of less than 2.5% (n=6). This method was applied to the determination of cellular uptake of phytosterols by Caco-2 cells. Recovery tests showed that phytosterols were extracted from the cell lysates by chloroform and determined by UPLC-UV with a recovery rate of more than 80.2% and an RSD of less than 11.3% (n=3). When Caco-2 cells were incubated with phytosterols at 37°C, their uptake was increased with time in a concentration-dependent manner. This method will be useful for the simultaneous determination of cellular phytosterols in an in vitro intestine model.     

Immunocytochemical and Biochemical Analysis of Carbonic Anhydrase in Primary Astrocyte Cultures from Rat Brain

Carbonic anhydrase (CA) was studied in primary monolayer cultures from neonatal rat cerebral hemispheres with both immunocytochemical and biochemical techniques. In such cultures, which consist predominantly of astrocytes, immunocytochemical staining for CA using antibody raised against the type II enzyme from rat erythrocytes resulted in positive staining of the flat, glial fibrillary acidic protein-positive, astrocytic monolayer. Smaller, process-bearing, round cells that grew on top of the astrocytes stained intensely for CA. We estimated that these cells represented 1% or less of the total cells in the cultures, and they have been identified by others as oligodendrocytes. The intensity of the staining of astrocytes for CA could be increased to that observed in oligodendrocytes when the astrocytes were made to round up and form processes by treatment with 2',3'-dibutyryl cyclic AMP. Enzymatic assays showed that CA activity of the cultures after 3 weeks of growth was 2.5- to 5-fold less than that found for cerebral homogenates from perfused 3-week-old rat brains. However, both activities were totally inhibited by acetazolamide with an I50 of 10(-8) M, confirming that both rat brain and the astrocyte cultures possess the high-activity type II enzyme. CA-II activity was unaffected by treatment of the cultures with a method reported to remove oligodendrocytes. Thus, the immunocytochemical and biochemical studies reported here demonstrate that astroglial cells in primary cultures from neonatal rat brain contain CA-II.     

Isolation of Chinese hamster ovary cells with reduced poly(ADP-ribose) polymerase activity

The biological function of poly(ADP-ribose) polymerase in DNA repair, cell-cycle regulation and cellular differentiation has yet to be defined. Isolation of cells which are deficient in poly(ADP-ribose) synthesis would greatly facilitate the determination of the biological role of this enzyme. A method is described for isolating Chinese hamster ovary (CHO) cells deficient in the poly(ADP-ribose) polymerase activity by direct screening of colonies for enzyme activity. Colonies with decreased production of poly(ADP-ribose) are recovered from nylon replicas for further analysis. Using this method we have isolated a series of CHO cells which have 50% or less poly(ADP-ribose) polymerase activity. These mutants have normal generation times and are 20% more sensitive to the effects of DNA (m)ethylating agents than the parental cell. However, these mutants display normal sensitivity to gamma-rays.     

Evidence that natural cytotoxicity and antibody-dependent cellular cytotoxicity are mediated in humans by the same effector cell populations.

The present study strongly suggests that, in humans, natural killer (NK) activity and antibody-dependent cell-mediated cytotoxicity (ADCC) are mediated by the same effector cell population. This is supported by two different experimental approaches. First, competition for NK effector cells was accompanied by simultaneous inhibition of ADCC activity. Target cells sensitive to NK activity were capable of inhibiting specifically an ADCC assay in cold target competition experiments. Second, specific removal of NK cells on monolayers formed by target cells sensitive to NK activity caused simultaneous depletion of ADCC effector cells. In association with the removal on the monolayers of effector cells for ADCC as well as NK activity, we also found a significant depletion of cells bearing Fc gamma receptors.     

Carboanhydrase activity in halophilic anaerobes from soda lakes

The activity and cellular localization of carboanhydrase (CA) in two alkaliphilic anaerobes growing in soda lakes at pH 9-10 was studied. CA activity in the cell extracts of the acetogenic bacterium Natroniella acetigena was comparable to that of the neutrophilic acetogens. Hydrogenotrophically grown cells of Desulfonatronum lacustre exhibited higher CA activity compared to the cells grown on media with formate. High CA activity in the cytoplasmic fraction and the absence of high activity in the extracellular fraction were demonstrated. We propose that the cytoplasmic CA in alkaliphilic sulfate-reducers participates in conversion of bicarbonate to CO2, which is reduced in the cell to acetate via the acetyl-CoA pathway.     

Acute radiation effects on the content and release of plasminogen activator activity in cultured aortic endothelial cells

Confluent monolayers from three lines of bovine aortic endothelial cells were exposed to a single dose of 10 Gy of 60Co gamma rays. Seventy-two hours later, the morphology of the irradiated and sham-irradiated monolayers was examined, and cellular DNA and protein contents were determined. In addition, the release of plasminogen activator (PA) activity into the culture media and PA activity in the cell lysates were assayed. Irradiated monolayers maintained their cobblestone appearance, but individual endothelial cells were enlarged considerably compared to sham-irradiated cells. DNA and protein contents in the irradiated monolayers were reduced to 43-50% and 72-95% of the control levels, respectively. These data indicate that radiation induced cell loss (detachment and/or lysis) from the monolayer, with hypertrophy of surviving (attached) cells to preserve the continuity of the monolayer surface. Total PA activity (lysate plus medium) in the irradiated dishes was reduced to 50-75% of the control level. However, when endothelial PA activity was expressed on the basis of DNA content, the irradiated monolayers from two of the three cell lines contained significantly more PA activity than did sham-irradiated monolayers. Most importantly, the percentage of the total PA activity released into the culture medium by irradiated cells (5-22%) was significantly (P less than 0.001) lower than that released by sham-irradiated cells (23-68%). These data suggest that fibrinolytic defects observed in irradiated tissues in situ may be attributable at least in part to a radiation-induced inhibition of PA release by vascular endothelial cells.     

Calcein as a fluorescent probe for ferric iron. Application to iron nutrition in plant cells

The recent use of calcein (CA) as a fluorescent probe for cellular iron has been shown to reflect the nutritional status of iron in mammalian cells (Breuer, W., Epsztejn, S., and Cabantchik, Z. I. (1995) J. Biol. Chem. 270, 24209-24215). CA was claimed to be a chemosensor for iron(II), to measure the labile iron pool and the concentration of cellular free iron(II). We first study here the thermodynamic and kinetic properties of iron binding by CA. Chelation of a first iron(III) involves one aminodiacetic arm and a phenol. The overall stability constant log beta111 of FeIIICAH is 33. 9. The free metal ion concentration is pFeIII = 20.3. A (FeIII)2 CA complex can be formed. A reversible iron(III) exchange from FeIIICAH to citrate and nitrilotriacetic acid is evidenced when these ligands are present in large excess. The kinetics of iron(III) exchange by CA is compatible with metabolic studies. The low reduction potential of FeIIICAH shows that the ferric form is highly stabilized. CA fluorescence is quenched by 85% after FeIII chelation but by only 20% using FeII. Real time iron nutrition by Arabidopsis thaliana cells has been measured by fluorimetry, and the iron buffer FeIIICAH + CA was used as source of iron. As a siderophore, FeIIICAH promotes cell growth and regreening of iron-deficient cells more rapidly than FeIIIEDTA. We conclude that CA is a good chemosensor for iron(III) in cells and biological fluids, but not for Fe(II). We discuss the interest of quantifying iron buffers in biochemical studies of iron, in vitro as well as in cells.     

Sensing minute changes in biological cell monolayers with THz differential time-domain spectroscopy

We used terahertz differential time-domain spectroscopy (THz-DTDS) to measure minute changes of bovine lung microvessel endothelial cells (BLMVEC) in response to vascular endothelial growth factor (VEGF). These changes were reflected by alterations in THz wave attenuations and THz dielectric properties of the treated cells. The VEGF-induced THz attenuations of cell monolayers correlated well with changes in transendothelial resistance, as measured using electric cell-substrate impedance sensing (ECIS). However, the morphological differences that gave rise to these changes were not observed with standard optical phase contrast microscopy. We conclude that THz-DTDS is a highly sensitive, non-invasive, powerful new tool to measure minute changes in the morphology of live, cultured cell monolayers. This method enables spectroscopic investigations of cells in the THz band, providing information unavailable through other conventional methods such as optical phase contrast microscopy and ECIS.     

Calcium, carbonic anhydrase and gastric acid secretion

Previous data concerning the action of calcium (Ca) on gastric acid secretion (GAS) indicated that calcium ions increase GAS elicited by gastrin released through a vagal mechanism, and also by a direct effect on parietal cells. Our research showed that the stimulating effect of calcium on gastric acid secretion can be antagonized by verapamil administration, which reduces gastric acid secretion . In the present study we followed the effect induced by administration of calcium and Ca-chelating agents (disodium EDTA) on gastric acid secretion and on carbonic anhydrase (CA) activity. We selected two groups of healthy volunteers: Group I (n=21) received a single i.v. dose of CaCl2 (15 mg/kg b.w.), whereas Group II (n=22) received a single i.v. dose of disodium EDTA (5 mg/kg b.w.). We determined blood calcium before and after treatment, gastric acid secretion at 2 hours. erythrocyte CA II activity, and CA IV activity in membrane parietal cells, which were isolated from gastric mucosa obtained by endoscopic biopsy. Assessment of carbonic anhydrase activity was achieved by the stopped-flow method. In Group I calcium administration increased blood calcium, HCl output, CA II and CA IV activity as compared to initial values. In Group II, disodium EDTA reduced blood calcium, HCl output, CA II and CA IV activity as compared to initial values. The results demonstrated that increased blood calcium and GAS values after calcium administration correlated with the increase of erythrocyte CA II and parietal cell CA IV activity, while disodium EDTA induced a reversed process. Our results also show that cytosolic CA II and membrane CA IV values are sensitive to calcium changes and they directly depend on these levels. Our data suggest that intra- and extracellular pH changes induced by carbonic anhydrase might account for the modulation of the physiological and pathological secretory processes in the organism.     

Combination of an enzymatic method and HPLC for the quantitation of cholesterol in cultured cells.

The study of the cellular events that lead to the foam cell formation requires the development of fast, accurate, and sensitive methods to quantify cholesterol in cultured cells. Here we describe a procedure that allows the rapid determination of free and total cholesterol in a reduced number of cells, which makes it very suitable for cholesterol determination in cell cultures. The method consists of the enzymatic conversion of cholesterol to cholest-4-ene-3-one by cholesterol oxidase followed by the analysis of the sample by high performance liquid chromatography (HPLC) to detect this oxidized product. Due to the relatively high wavelength at which cholest-4-ene-3-one has its maximum absorption (240 nm), other cellular components do not interfere with the chromatographic procedure and prior lipid extraction is not required. Moreover, the duration of each chromatogram is about 3 min, contributing to the celerity of the method. All the cholesteryl esters used (oleate, palmitate, stearate and linoleate) were quantitatively hydrolyzed by incubation with cholesterol esterase; this was observed to occur with both pure standards and in cell homogenates. Sensitivity is enough to allow the determination of free and total cholesterol in less than 5 x 10(3) cells. We have applied this method to human monocyte-derived macrophages and the values obtained for free and total cholesterol are in close agreement with published data.     

Determinations of catechols in small volumes of plasma using ion-pair reversed phase liquid chromatography/electrochemistry

A method with improved sensitivity for detection of catechols (CA) in small volumes of plasma using an ion-pair reversed phase HPLC system with electromechemical detection is presented. Fast isocratic separations were obtained by using 7.5 cm x 4.6 mm (i.d.) reversed phase columns with 3C18 3 micron silica particles. The CA:s L-DOPA, Noradrenaline (NA), Adrenaline (A), Dihydroxybenzylamine (DHBA, i.s.), DOPAC and Dopamine (DA) were separated in less than 4 min. The performance of three different electrochemical cells was compared with respect to hydrodynamic voltammogram, band broadening effect, linearity and detection limit. The sample preparation procedure using alumina extraction of CA:s, was modified to improve recoveries and decrease dilution factors. A modified carbon paste cell (CP-O) gave a response 4-8 times higher than what is previously reported for GC cells. Detection limits were: L-DOPA 80, NA 1.25, A 1.25, DHBA 0.4, DOPAC 1.25 and DA 0.6 pg/injection. Application to plasma from rat and fish (cod) under rest, exercise and stress is reported. The method allows determination of CA:s in small volumes of plasma (less than 500 microliter) obtained several times a day from the same animal even if it is small (less than 1/2 kg), is under rest and parts of the plasma sample are to be used for analysis of other parameters than CA:s.     

Individual-based and continuum models of growing cell populations: a comparison

In this paper we compare two alternative theoretical approaches for simulating the growth of cell aggregates in vitro: individual cell (agent)-based models and continuum models. We show by a quantitative analysis of both a biophysical agent-based and a continuum mechanical model that for densely packed aggregates the expansion of the cell population is dominated by cell proliferation controlled by mechanical stress. The biophysical agent-based model introduced earlier (Drasdo and Hoehme in Phys Biol 2:133-147, 2005) approximates each cell as an isotropic, homogeneous, elastic, spherical object parameterised by measurable biophysical and cell-biological quantities and has been shown by comparison to experimental findings to explain the growth patterns of dense monolayers and multicellular spheroids. Both models exhibit the same growth kinetics, with initial exponential growth of the population size and aggregate diameter followed by linear growth of the diameter and power-law growth of the cell population size. Very sparse monolayers can be explained by a very small or absent cell-cell adhesion and large random cell migration. In this case the expansion speed is not controlled by mechanical stress but by random cell migration and can be modelled by the Fisher-Kolmogorov-Petrovskii-Piskounov (FKPP) reaction-diffusion equation. The growth kinetics differs from that of densely packed aggregates in that the initial spread, as quantified by the radius of gyration, is diffusive. Since simulations of the lattice-free agent-based model in the case of very large random migration are too long to be practical, lattice-based cellular automaton (CA) models have to be used for a quantitative analysis of sparse monolayers. Analysis of these dense monolayers leads to the identification of a critical parameter of the CA model so that eventually a hierarchy of three model types (a detailed biophysical lattice-free model, a rule-based cellular automaton and a continuum approach) emerge which yield the same growth pattern for dense and sparse cell aggregates.     

Carbonic anhydrase activity in halophilic anaerobes from soda lakes

The activity and cellular localization of carbonic anhydrase (CA) in two alkaliphilic anaerobes growing in soda lakes at pH 9–10 were studied. CA activity in the cell extracts of the acetogenic bacterium Natroniella acetigena was comparable to that of neutrophilic acetogens. Hydrogenotrophically grown cells of Desulfonatronum lacustre exhibited higher CA activity compared to the cells grown on medium with formate. High CA activity in the cytoplasmic fraction and the absence of high activity in the extracellular fraction were demonstrated. We propose that the cytoplasmic CA in alkaliphilic sulfate-reducers participates in conversion of bicarbonate to CO₂, which is reduced in the cell to acetate via the acetyl-CoA pathway.     

Specific versus nonspecific target cell destruction by T lymphocytes sensitized in vitro. I. Kinetics

An in vitro culture method giving a high cell recovery rate of 25–38% has been developed to generate a relatively pure population of T cell progeny reacting to xenogeneic cell antigen. The method is basically a modification of the Ginsburg method and utilizes mosaic monolayers that are comprised of syngeneic Lewis and xenogeneic BALB/c fibroblasts. Thoracic duct lymphocytes of Lewis rats were cultured for 6 days on these monolayers and the resulting lymphocyte population, rich in blast cells, was collected and separated free of contaminating fibroblasts by an additional 6 hr incubation without monolayers. Cytotoxic activity of these harvested lymphocytes was assessed by a modification of the Hellström method in which embryonic target fibroblasts, Lewis or BALB/c, were plated at a critical density of 2200 cells/cm² of culture area. This, coupled with the use of a newly developed culture medium, allowed the demonstration of specific as well as nonspecific activity of up to 100% suppression at 48 hr of test incubation. The specific activity was clarified and determined critically by comparing the activity of these lymphocytes to that of lymphocytes grown on syngeneic monolayers, though rich in blast cells, reacting only to the horse serum in the medium. The nonspecific activity was not due to the deterioration of test cultures and needed the continued presence of fully active blast cells for complete suppression of the target cell growth in the later phase of test cultures. By changing the ratio of lymphocytes to target cells and following the kinetics of target cell destruction, it was shown that specific activity effects an active killing of target cells, whereas the nonspecific activity was primarily the suppression of target cell growth. The specific activity was effective with far less lymphocytes per target cell; was nearly complete by 12 hr of test cultures while the nonspecific activity was complete at 48 hr. Yet, the increase in the specific activity was nearly proportional to that of nonspecific activity and even the nonspecific activity seemed to kill sensitive Lewis target cells at its full intensity. These observations are in accordance with an interpretation of the mechanism of the specific target cell destruction as involving contact and recognition followed by the release of lymphotoxin into or onto target cells.     

Ensemble place codes in hippocampus: CA1, CA3, and dentate gyrus place cells have multiple place fields in large environments

Previously we reported that the hippocampus place code must be an ensemble code because place cells in the CA1 region of hippocampus have multiple place fields in a more natural, larger-than-standard enclosure with stairs that permitted movements in 3-D. Here, we further investigated the nature of hippocampal place codes by characterizing the spatial firing properties of place cells in the CA1, CA3, and dentate gyrus (DG) hippocampal subdivisions as rats foraged in a standard 76-cm cylinder as well as a larger-than-standard box (1.8 m×1.4 m) that did not have stairs or any internal structure to permit movements in 3-D. The rats were trained to forage continuously for 1 hour using computer-controlled food delivery. We confirmed that most place cells have single place fields in the standard cylinder and that the positional firing pattern remapped between the cylinder and the large enclosure. Importantly, place cells in the CA1, CA3 and DG areas all characteristically had multiple place fields that were irregularly spaced, as we had reported previously for CA1. We conclude that multiple place fields are a fundamental characteristic of hippocampal place cells that simplifies to a single field in sufficiently small spaces. An ensemble place code is compatible with these observations, which contradict any dedicated coding scheme.     

Regulation of cytoskeletal structure in human mammary carcinoma cells MCF-7 by culture substrata

Three-dimensional cellular structures formed by MCF-7 human mammary carcinoma cells within collagen gels were isolated with collagenase and cultivated on plastic substratum to examine whether the cytoskeleton specific for cells forming cellular structures (S-type) changes to that specific for cells grown as monolayers (M-type). The cytoskeleton isolated as 0.05% Triton-insoluble fraction from the cellular structures after culture for 1 day on plastic was exclusively S-type. However, both types of cytoskeletons were observed in the cellular structures cultivated for 7 days on plastic as well as in the cells grown as monolayers for 2 days after dissociation of the cellular structures with trypsin. By use of an antibody raised against a 65-kD polypeptide that was specific for the M-type cytoskeleton, the presence of the polypeptide was found to be restricted to the cells grown out as monolayers from the edge of the cellular structures. In the cells grown for 2 days as monolayers, a mixture of cells both having and lacking the polypeptide was observed. After a 7-day culture of the dissociated cells as monolayers on plastic, however, most of the cells had M-type cytoskeletons. The present results show that the apparent change in the cytoskeleton of MCF-7 cells from S-type to M-type does not occur in cells involved in the three-dimensional cellular structures even in the absence of collagen gels, but that it occurs in cells which are grown as monolayers for at least 7 days on plastic substratum.     

A rapid fluorescent screening method for cellular sensitivity to anti-cancer compound

The tetrazolium salts (MTT, XTT, MTS, WST) based colorimetric assay or resazurin based fluorimetric assay are currently typical methods for cell sensitivity determination to anticancer compounds. We presented here a new rapid method for this purpose. This method uses a fluorescent dye named DCFH-DA which is previously taken as a intracellular probe for measurement of H(2)O(2) levels within a cell. The application basis for this method lies in two facts: the membrane permeable feature of the final metabolite of DCFH-DA inside a cell, and the linearity relationship between cell number and H(2)O(2) level. The results showed that there was a perfect association between cell number and fluorescent intensity determined by the DCFH-DA method, no matter whether using resuspended or adherent cells, and further 50% concentration of inhibition (IC(50)) comparison between data obtained by DCFH-DA method or MTT method using a positive known anticancer compound Baicalin showed that there were no significant differences in cellular sensitivity determination to compound Baicalin though there existed a relatively higher coefficient of variation of IC(50) by the DCFH-DA method than that by the MTT method. Thus our data indicate that DCFH-DA might not only be a fine reagent for determination of H(2)O(2) levels in cells but also an ideal fluorescent dye for cellular sensitivity test of anti-cancer compounds, and may be suitable for primary high-throughput drugs screening.