Alan K. Keenan

Modulation of human endothelial cell permeability by combinations of the cytokines interleukin-1 α/β, tumor necrosis factor-α and interferon-γ

The permeability of human umbilical vein endothelial cell (HUVEC) monolayers to [125I]-labelled bovine serum albumin (BSA) was examined following pretreatment of the cells with various cytokines. The electrical resistance measured across untreated, confluent, intact HUVEC monolayers was 18.2 +/- 3.8 omega.cm2 (mean +/- S.D. of 4 observations). Human recombinant (hr) interleukin-1 alpha/beta (IL-1 alpha/beta), hr tumor necrosis factor-alpha (TNF-alpha), and hr interferon-gamma (IFN-gamma) each increased HUVEC monolayer permeability in a time- and dose-dependent manner. These effects were inhibitable by neutralizing antibodies (nAb) to the corresponding cytokines, and were not due to contamination by endotoxin (abolition of cytokine effect by heat treatment, and no effect on cytokine action of the endotoxin inhibitor polymyxin B). The effects of these cytokines were not due to endothelial cell (EC) interleukin-6 (IL-6) induction (IL-6 shown not to increase permeability) and the effect of hrTNF-alpha could not be accounted for by induction of IL-1 (effect not inhibited by hrIL-1 alpha/beta nAb). The effects of three different combinations of the cytokines (each combination at two different concentrations) on HUVEC monolayer permeability were also examined. hrIFN-gamma with hrTNF-alpha or hrIL-1 alpha/beta gave an increase in permeability (at both concentration combinations) greater than that seen with either cytokine alone. hrTNF-alpha and hrIL-1 alpha/beta in combination however produced an enhanced effect only at low concentrations, high concentrations in combination producing an effect no greater than either agent alone. These results highlight the importance of investigating actions of cytokine combinations on in vitro models of endothelial cell activation.

Interaction of soft condensed materials with living cells: Phenotype/transcriptome correlations for the hydrophobic effect

The assessment of biomaterial compatibility relies heavily on the analysis of macroscopic cellular responses to material interaction. However, new technologies have become available that permit a more profound understanding of the molecular basis of cell–biomaterial interaction. Here, both conventional phenotypic and contemporary transcriptomic (DNA microarray-based) analysis techniques were combined to examine the interaction of cells with a homologous series of copolymer films that subtly vary in terms of surface hydrophobicity. More specifically, we used differing combinations of N-isopropylacrylamide, which is presently used as an adaptive cell culture substrate, and the more hydrophobic, yet structurally similar, monomer N-tert-butylacrylamide. We show here that even discrete modifications with respect to the physiochemistry of soft amorphous materials can lead to significant impacts on the phenotype of interacting cells. Furthermore, we have elucidated putative links between phenotypic responses to cell–biomaterial interaction and global gene expression profile alterations. This case study indicates that high-throughput analysis of gene expression not only can greatly refine our knowledge of cell–biomaterial interaction, but also can yield novel biomarkers for potential use in biocompatibility assessment.

Poly(N-isopropylacrylamide) co-polymer films as potential vehicles for delivery of an antimitotic agent to vascular smooth muscle cells

INTRODUCTION Local delivery of antimitotic agents is a potential therapeutic strategy for protection of injured coronary vasculature against intimal hyperplasia and restenosis. This study sought to establish the principle that thermoresponsive poly(N-isopropylacrylamide) co-polymer films can be used to deliver, in a controlled manner, an antimitotic agent to vascular smooth muscle cells (VSMC). METHODS A series of co-polymer films was prepared, using varying ratios (w/w) of N-isopropylacrylamide (NiPAAm) monomer to N-tert-butylacrylamide (NtBAAm) and loaded with the antimitotic agent colchicine (100 nmol/film) at room temperature. RESULTS The extent of colchicine release at 37 degrees C was inversely proportional to the amount of NtBAAm in co-polymer films: release after 48 h from 85:15, 65:35 and 50:50 (NiPAAm:NtBAAm) films was 26, 17 and 0.5 nmol, respectively. In cytotoxicity studies, when medium incubated with co-polymers for 24 h (in the absence of colchicine) was further incubated with target bovine aortic smooth muscle cells (BASMC), no loss of cell viability occurred. Colchicine released from all three co-polymer films significantly inhibited proliferation and random migration of BASMC: 100 nM colchicine (released from 65:35 NiPAAm:NtBAAm) reduced cell proliferation to 25.7+/-1.7% of levels seen in the absence of colchicine (control) and random cell migration to 37.7+/-5.7% of control (mean+/-S.E.M., n = 3, P < .01 and P < .05, respectively). The magnitudes of these effects were comparable to those seen in separate experiments with native colchicine and were observed in samples of released colchicine which had been stored at -20 degrees C for up to 6 months. CONCLUSIONS This study has shown that the release of the antimitotic agent colchicine, from NiPAAm/NtBAAm co-polymer films can be manipulated by changes in co-polymer composition. Furthermore, such drug released at 37 degrees C retains comparable bioactivity to that of native colchicine.

Investigation of vascular endothelial growth factor effects on pulmonary endothelial monolayer permeability and neutrophil transmigration

This study sought to determine whether vascular endothelial growth factor (VEGF)-induced permeabilisation of pulmonary endothelium to macromolecules could be related to a permissive role for neutrophil-derived VEGF in neutrophil transmigration. Treatment of human pulmonary artery endothelial cell (HPAEC) monolayers with 1, 10 or 100 ng/ml VEGF for 15 min or 1, 10 ng/ml for 90 min significantly increased endothelial permeability to trypan blue-labelled albumin (TB-BSA). These increases were correlated with changes in the cellular distribution of F-actin, as visualised by rhodamine-phalloidin staining: increased stress fibre formation, cellular elongation and formation of intercellular gaps after 15 min; at 90 min, there was also evidence of microspike formation and extension of spindle processes from the cell surface. Treatment of human neutrophil suspensions with 200 nM phorbol myristyl acetate (PMA), n-formyl-methionyl leucylphenylalanine (fMLP, 10 nM), interleukin-8 (IL-8, 10 nM) (but not with leukotriene B(4) (LTB(4)) 100 nM), for 30 min caused significant extracellular release of neutrophil VEGF stores. A permissive role for neutrophil-derived VEGF in facilitating migration across HPAEC monolayers was assessed in experiments using a functional blocking antihuman VEGF antibody. In the presence of this antibody (10 microg/ml), neutrophil migration in response to fMLP (10 nM), IL-8 (10 nM) or LTB(4) (100 nM) was not significantly different to that in the absence of antibody. We conclude that neutrophil-derived VEGF does not play a functional role in facilitating neutrophil migration across pulmonary vascular endothelium, despite its ability to induce cytoskeletal changes and enhance endothelial macromolecular permeability.

Evidence for modulation of hydrogen peroxide-induced endothelial barrier dysfunction by nitric oxide in vitro

Acute effects of the nitric oxide (NO) donors sodium nitroprusside and glyceryl trinitrate on hydrogen peroxide (H2O2)-induced increases in endothelial monolayer permeability to trypan blue-labelled bovine serum albumin have been investigated in vitro. Exposure of bovine pulmonary artery endothelial cell monolayers to 0.2 mM H2O2 for 20 min caused a significant increase in percentage trypan blue-labelled albumin transfer from the lumenal to the ablumenal compartment (basal 6.0 +/- 0.6 to 25.4 +/- 0.9%, n = 4, P < 0.0005). In separate experiments 100 microM sodium nitroprusside significantly enhanced the effect of 0.2 mM H2O2 (from 7.4 +/- 1.4 to 11.9 +/- 1.5%, n = 9, P < 0.0001) but did not alter albumin transfer in the absence of H2O2. This additive effect appeared to be due to NO release from sodium nitroprusside, since nitrite concentration in the medium overlying cells treated with 100 microM sodium nitroprusside was 19.9 +/- 1.8 microM (n = 12). Significantly less nitrite (3.5 +/- 0.5 microM, n = 12, P < 0.0001) was found in the medium overlying cells treated with 100 microM glyceryl trinitrate, which in contrast to sodium nitroprusside, inhibited the permeability increase caused by H2O2 (from 15.6 +/- 3.3 to 13.8 +/- 3.1%, n = 6, P < 0.001). Furthermore 10 microM sodium nitroprusside, which released comparable amount of nitrite (4.5 +/- 0.4 microM, n = 6) to 100 microM glyceryl trinitrate, also inhibited the permeability increase caused by H2O2 (from 20.7 +/- 0.4 to 19.4 +/- 0.3%, n = 9, P < 0.01). We conclude that relatively large amounts of NO released from 100 microM sodium nitroprusside exacerbate the barrier dysfunction caused by H2O2, while lower amounts of NO give a small amount of cytoprotection.

Comparative potencies of 3,4-methylenedioxymethamphetamine (MDMA) analogues as inhibitors of [3H]noradrenaline and [3H]5-HT transport in mammalian cell lines

Illegal ‘ecstasy’ tablets frequently contain 3,4‐methylenedioxymethamphetamine (MDMA)‐like compounds of unknown pharmacological activity. Since monoamine transporters are one of the primary targets of MDMA action in the brain, a number of MDMA analogues have been tested for their ability to inhibit [3H]noradrenaline uptake into rat PC12 cells expressing the noradrenaline transporter (NET) and [3H]5‐HT uptake into HEK293 cells stably transfected with the 5‐HT transporter (SERT).

Role of hemin in the modulation of H2O2-mediated endothelial cell injury

Heme oxygenase (HO) has been primarily regarded as the rate-limiting enzyme in the degradation of heme. However it has recently been proposed that the inducible isoform, HO-1 (EC 1.14.99.3), functions as a stress-responsive antioxidant enzyme, with the capacity to protect against oxidant-mediated vascular injury. This study used an in vitro model of endothelial permeability to determine the effects of the HO-1-inducing agent hemin on noncytotoxic endothelial injury mediated by acute oxidant stress. Effects of hemin on oxidant-mediated cytotoxicity in a number of endothelial cell types were also investigated. A 20-min exposure of human umbilical vein endothelial cell (HUVEC) monolayers to H(2)O(2) resulted in a significant concentration-dependent increase in permeability, which was reversible 48 h later. Pretreatment of monolayers with hemin for 2 h followed by 18 h in complete medium resulted in HO-1 induction and the attenuation of H(2)O(2)-mediated increases in endothelial permeability, and significantly improved the restoration of endothelial barrier function 48 h later. In HUVEC and in the human microvascular endothelial cell line HMEC-1, hemin treatment as above resulted in protection against cytotoxicity, but not in bovine aortic endothelial cells (BAECs), where such toxicity was potentiated. This potentiation was inhibited by incubation with the HO inhibitor tin protoporphyrin IX, supporting a role for HO-1 in the potentiation of the cytotoxic response. When the exposure time of BAEC to hemin was extended to 24 h, H(2)O(2)-mediated cytotoxicity was attenuated. We conclude that hemin treatment is cytoprotective against noncytotoxic endothelial injury in vitro, under conditions that may not offer global protection against cytotoxic injury to vascular endothelium. This would indicate that HO-1 induction associated with cytotoxic injury in vivo is not always beneficial and therefore that the use of hemin as a therapeutic agent to offset oxidant injury in vascular endothelium should be undertaken with caution.

Inhibition by fatty acids of cyclic AMP-dependent protein kinase activity in brush border membranes isolated from human placental vesicles.

1 The inhibitory effects of arachidonic acid (AA) and a number of structurally related fatty acids on cyclic AMP‐dependent protein kinase activity have been investigated in brush border membranes (BBM) prepared from human placental vesicles. 2 BBM vesicles were characterized by electron microscopy and displayed enrichment of the appropriate marker enzymes, alkaline phosphatase and γ‐glutamyltranspeptidase; BBM were prepared by vesicle lysis in hypotonic medium. 3 Cyclic AMP‐dependent protein kinase (PKA) activity was measured in BBM. At 1 μm, cyclic AMP stimulated a 4.2 ± 0.06 fold increase over basal levels of [32P]‐phosphate incorporation into the synthetic substrate kemptide and this effect was abolished by a selective PKA inhibitor. By use of synergistic pairs of site‐selective cyclic AMP analogues, the kinase was identified as the type II enzyme. 4 Cyclic AMP‐stimulated PKA activity was inhibited by 10 μm AA and this effect was significantly enhanced by nordihydroguaiaretic acid (NDGA) + indomethacin (Indo), inhibitors of the lipoxygenase and cyclo‐oxygenase pathways of AA metabolism respectively. 5 Oleic acid, elaidic acid, but not caprylic or palmitic acids, also significantly inhibited PKA activity and this effect was again enhanced by NDGA + Indo. While arachidonyl alcohol alone was not inhibitory, in the presence of the metabolic inhibitors a significant reduction in stimulated activity was observed. 6 The commercially available PKA type II holoenzyme (activated by cyclic AMP), but not the free catalytic subunit, was inhibitable by AA, oleic or elaidic acids. 7 These results suggest that PKA localized to the brush border membrane of human placental vesicles is inhibited by fatty acids which may compete with cyclic AMP for binding to the kinase regulatory subunit. The reported inhibition by fatty acids of cyclic AMP‐dependent Cl− secretion in epithelial cells may therefore be due in part to negative regulation of a Cl− channel‐associated PKA.

Atrial natriuretic factor recognizes two receptor subtypes in endothelial cells cultured from bovine pulmonary artery

In this study specific high affinity binding sites for atrial natriuretic factor (rANF(99–126)) have been identified on cultured endothelial cells of bovine pulmonary artery origin (BPAEC). A time‐dependent rise in cellular cGMP levels stimulated by rANF(99–126) was followed by release of the nucleotide into the incubation medium. The use of truncated, ring‐deleted and linear atrial peptide analogs in competitive displacement analysis and measurement of cGMP accumulation indicated that only a minor proportion (5–11%) of the available receptor pool was of the ANF‐B receptor subtype, linked to guanylate cyclase, with the remaining major proportion possibly of the ANF‐C (clearance) receptor subtype. The existence of two ANF receptor subtypes in this cell culture model would suggest a significant role for the circulating peptide in modulation of pulmonary endothelial cell function, which would influence or complement its direct actions on the underlying vasculature of the pulmonary circulation.

Cytotoxicity-Associated Effects of Reactive Oxygen Species on Endothelin-1 Secretion by Pulmonary Endothelial Cells

In this study bovine pulmonary artery endothelial cells (BPAEC) were used as a model system to investigate the effects of the hypoxanthine-xanthine oxidase (HXXO) oxygen radical donor system on ET-1 secretion into pulmonary vasculature. Incubation of BPAEC with HXXO for 4 h caused a significant reduction in ET-1 secretion, which was significantly offset by allopurinol or catalase, but not by Cu/Zn superoxide dismutase (SOD). ET-1 secretion was also reduced by H2O2, and this effect was again significantly offset by catalase. XO alone also reduced ET-1 secretion, but to a significantly lesser degree than did HXXO, and this effect was not offset by allopurinol, catalase, or SOD. None of the oxidant treatments were associated with a loss of immunoreactive ET-1 from endothelial cell medium containing synthetic peptide. The HXXO- and H2O2-mediated reductions in ET-1 secretion were accompanied by evidence of reduced cell viability. This loss of viability was absent when cells were treated with HXXO + catalase, allopurinol, or mercaptopropionyl glycine, but not when SOD was present. We conclude that under conditions of oxidative stress, the pulmonary vascular endothelium responds by secreting less ET-1. This may be relevant to its vasodilator functions in the pulmonary vasculature, which would therefore be compromised when the endothelium is exposed to oxidant stress.