M Allegra

F D'acquisto

L Tesoriere

M A Livrea

Mauro Perretti
email: m.perretti@qmul.ac.uk

Cross-talk between minimally-primed HL-60 cells and resting HUVEC reveals a crucial role for adhesion over extracellularly-released oxidants

Keywords: neutrophils, priming, endothelial cells, inflammation, adhesion, oxidants

This study demonstrates that a long-lasting co-culture of neutrophil surrogates (HL-60 cells), minimally-primed by platelet activating factor (PAF), and resting endothelial cells (EC) results in the elaboration of an hyper-adhesive endothelial surface, as measured by the increase in the expression of endothelial adhesion molecules E-Selectin, VCAM-1, and ICAM-1. This endothelial dysfunction is mediated by the activation of the redox-sensitive transcription factor NF-B through an exclusive adhesion-driven mechanism active in the endothelial cell: reactive oxygen and nitrogen species, extracellularly-released by minimallyprimed HL-60 cells, are not involved in the induction of the endothelial dysfunction.

By exploring for the first time the potential for minimally-primed neutrophil surrogates to induce endothelial dysfunction, this study suggests a novel mechanism which may be operative in pathologies, mediated by minimally-primed neutrophils, such as hyperdyslipidemia and cardiovascular complications.

A c c e p t e d M a n u s c r i p t

Introduction

Given the impressive array of their harmful weapons, neutrophils (PMNs) have long been considered effective but indiscriminate attackers with a low control of their responses hence with a great potential for damaging the host tissue, should these weapons become uncontrolled and/or misdirected [START_REF] Swain | Neutrophil priming in host defense: role of oxidants as priming agents[END_REF]. In recent years, however, it has become increasingly clear that the PMN response to pro-inflammatory stimuli is finely tuned to provide an appropriate level of reactivity. One of the regulatory mechanisms to control neutrophil responses is priming [START_REF] Swain | Neutrophil priming in host defense: role of oxidants as priming agents[END_REF]. PMN activation, indeed, is not an all-or-none process; rather it can be modeled as a continuum from a nonresponsive to a fully activated phenotype. Within this continuum, PMN can exist in one of three functional states: quiescent, primed or activated [START_REF] Swain | Neutrophil priming in host defense: role of oxidants as priming agents[END_REF].

Under non-infectious conditions PMNs are quiescent, exhibiting little or no release of oxidants; in this state, when first encountering a stimulus they reach a primed status. Upon encountering a second appropriate stimulus, PMNs would then proceed to full activation releasing oxidants, granule contents and inflammatory mediators in the surrounding milieu.

Noteworthy, if the PMN has been primed, its functional response (ROS production, chemotaxis) to the second stimulus will be amplified, in comparison to that of unprimed (quiescent) cells. Importantly, the priming agent itself does not cause any noticeable functional response [START_REF] Swain | Neutrophil priming in host defense: role of oxidants as priming agents[END_REF][START_REF] Mccall | Human toxic neutrophils. 3. Metabolic characteristics[END_REF][START_REF] Guthrie | Priming of neutrophils for enhanced release of oxygen metabolites by bacterial lipopolysaccharide. Evidence for increased activity of the superoxide-producing enzyme[END_REF].

Interaction between PMNs and endothelial cells (EC) has been extensively investigated due to the crucial role these cells play in vascular biology and inflammatory processes. Indeed PMNs may trigger an endothelial injury seriously compromising the function of vasculature in A c c e p t e d M a n u s c r i p t 4 several inflammatory pathologies as well as in relation to the inflammatory processes operative in atherosclerosis, diabetes and ischemia-reperfusion injury [START_REF] Brocq | Endothelial dysfunction: from molecular mechanisms to measurement, clinical implications, and therapeutic opportunities[END_REF][START_REF] Ross | Atherosclerosis--an inflammatory disease[END_REF][START_REF] Kaminski | Oxidative stress and neutrophil activation--the two keystones of ischemia/reperfusion injury[END_REF].

While it has extensively been demonstrated that fully activated PMNs do induce endothelial dysfunction [START_REF] Wang | The signaling pathways induced by neutrophilendothelial cell adhesion[END_REF], it is now becoming evident that also primed cells can be direct mediators of EC dysfunction. For instance, PMNs from patients with risk of developing atherosclerosis and cardiovascular complications (e.g. essential hypertension, diabetes mellitus type II or chronic renal failure) display a certain degree of priming that correlates positively with both the magnitude and type of the disease, leading to an oxidative imbalance within the endothelium [START_REF] Kristal | Participation of peripheral polymorphonuclear leukocytes in the oxidative stress and inflammation in patients with essential hypertension[END_REF][START_REF] Sela | The involvement of peripheral polymorphonuclear leukocytes in the oxidative stress and inflammation among cigarette smokers[END_REF][START_REF] Shurtz-Swirski | Involvement of peripheral polymorphonuclear leukocytes in oxidative stress and inflammation in type 2 diabetic patients[END_REF][START_REF] Sela | Primed peripheral polymorphonuclear leukocyte: a culprit underlying chronic low-grade inflammation and systemic oxidative stress in chronic kidney disease[END_REF][START_REF] Jacobi | Exogenous superoxide mediates pro-oxidative, proinflammatory, and procoagulatory changes in primary endothelial cell cultures[END_REF][START_REF] Jacobi | Priming of polymorphonuclear leukocytes: a culprit in the initiation of endothelial cell injury[END_REF][START_REF] Mazor | Primed polymorphonuclear leukocytes constitute a possible link between inflammation and oxidative stress in hyperlipidemic patients[END_REF]. This has led to propose that loose -but continuous -interaction between primed neutrophils and EC may induce a chronic threshold injury to the endothelium and a systemic oxidative stress able to initiate atherosclerosis and cardiovascular complications [START_REF] Kristal | Participation of peripheral polymorphonuclear leukocytes in the oxidative stress and inflammation in patients with essential hypertension[END_REF][START_REF] Sela | The involvement of peripheral polymorphonuclear leukocytes in the oxidative stress and inflammation among cigarette smokers[END_REF][START_REF] Shurtz-Swirski | Involvement of peripheral polymorphonuclear leukocytes in oxidative stress and inflammation in type 2 diabetic patients[END_REF].

Conversely, EC-derived factors can also contribute to maintain and/or increment PMN activation.

Most of the studies have so far focused on the dysfunctioning effect produced in EC by fully activated or maximally primed PMNs [START_REF] Kristal | Participation of peripheral polymorphonuclear leukocytes in the oxidative stress and inflammation in patients with essential hypertension[END_REF][START_REF] Shurtz-Swirski | Involvement of peripheral polymorphonuclear leukocytes in oxidative stress and inflammation in type 2 diabetic patients[END_REF][START_REF] Jacobi | Priming of polymorphonuclear leukocytes: a culprit in the initiation of endothelial cell injury[END_REF]. While the evidence of an endothelial damage by maximally primed PMN can support a pathogenic role for these cells, investigating the interaction between minimally-primed neutrophils and resting EC may help to understand the very early events promoting endothelial injury, possibly occurring before overt EC damage.

Materials and methods

Chemicals

Unless otherwise stated, all chemicals were from Sigma-Aldrich (Poole, UK), and of the highest available purity grade.

2.2.Cell cultures

Primary human umbilical vein ECs (HUVEC) were obtained from Lonza (Wokingham, UK) and cultured in Endothelial Growth Medium (Lonza, UK) at 37°C in a humidified atmosphere with 5% CO 2 ; cells were sub-cultured by trypsinization and used for the investigation up to passage four. HL-60 cells were obtained from European Type Culture Collection (ECACC, Sigma-Aldrich, Poole, UK) and maintained in RPMI-1640 with 10% heat-inactivated foetal bovine serum (Invitrogen, Paisley, UK), 1% L-glutamine, 1% non essential amino acids, 0.5% Gentamicin in a 5% CO 2 atmosphere at 37°C; cellular density was maintained between 2 and 9x10 5 cells/ml. Cell number and viability were determined by Trypan blue exclusion. Differentiated HL-60 (dHL-60) cells were obtained by 5-day culturing in a medium containing 1% (v/v) dimethyl sulfoxide, as previously reported [START_REF] Harris | Human leukemic models of myelomonocytic development: a review of the HL-60 and U937 cell lines[END_REF]. On day five, cells were harvested and re-suspended in fresh medium.

Assessment of HL-60 cell differentiation into neutrophil-like cells

CD11b expression was evaluated as marker of HL-60 cell differentiation into PMN-like cells [START_REF] Trayner | Changes in antigen expression on differentiating HL60 cells treated with dimethylsulphoxide, all-trans retinoic acid, alpha1,25-dihydroxyvitamin D3 or 12-O-tetradecanoyl phorbol-13-acetate[END_REF]. Both HL-60 and dHL-60 cells were washed with a buffer containing PBS and 0.1% bovine serum albumin. Aliquots (1-5 x10 4 cells in 20µl) were added to a 96-well plate and incubated at 4°C with 3µg/well of a mouse anti-human CD11b phycoerythrin-conjugated monoclonal antibody (Immunotech, Marseille, France). After 1 h at 4°C, cells were washed and analyzed for fluorescence on a Becton Dickinson FACScan (NJ, USA) using a Cell Quest A c c e p t e d M a n u s c r i p t 6 software. At least 10,000 events were analyzed for each sample. Data are expressed as median fluorescence intensity units measured in the FL2 (red) channel with a 488 nm (excitation) and 585 nm (emission) filter set.

Assessment of dHL-60 cell priming

Platelet activator factor (PAF) is a lipid mediator produced by enzymatic cleavage of membrane phospholipids [START_REF] Swain | Neutrophil priming in host defense: role of oxidants as priming agents[END_REF]. In addition to its activating effects on neutrophils (at a concentration > 10 -6 M), PAF may also exert many priming-type actions, evident at concentrations between 10 -7 -10 -9 M, without stimulating PMN to produce significant amounts of oxidants [START_REF] Gay | Modulation of neutrophil oxidative responses to soluble stimuli by platelet-activating factor[END_REF]. Thus, differentiated HL-60 cells were pre-treated with PAF (10 -10 -10 -5 M) for 30 min at 37°C and then activated with phorbol 12-myristate 13-acetate (PMA, 0.3x10 -7 M at 37°C for 60 min). Superoxide release was evaluated to assess the degree of priming.

Measurements of the rate of superoxide release are based on SOD inhibitable reduction of 80µM cytochrome C to its ferrous form. The rate of O 2 -. release was monitored from 10 6

either HL-60 or dHL-60 cells at 22°C up to 90 min, applying a protocol previously described in detail [START_REF] Sela | Primed peripheral polymorphonuclear leukocyte: a culprit underlying chronic low-grade inflammation and systemic oxidative stress in chronic kidney disease[END_REF].

HUVEC dysfunction

Given the minimal priming state of PMN, a long lasting co-culture was required to visualize changes in cell phenotype. Primary PMN are end-stage cells that cannot be cultured for long periods of time (>4h) without losing responsiveness to various stimuli or becoming apoptotic [START_REF] Omori | Priming of neutrophil oxidative burst in diabetes requires preassembly of the NADPH oxidase[END_REF]. HL-60 cells are widely reported to be similar to human neutrophils in morphology, expression of receptors, superoxide generation, chemotaxis and, unlike primary neutrophils, can be cultured for long periods of time [START_REF] Omori | Priming of neutrophil oxidative burst in diabetes requires preassembly of the NADPH oxidase[END_REF]. Intracellular levels of ROS and RNS were assessed using the non-fluorescent probe 2',7'dichlorodihydrofluorescein diacetate (DCDHF-DA) as previously reported [START_REF] Royall | Evaluation of 2',7'-dichlorofluorescin and dihydrorhodamine 123 as fluorescent probes for intracellular H2O2 in cultured endothelial cells[END_REF]. In brief, either EC or dHL-60 cells were cultured as described above. At selected time points, cells were washed with warm PBS, 100 µM DCDHF-DA (Molecular Probes, OR, USA) was added to the culture system and cells were incubated at 37°C in a humidified atmosphere with 5% CO 2 . After 45 minutes cells were washed with PBS, harvested, diluted in a FACS buffer containing PBS and 0.1% bovine serum albumin, and analyzed for fluorescence on a Becton Dickinson FACScan using a Cell Quest software. At least 20,000 events were analyzed for each sample. Data are expressed as median fluorescence intensity units as measured in the FL1 channel.

NF-B luciferase assay

NF-B activity was examined by transfecting HUVEC with pNF-B-Luc luciferase construct (Stratagene, CA, USA). Transfections were carried out using Amaxa Nucleofector Technology (Lonza, UK). Briefly, HUVEC were passaged two days before nucleofection and, on the day of the experiment, 100 µl of a cell suspension at 0.5x10 6 cell/ml was combined with 3µg of pNF-B-Luc luciferase construct, transfected according to the manufacturer protocol and incubated for 12 h in a 5% CO 2 atmosphere at 37°C. Transfected HUVEC were thereafter co-cultured with PAF-primed dHL-60 cells and the specific treatments applied as detailed above. After 6 h, cells were washed with PBS and lysed for 5 min at 4°C using a lysis buffer according to the manufacturer's instructions (Promega, WI, USA). Luciferase activity is expressed as relative luminescence units (RLU) x10 3 following reading in a TD/2020 luminometer (Turner Biosystems, CA, USA). Differences among experimental groups were assessed using one-way analysis of variance and, once significant, applying the Dunnett test to selected experimental groups. A probability value less than 0.05 was considered significant.

Results

3.1.

Priming effect of PAF on dHL-60 cells.

Primed PMN are pre-activated cells such that exposure to a subsequent inflammatory stimulus generates an enhanced response with respect to resting cells. Preliminary assays were carried out to establish the concentration of PAF required to yield minimally-primed dHL-60 cells. As shown in Figure 1, stimulation with PAF < 10 -7 M did not prime cells, as they did not release any significant amount of O 2 .

-above the levels of control cells exposed to PMA. In contrast, PAF concentrations between 10 -7 -10 -5 M primed dHL-60 cells in a concentration-dependent fashion, as judged by the increment of PMA-stimulated superoxide release (Figure 1). No release of O 2 .-was measured when cells were not treated with PMA, irrespective of the PAF concentration used for priming (not shown). Thus, 10 -7 M PAF was regarded to be the minimal concentration required to significantly prime, without activating, dHL-60 cells. We then investigated the mechanisms by which minimally-primed dHL-60 cells might cause the HUVEC activation. Since changes in endothelial adhesion molecule expression can be induced by changes of the endocellular redox milieu [START_REF] Dworakowski | NADPH oxidase-derived reactive oxygen species in the regulation of endothelial phenotype[END_REF], oxidative stress inside EC was then evaluated. Co-incubation of minimally-primed dHL-60 cells with resting HUVEC induced a time-dependent increase of reactive species (ROS/RNS) inside EC that peaked at 4 h, returning to basal levels >12 hours (Figure 3A).

Closer monitoring of the time-course of E-Selectin, VCAM-1 and ICAM-1 expression in the same conditions, provided evidence that the increase of the adhesion molecule expression started only after a critical ROS/RNS level was achieved (i.e. 4 h), with a maximum expression at 16 h (Figure 3A, inset). Consistent with this, pretreatment of HUVEC with Nacetyl-cysteine (NAC), a cell-permeable thiol radical scavenger widely used to assess the role of reactive species in gene expression [START_REF] Schreck | Reactive oxygen intermediates as apparently widely used messengers in the activation of the NF-kappa B transcription factor and HIV-1[END_REF], abrogated the expression of ICAM-1, VCAM-1 and E-selectin induced by minimally-primed dHL60 (Figure 3B). Conversely, co-culture of HUVEC with minimally-primed dHL-60 cells caused a significant increase (~ 3-fold) in the activation of this transcription factor within the EC (Figure 4A).

HUVEC dysfunction is mediated by cell-cell interaction and not by extracellularlyreleased oxidants.

It has been reported that PMN-induced EC activation can be triggered by oxidants released by adherent PMN, adhesion molecule ligation, or both [START_REF] Wang | The signaling pathways induced by neutrophilendothelial cell adhesion[END_REF]. In order to investigate which event was to be considered crucial for the EC activation measured in our system we determined the effect of superoxide dismutase and catalase on NF-B activation and ICAM-1 upregulation in HUVEC . No differences in both NF-B activation and ICAM-1 up-regulation were observed between HUVEC stimulated in the absence or in the presence of these two antioxidant enzymes (Figure 4B), suggesting that released oxidants, whenever released by adherent PMN, were ineffective on the modulation of redox-regulated signaling pathways.

To investigate the role of physical adhesion between HUVEC and minimally-primed dHL-60 cells on the process leading to endothelial dysfunction, HUVEC were co-cultured with minimally-primed dHL-60 cells, either in the absence or in the presence of a Transwell TM 3.6. Cross-talk between minimally-primed dHL-60 cells and resting HUVEC.

Though priming pre-disposes neutrophils to a prompt activation, primed PMN by themselves are not capable of producing reactive species. When co-cultured with resting HUVEC for 16h, minimally-primed dHL-60 cells showed a significantly increase in the endocellular levels of ROS/RNS, peaking at 4h. On the other hand, when they were prevented to adhere to EC, by being cultured inside a Transwell TM inserts, no intracellular production of ROS/RNS was detected (Figure 5). This provides evidence that adhesion-driven signals establish a real cross-talk between EC and dHL-60 cells, yielding EC activation. In our experimental settings cell adhesion represents the "second stimulus" that leads primed-neutrophils to full activation.

Discussion

This study investigate endothelial dysfunction mediated by primed PMN, and explores mechanistic aspects of minimally-primed neutrophil surrogates to potentially induce EC activation. Our results support previous studies on the pathogenic role of primed neutrophils [START_REF] Swain | Neutrophil priming in host defense: role of oxidants as priming agents[END_REF][START_REF] Kristal | Participation of peripheral polymorphonuclear leukocytes in the oxidative stress and inflammation in patients with essential hypertension[END_REF][START_REF] Sela | The involvement of peripheral polymorphonuclear leukocytes in the oxidative stress and inflammation among cigarette smokers[END_REF][START_REF] Shurtz-Swirski | Involvement of peripheral polymorphonuclear leukocytes in oxidative stress and inflammation in type 2 diabetic patients[END_REF][START_REF] Sela | Primed peripheral polymorphonuclear leukocyte: a culprit underlying chronic low-grade inflammation and systemic oxidative stress in chronic kidney disease[END_REF][START_REF] Jacobi | Exogenous superoxide mediates pro-oxidative, proinflammatory, and procoagulatory changes in primary endothelial cell cultures[END_REF][START_REF] Jacobi | Priming of polymorphonuclear leukocytes: a culprit in the initiation of endothelial cell injury[END_REF][START_REF] Mazor | Primed polymorphonuclear leukocytes constitute a possible link between inflammation and oxidative stress in hyperlipidemic patients[END_REF] where it has been shown that minimally-primed neutrophils are able to induce overexpression of E-Selectin, VCAM-1 and ICAM-1, thus promoting an activating phenotype of EC. However, several novel observations were made here.

A c c e p t e d M a n u s c r i p t 13 First, minimally-primed neutrophil surrogates can activate EC as long as they are co-cultured for a time period considerably higher (16 h) than that reported in studies employing highlyprimed neutrophils from hemodialysis, diabetic or hyperlipidemic patients (15 min) [START_REF] Shurtz-Swirski | Involvement of peripheral polymorphonuclear leukocytes in oxidative stress and inflammation in type 2 diabetic patients[END_REF][START_REF] Jacobi | Priming of polymorphonuclear leukocytes: a culprit in the initiation of endothelial cell injury[END_REF][START_REF] Mazor | Primed polymorphonuclear leukocytes constitute a possible link between inflammation and oxidative stress in hyperlipidemic patients[END_REF]. Secondly, minimally-primed dHL-60 cells neither alter EC viability nor induce irreversible lethal events that would result in cell detachment and apoptosis, outcomes reported when highly primed PMN are used [START_REF] Shurtz-Swirski | Involvement of peripheral polymorphonuclear leukocytes in oxidative stress and inflammation in type 2 diabetic patients[END_REF][START_REF] Jacobi | Priming of polymorphonuclear leukocytes: a culprit in the initiation of endothelial cell injury[END_REF][START_REF] Mazor | Primed polymorphonuclear leukocytes constitute a possible link between inflammation and oxidative stress in hyperlipidemic patients[END_REF]. This is coherent with the threshold degree of priming employed here, at variance from that used in previously published studies.

At a mechanistic level, our data demonstrate for the first time that minimally-primed HL-60 generate endothelial intracellular signals, namely variation in the endothelial redox mileu and downstream activation of NF-B, resulting in EC dysfunction.

In endothelial cells, the activation of NF-B by oxygen radicals is a cardinal event leading to up-regulation of several adhesion molecules including ICAM-1 and VCAM-1. Furthermore, treatment of TNF--stimulated cells with antioxidants such NAC and pyrolidine dithiocarbamate reduces Ser32 phosphorylation leading to a reduction in NF-B activity in endothelial cells. Our results are consistent with these findings and show a strong reduction of E-selectin, ICAM-1 and VCAM-1 upregulation in HUVEC co-cultured with minimallyprimed dHL-60 in presence of NAC providing a direct link between activation of NF-B and the increased expression of these adhesion molecules.

More importantly, if integrated in the wider frame of knowledge related to the pathogenic role of primed neutrophils, our results also suggest that EC dysfunction can be the result of a continuous interaction of minimally-primed PMN with endothelium, finally leading to a clear injury. Moreover, we suggest that the extent of neutrophil priming is crucial to influence the way the EC response can be triggered. Indeed, we show that when neutrophils are minimally- Finally, a pivotal function for physical adhesion between EC and minimally-primed dHL60 was found. Adhesion to the monolayers led to production of reactive species in the neutrophil-like cells too, stimulating a real cross-talk between these two cell types instead of a mere "one-way" activation of EC.

In conclusion, the present study reveals a function for intracellular, but not extracellularlyreleased, oxidants and, more importantly, describes how a minimal degree of leukocyte activation might be sufficient to elicit these intracellular responses which would, then, impact

  With this in mind, we have created a novel experimental model of resting EC/minimallyprimed PMN interaction to investigate whether, and to what extent, this cross-talk can lead to endothelial activation.

5

 5

A c c e p t e d M a n u s c r i p t 7 HUVEC 1 (

 71 were grown on 6-well plates and when confluent incubated with or without 1x106 dHL-60 cells for 16 h, with or without 1x10 -7 M PAF. When required, superoxide dismutase (SOD) and catalase (CAT) were added in combination (200 U/ml each enzyme) to the culture medium. In the experiments employing cell-permeable antioxidant, HUVEC were pretreated for 2 h with N-acetyl-cysteine (NAC, 30 mM), washed with fresh medium and then cocultured for 16 h with dHL-60 cells as above described. In the experiments designed to prevent dHL-60 cell adhesion to HUVEC, Transwell polycarbonate membranes inserts (24 mm diameter, 0.4 µm pore size, Corning B.V. Life Sciences (Amsterdam, The Netherlands) were employed: confluent HUVEC were co-cultured for 16 h with PAF-primed dHL-60 cells added in the Transwell insert.In all cases, at the end of the incubation time HUVEC were washed with PBS, harvested with cell dissociation medium and diluted in a washing buffer containing PBS, 0.1% bovine serum albumin and 1 mM CaCl 2 . Aliquots (0.1 to 0.5 x 10 5 cells in 20µl) were added to a 96-well plate and incubated with 2µg/well of a mouse anti-human ICAM-1 (CD54), PECAM-CD31) or E-selectin (CD62E) monoclonal antibody (clones 15.2, 13.5 and18.6, respectively; AbD Serotec, Kidlington, UK). After 1 h at 4°C, cells were washed and incubated with 20 µl of a rabbit anti-mouse F(ab') 2 polyclonal FITC-conjugated secondary antibody (Serotec, Kidlington, UK) for a further 1 h. Finally, samples were washed and analyzed for fluorescence on a Becton Dickinson FACScan using a Cell Quest software. HUVEC and dHL-60 cells were clearly distinguished for their forward and side scatter characteristics. At least 20,000 events were analyzed for each sample. Data are expressed as median fluorescence intensity units, measured in the FL1 (green) channel with a 488 nm (excitation) and 530 nm (emission) filter set.

A c c e p t e d M a n u s c r i p t 8 2. 6 .

 86 Detection of intracellular reactive oxygen and nitrogen species

A c c e p t e d M a n u s c r i p t 9 2. 8 .

 98 Statistical AnalysisData are reported as mean ± SEM of n experiments performed in triplicate or quadruplicate.

3. 2 .

 2 Co-culture of minimally-primed dHL-60 cells with resting HUVEC causes EC dysfunction.Next we investigated the effects of a long-lasting co-culture of minimally-primed dHL-60 cells with resting HUVEC on the expression of the major EC adhesion molecules. FlowA c c e p t e d M a n u s c r i p t 10 cytometry allowed endothelial cells and adherent leukocytes to be distinguished in view of their different scatter profiles: HUVEC showed a spread distribution with high FSC and SSC values (Figure2Aand B) whereas dHL-60 cells appeared as a lower and more compact population (Figure2B). Co-incubation of minimally-primed dHL60 cells with HUVEC for 16 h induced a significant increase of E-Selectin, VCAM-1 and ICAM-1 levels (P < 0.01) (Figure1D-F, respectively) whereas the expression of PECAM-1 did not show any significant change (Figure1C), demonstrating that the HUVEC dysfunction was not consequent to non-specific loss of cell viability or phenotype.3.3. Minimally-primed dHL-60 cell-induced endothelial dysfunction is mediated by a redoxdependent mechanism.

A c c e p t e d M a n u s c r i p t 11 3. 4 .

 114 Activation of NF-B in HUVEC by minimally-primed dHL-60 cellsIt is well established that endothelial adhesion molecule expression is under control of several redox-regulated transcription factors, including the master minder NF-B[START_REF] Liu | NF-kappa B activation as a pathological mechanism of septic shock and inflammation[END_REF]. To determine whether this possible cascade of events was indeed occurring in our experimental settings, we transfected HUVEC were with a NF-B reporter construct prior to co-culture with minimallyprimed dHL-60 cells. Luciferase activity in HUVEC co-cultured with control un-primed dHL-60 cells was not significantly different than that observed in HUVEC alone (not shown).

A c c e p t e d M a n u s c r i p t 12 insert

 12 , a device that allows EC and dHL-60 cells to exchange soluble molecules, but prevents the physical contact between the two cell types. As shown in Figure4B, physical separation between cells abolished both NF-B activation and ICAM-1 up-regulation, indicating that cell adhesion is necessary and sufficient to trigger the signaling events conducive to EC dysfunction.

A c c e p t e d M a n u s c r i p t 14 primed,

 14 EC activation is achieved through adhesion-driven signals whereas, as elsewhere reported[START_REF] Shurtz-Swirski | Involvement of peripheral polymorphonuclear leukocytes in oxidative stress and inflammation in type 2 diabetic patients[END_REF], soluble factors released by these cells overcome direct adhesive effects when highly-primed PMN are used.

  on the cell phenotype. Based on these results we propose a new mechanism of EC dysfunction that maybe relevant to clinical states where a persistent threshold level of neutrophil activation occurs, without overt cell activation or neutrophil-dependent cell injury. Indeed, since initial endothelial dysfunction is a fundamental event in hyperdyslipidemiaassociated and inflammation-based diseases such as atherosclerosis and cardiometabolic complications, our findings may possibly suggest new pharmacological targets helpful to the development of preventive strategies.

A c c e p t e d M a n u s c r i p t 15 Figure legends Figure 1 .

 15legends1 Figure legendsFigure 1. Effect of PAF on dHL-60 cell-released O 2 -. . Cell were exposed to the indicated

Figure 2 .

 2 Figure 2. Flow cytometry analysis of endothelial PECAM-1, E-selectin, VCAM-1 and

Figure 3 .

 3 Figure 3. Time-course of EC dysfunction. (A) Kinetics of endothelial ROS/RNS increase

Figure 4 .

 4 Figure 4. NF-B activation in EC. (A) Effect of PAF-primed dHL-60 cells on endothelial

Figure 5 .

 5 Figure 5. Time course of intracellular ROS/RNS generation in dHL-60 cells. Differentiated HL-60 cells were primed with 10 -7 M PAF and either left alone (), or cocultured with HUVEC in the absence () or in the presence () of Transwell  inserts. At

Figure 2

 2 Figure 2 Click here to download high resolution image

Figure 3

 3 Figure 3 Click here to download high resolution image

Figure 4

 4 Figure 4 Click here to download high resolution image

  Figure 5 Click here to download high resolution image

Acknowledgements

This study was supported by the William Harvey Research Foundation.


Katherine Ann Scott

John Barnes

Roger Clive Whitehead

Ian James Stratford

Karen Ann Nolan
email: karen.nolan-2@manchester.ac.uk

Inhibitors of NQO1: Identification of compounds more potent than dicoumarol without associated off-target effects

Keywords: NQO1, Dicoumarol, Coumarins, Superoxide, Apoptosis

The enzyme NAD(P)H quinone oxidoreductase (NQO1) can function both as a detoxifying enzyme as well as chaperone protein. The latter property has been extensively characterized by the use of dicoumarol which inhibits the chaperone properties of NQO1 in cells. However, the use of this compound is compromised by its multiple "off-target" effects. Coumarin-based compounds that are more potent than dicoumarol as inhibitors of NQO1 in cells have been identified (Nolan et al., Biochem Pharmacol 2010;80:977-81). The purpose of the work reported here is to evaluate the off-target effects of these compounds when compared to dicoumarol. A range of these substituted coumarins are identified that are significantly less toxic than dicoumarol in a panel of nine cell lines. Further a number of the compounds generate much less intracellular superoxide, and many of them also show a reduced ability to induce apoptosis when compared to dicoumarol. None of these effects correlate with the ability of the compounds to inhibit the enzymatic activity of NQO1 in cells. In conclusion, potent inhibitors of NQO1 have been identified that will be more pharmacologically useful than dicoumarol for probing the function of NQO1 in cells and tissues.

A c c e p t e d M a n u s c r i p t

Introduction

Human NAD(P)H quinone oxidoreductase (NQO1, DT-diaphorase, Quinone Reductase 1, Vitamin K Reductase, E.C.1.6.99.2.) is a flavoprotein with 274 residues per monomer and a molecular mass of 30,867 Da. NQO1 exists as a homodimer with one molecule of noncovalently bound flavin adenine dinucleotide (FAD) per monomer [START_REF] Li | The three-dimensional structure of NAD(P)H:quinone reductase, a flavoprotein involved in cancer chemoprotection and chemotherapy: mechanism of the two-electron reduction[END_REF]. It has two active sites, which catalyse the obligate two-electron reduction of quinones, quinone epoxides and quinone-imines to hydroquinones [START_REF] Siegel | NAD(P)H:Quinone Oxidoreductase 1: Role as a Superoxide Scavenger[END_REF]. The enzyme is unusual in that it has the capacity to utilise both the pyrimidine nucleotides NADH and NADPH, without preference, as reducing co-substrates [START_REF] Lind | DT-diaphorase: purification, properties, and function[END_REF]. The catalytic cycle of NQO1 functions via a "ping-pong" mechanism in two distinct steps: hydride transfer from the NAD(P)H to the FAD cofactor, followed by release of NAD(P) + and hydride transfer from the reduced cofactor to the quinone substrate [START_REF] Faig | Structures of recombinant human and mouse NAD(P)H:quinone oxidoreductases: species comparison and structural changes with substrate binding and release[END_REF]. This two electron reduction mechanism also means that NQO1 has antioxidant properties, as it will prevent reduction of quinones by one-electron reductases. The latter would cause the formation of semiquinones with concomitant formation of reactive oxygen species [START_REF] Siegel | NAD(P)H:Quinone Oxidoreductase 1: Role as a Superoxide Scavenger[END_REF].

An additional function of NQO1 is its clear ability to act as a chaperone protein. It has been described as the "gate keeper" of the 20S proteosome [START_REF] Asher | 20S proteasomes and protein degradation "by default[END_REF] and regulates the degradation of certain onco-proteins such as p53, p73α and ornithine decarboxylase and proteins important in the regulation of mRNA translation [START_REF] Asher | The crystal structure of NAD(P)H quinone oxidoreductase 1 in complex with its potent inhibitor dicoumarol[END_REF][START_REF] Alard | NAD(P)H Quinone-Oxydoreductase 1 Protects Eukaryotic Translation Initiation Factor 4GI[END_REF]. The ability of NQO1 to stabilize proteins is dependent upon NADH [START_REF] Asher | A mechanism of ubiquitinindependent proteasomal degradation of the tumor suppressors p53 and p73[END_REF] which suggests that the binding of the enzyme to its client protein(s) is most efficient when the protein FAD is in its reduced form. The anticoagulant dicoumarol (3,3'-methylenebis(4-hydroxycoumarin), has historically been regarded as the most potent inhibitor of NQO1 [START_REF] Ernster | Purification and some properties of a highly dicumarol-sensitive liver diaphorase[END_REF]; it acts through competitive binding with NAD(P)H and A c c e p t e d M a n u s c r i p t 4 prevents the two-electron transfer to FAD from occurring [START_REF] Ernster | DT diaphorase[END_REF]. Hence, addition of dicoumarol to cells has been shown to target p53 and other client proteins for degradation [6-8, 11, 12].

NQO1 is constitutively expressed in a variety of tissues throughout the body, but is overexpressed in many solid tumours [START_REF] Schlager | Cytosolic NAD(P)H:(quinone-acceptor) oxidoreductase in human normal and tumor tissue: effects of cigarette smoking and alcohol[END_REF][START_REF] Workman | The experimental development of bioreductive drugs and their role in cancer therapy[END_REF]. These observations have made NQO1 a potential target for the activation of certain bioreductive anticancer agents [START_REF] Workman | Enzyme-directed bioreductive drug development revisited: a commentary on recent progress and future prospects with emphasis on quinone anticancer agents and quinone metabolizing enzymes, particularly DT-diaphorase[END_REF] such as mitomycin C [START_REF] Cummings | Enzymology of mitomycin C metabolic activation in tumour tissue: implications for enzyme-directed bioreductive drug development[END_REF], EO9 (5-aziridinyl-3-hydroxymethyl-2-(3-hydroxyprop-1-enyl)-1methylindole-4,7-dione) [START_REF] Robertson | The sensitivity of human tumour cells to quinine bioreductive drugs: what role for DT-diaphorase?[END_REF], streptonigrin [START_REF] Gustafson | Expression of human NAD(P)H: quinone oxidoreductase (DT-diaphorase) in Chinese hamster ovary cells: effect on the toxicity of antitumor quinones[END_REF], RH1 ((2,5-diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone) [START_REF] Winski | A new screening system for NAD(P)H:quinone oxidoreductase (NQO1)-directed antitumor quinones: identification of a new aziridinylbenzoquinone, RH1, as a NQO1-directed antitumor agent[END_REF] and β-lapachone [START_REF] Ough | Efficacy of betalapachone in pancreatic cancer treatment: exploiting the novel, therapeutic target NQO1[END_REF].

Dicoumarol is often used as a pharmacological inhibitor to study the function of NQO1 in cells. This has not only been to establish the importance of the enzyme for the activation of bioreductive drugs [START_REF] Robertson | Factors affecting sensitivity to EO9 in rodent and human tumour cells in vitro: DT-diaphorase activity and hypoxia[END_REF][START_REF] Plumb | DT-diaphorase protects cells from the hypoxic cytotoxicity of indoloquinone EO9[END_REF], but also to identify the importance of NQO1 for determining protein stability [START_REF] Asher | P53 hot-spot mutants are resistant to ubiquitin-independent degradation by increased binding to NAD(P)H:quinone oxidoreductase 1[END_REF]. However, the actions of dicoumarol are compromised by extensive protein binding [START_REF] Garten | Comparative study of the binding of coumarin anticoagulants and serum albumins[END_REF] and confounding "off-target" effects such as mitochondrial uncoupling [START_REF] Collier | The mitochondrial uncoupler dicumarol disrupts the MTT assay[END_REF][START_REF] Gonzalez-Aragon | Dicoumarol impairs mitochondrial electron transport and pyrimidine biosynthesis in human myeloid leukemia HL-60 cells[END_REF] and the increased production of intracellular superoxide [START_REF] Du | Mitochondrial production of reactive oxygen species mediates dicumarol-induced cytotoxicity in cancer cells[END_REF][START_REF] Nolan | Coumarinbased inhibitors of human NAD(P)H:quinone oxidoreductase-1. Identification, structure-activity, off-target effects and in vitro human pancreatic cancer toxicity[END_REF]. These off-target effects can interfere with the interpretation of the function of NQO1 in cells. An example is where dicoumarol-induced production of superoxide was thought to be the basis for toxicity towards pancreatic cancer cells and this phenomena was considered to be NQO1 dependent [START_REF] Lewis | Treatment of pancreatic cancer cells with dicumarol induces cytotoxicity and oxidative stress[END_REF][START_REF] Cullen | Dicumarol inhibition of NADPH:quinone oxidoreductase induces growth inhibition of pancreatic cancer via a superoxide-mediated mechanism[END_REF]. However, different research group have subsequently presented data to show that these observations are not solely due to the inhibition of NQO1 but more importantly due to other properties of dicoumarol [START_REF] Nolan | Coumarinbased inhibitors of human NAD(P)H:quinone oxidoreductase-1. Identification, structure-activity, off-target effects and in vitro human pancreatic cancer toxicity[END_REF][START_REF] Reigan | Development of indolequinone mechanism-based inhibitors of NAD(P)H:quinone oxidoreductase 1 (NQO1), NQO1 inhibition and growth inhibitory activity in human pancreatic MIA PaCa-2 cancer cells[END_REF][START_REF] Dehn | 5-Methoxy-1,2dimethyl-3-[(4-nitrophenoxy)methyl]-indole-4,7-dione, a mechanism-based inhibitor of NAD(P)H:quinine oxidoreductase 1, exhibits activity against human pancreatic cancer in vitro and in vivo[END_REF]. Thus, it is therefore of interest to develop novel inhibitors of NQO1 which retain the NQO1 inhibitory potency of dicoumarol but lack its unfavourable off-target effects.

A c c e p t e d M a n u s c r i p t

The synthesis of a series of novel substituted coumarin-based compounds with improved inhibitory potency in both isolated enzyme systems and cells has recently been reported [START_REF] Nolan | Coumarinbased inhibitors of human NAD(P)H:quinone oxidoreductase-1. Identification, structure-activity, off-target effects and in vitro human pancreatic cancer toxicity[END_REF][START_REF] Nolan | Synthesis and biological evaluation of coumarin-based inhibitors of NAD(P)H: quinone oxidoreductase-1 (NQO1)[END_REF][START_REF] Nolan | Phamacological inhibitors of NAD(P)H quinone oxidoreductase, NQO1: Structure/activity relationships and functional activity in tumour cells[END_REF]. This series of compounds have now been evaluated for their "off-target" effects when compared with dicoumarol. This paper provides an evaluation of the toxicity of these compounds in vitro, effects on cell cycle, and their abilities to cause apoptosis and generate superoxide. The major aim of the study was to identify compounds that may be more pharmacologically acceptable than dicoumarol; these will be compounds that have a similar or greater NQO1 inhibitory potency to dicoumarol, but have a lower level of cellular toxicity and fewer off-target effects.

Materials and Methods

Reagents, chemicals and NQO1 inhibitors

Unless otherwise stated, all reagents and chemicals were supplied by Sigma Aldrich (Poole, Dorset, UK). The NQO1 inhibitors have been previously described [START_REF] Nolan | Coumarinbased inhibitors of human NAD(P)H:quinone oxidoreductase-1. Identification, structure-activity, off-target effects and in vitro human pancreatic cancer toxicity[END_REF][START_REF] Nolan | Synthesis and biological evaluation of coumarin-based inhibitors of NAD(P)H: quinone oxidoreductase-1 (NQO1)[END_REF][START_REF] Nolan | Phamacological inhibitors of NAD(P)H quinone oxidoreductase, NQO1: Structure/activity relationships and functional activity in tumour cells[END_REF]. They comprise two series of substituted coumarins. The first includes a set of substituted dicoumarol analogues (the symmetric, S series, where dicoumarol is designated S1). The second group of compounds have one of the 4-hydroxycoumarol rings replaced by an aromatic ring system (the asymmetric, AS series). Structures are given in Figure 1 and Table 1. The compounds were dissolved in either DMSO (Fisher Scientific, Leicestershire, UK) or 0.13M NaOH (VWR, Leicestershire, UK) to give a final concentration of 25mM.

A c c e p t e d M a n u s c r i p t 6

Cell lines and cell culture

Human carcinoma cells with varying levels of NQO1 expression were selected for use. Cell lines were maintained at 37C in a humidified incubator with an atmosphere of air plus 5% supplemented with 10% (v/v) FCS, 2.5% (v/v) horse serum (Invitrogen, Paisley, UK) and 2mM L-glutamine. In the experiments using the MDA-MB-231, MDA-MB-468 and T47D cells which had been stably transfected with NQO1 (designated-DTD) [35], puromycin was added to the culture medium at a concentration of 5µg/ml in order to maintain selection.

Determining the activity of NQO1 in cells

Cells at a concentration of 1.5 x 10 5 were seeded into 10cm dishes and left to reach approximately 70% confluence. They were then washed in PBS (OXOID, Hampshire, UK) and scraped into phosphate buffer (pH 7.5) containing 5µM FAD and 250mM sucrose. The cells were sonicated twice for 5 seconds on ice, centrifuged at 13,000rpm (16,200g) for 15 minutes at 4C, and the supernatants collected and stored at -80C. Protein concentrations were determined using the Bicinchoninic acid (BCA) protein assay [36]. The assay for NQO1 enzyme activity has been described previously [START_REF] Nolan | Coumarinbased inhibitors of human NAD(P)H:quinone oxidoreductase-1. Identification, structure-activity, off-target effects and in vitro human pancreatic cancer toxicity[END_REF]. Briefly, 10µl of cell lysate was mixed with 490µl of 50mM phosphate buffer at pH 7.5 containing 200μM NADH, 70μM cytochrome c and 20μM menadione. Reactions were carried out at 25°C and cytochrome c reduction was followed at 550nm in a Beckman DU650 spectrophotometer. Dicoumarol A c c e p t e d M a n u s c r i p t 7 (100µM) was added to a parallel reaction mixture and the difference in the rates of reaction in the presence and absence of dicoumarol was then used to define the activity of NQO1 in the cellular lysates.

Assays of toxicity

The MTT proliferation assay [START_REF] Mosmann | Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays[END_REF] was used as a surrogate measure of the toxicity of the dicoumarol analogues. Cells were seeded at 7.5 x 10 3 cells/well into 96-well plates and were exposed to increasing concentrations of dicoumarol (S1) or each inhibitor from the S and AS series for 96 hours. The number of surviving cells was then determined using the MTT assay. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was dissolved in sterile PBS and added to the wells at a final concentration of 1.5mM. Cells were incubated with MTT for 4 hours, at which time the resulting formazan crystals were solubilised with DMSO and absorbance read at 540nm. Values of IC 50 were calculated as the drug concentration required to reduce optical density by 50% relative to vehicle treated control cells.

In some experiments, A549 or MIA PaCa-2 cells were treated with EO9 (Apaziquone, synthesized in-house) with and without various non-toxic concentrations of the S and AS compounds for 3 hours. EO9 and the compounds were then removed and cells left in growth medium for 96 hours prior to addition of MTT for assessment of toxicity. As this has been described in detail previously [START_REF] Nolan | Phamacological inhibitors of NAD(P)H quinone oxidoreductase, NQO1: Structure/activity relationships and functional activity in tumour cells[END_REF], the ability of the putative enzyme inhibitors to protect against the toxicity of EO9 in cells is a surrogate measure for their ability to inhibit cellular NQO1.

A c c e p t e d M a n u s c r i p t 8 2.5. Intracellular superoxide production MIA PaCa-2 and MDA-MB-231 wild-type (WT) and DTD cells were grown to approximately 70% confluence, harvested using trypsin/EDTA, and 1 x 10 6 cells were resuspended in 1.5ml culture media in 5ml tubes. Dicoumarol or the other S and AS compounds were added to the cells (at varying concentrations up to 400µM) and incubated for 4 hours, with agitation every 20 minutes. Dihydroethidium was then added to each tube to give a final concentration of 10µM. The cells were incubated for another 30 minutes, centrifuged and the cell pellets were resuspended in 1ml ice-cold PBS. Samples were kept on ice and analysed immediately on a CyanADP flow cytometer using the Summit software package (Dako, Colorado, USA). Dihydroethidium is oxidised to 2-hydroxyethidium by superoxide, which then intercalates DNA and fluoresces red. This signal has been suggested to report the formation of intracellular superoxide [START_REF] Carter | Intracellular hydrogen peroxide and superoxide anion detection in endothelial cells[END_REF]. The oxidized product, 2hydroxyethidium, was excited by blue light (488nm, argon laser) and fluorescence was measured using a 613/20 bandpass filter. The use of dihydroethidium is considered to be a relatively specific method for measuring superoxide [START_REF] Carter | Intracellular hydrogen peroxide and superoxide anion detection in endothelial cells[END_REF]. However, it can be oxidized by other agents such as hydrogen peroxide in the presence of haem containing proteins [START_REF] Fernandes | Analysis of DHE-derived oxidation products by HPLC in the assessment of superoxide production and NADPH oxidase activity in vascular systems[END_REF].

These oxidation reactions can result in the production of different oxidation products with excitation and emission spectra overlapping with that of 2-hydroxyethidium [START_REF] Gonzalez-Aragon | Dicoumarol impairs mitochondrial electron transport and pyrimidine biosynthesis in human myeloid leukemia HL-60 cells[END_REF]. Thus, to confirm that oxidation of dihydroethidium occurred via reaction with superoxide, experiments were carried out in the presence of ambroxol (2-amino-3,5-dibromo-N-[trans-4hydroxycyclohexyl] benzylamine). Ambroxol is a, so-called, superoxide, dismutase mimetic and has been used previously [START_REF] Gonzalez-Aragon | Dicoumarol impairs mitochondrial electron transport and pyrimidine biosynthesis in human myeloid leukemia HL-60 cells[END_REF][START_REF] Bello | NAD(P)H:quinone oxidoreductase 1 expression, hydrogen peroxide levels, and growth phase in HeLa cells[END_REF] to confirm oxidation reactions occurring via superoxide.

In all experiments, cells were exposed to 50µM menadione as a positive control for generation of superoxide.

A c c e p t e d M a n u s c r i p t 9

Cell cycle analysis

Cells were seeded into 10cm dishes at a concentration of 1.5 x 10 5 cells per dish and left to adhere overnight. Cells were treated with 100, 200, 300 or 400µM of dicoumarol (S1) or the S or AS compounds and incubated for 24 hours. The cells were harvested using trypsin/EDTA, washed in PBS and then resuspended in 1ml ice-cold 70% (v/v) ethanol added drop-wise while using a vortex mixer to avoid clumping. The cells were then left in ethanol at 4C for a minimum of 24 hours. For cell cycle analysis, the cells were washed twice in PBS and finally resuspended in 500µl of PBS containing propidium iodide (PI, 25µg/ml) and RNase A (250µg/ml), and incubated at 37C in the dark for 30 minutes. Cells were then washed and resuspended in 500µl PBS and kept in the dark in preparation for analysis. Samples were analysed on a CyanADP flow cytometer using the Summit software package. Cell doublets were gated out using a plot of PI signal height against signal area and data was collected as histograms illustrating fluorescence intensity and hence DNA content of the cells. The percentage of cells in the different phases of the cell cycle were then calculated using gates positioned to encompass the respective phases of control cells in each individual experiment.

Western blot analysis of apoptotic markers

Exponentially growing cells were seeded into 6-well plates at a concentration of 1. ). Samples were sonicated on ice and the protein concentration was estimated using the BCA protein assay. Protein (40µg) was loaded for each sample, resolved on a 10% polyacrylamide gel and then transferred to a polyvinylidene (PVDF) membrane (Millipore, Hertfordshire, UK). Immunoblotting was carried out using a 1:1000 dilution of the cleaved PARP antibody (New England Biolabs, HertfordshireUK; anti-rabbit) or a 1:40000 dilution of the actin antibody. Primary antibodies were detected using the correct horse radish peroxidise (HRP)-conjugated secondary antibody, and the ECL detection system (GE Healthcare, Buckinghamshire, UK) was used to visualise the respective bands.

Results

Novel analogues of dicoumarol (S1) have been synthesized (the S and AS series in Figure 1 and Table 1) and shown to be potent inhibitors of recombinant NQO1 [START_REF] Nolan | Synthesis and biological evaluation of coumarin-based inhibitors of NAD(P)H: quinone oxidoreductase-1 (NQO1)[END_REF]. These molecules also show potent activity for inhibiting the activity of NQO1 in HT29 cells, with potency correlating well with that observed when measuring inhibition of recombinant enzyme in the presence of BSA [START_REF] Nolan | Phamacological inhibitors of NAD(P)H quinone oxidoreductase, NQO1: Structure/activity relationships and functional activity in tumour cells[END_REF]. These published data are given in Table 1. Inhibition of cellular NQO1 was characterized by the ability of the S and AS compounds to inhibit the cytotoxic activity of the indolequinone EO9, an agent known to be exquisitely dependent on NQO1 for toxicity under aerobic conditions [START_REF] Robertson | Factors affecting sensitivity to EO9 in rodent and human tumour cells in vitro: DT-diaphorase activity and hypoxia[END_REF][START_REF] Plumb | DT-diaphorase protects cells from the hypoxic cytotoxicity of indoloquinone EO9[END_REF]. Table 1 also shows the ability of the S and AS compounds to inhibit NQO1 in A549 and MIA PaCa-2 cells. Using the EO9 toxicity end-point as a surrogate measure of NQO1 inhibition, it is apparent that S3 is the most active compound in each cell line, being 2.5 to 10-fold more potent than dicoumarol for protecting against EO9 toxicity. In the S series, S10 and S13 are up to 2-fold more efficient than dicoumarol (S1), whereas the remaining compounds are less effective. On the contrary, in the AS series, all the A c c e p t e d M a n u s c r i p t 11 compounds are at least as efficient as dicoumarol, with some (AS2 and AS14) showing 2-to 4-fold greater potency in the different cell lines. With the knowledge that these compounds are functionally active as inhibitors of NQO1 in cells, it is now possible to assess any "offtarget" activity and relate this to NQO1 inhibition.

The toxicity of the dicoumarol analogues in cell lines expressing varying levels of NQO1

The activity of NQO1 in the cell lines used in the present work is given in Table 2. Cell lines were chosen to have varying NQO1 activity. The MDA-MB-231, MDA-MB-468 and T47D WT cells are null for NQO1 due to a homozygous polymorphism (NQO1*2/*2) [START_REF] Anwar | Interaction of the Molecular Chaperone Hsp70 with Human NAD(P)H:Quinone Oxidoreductase 1[END_REF][START_REF] Dehn | Development of a New Isogenic Cell-Xenograft System for Evaluation of NAD(P)H:Quinone Oxidoreductase-Directed Antitumor Quinones: Evaluation of the Activity of RH1[END_REF][START_REF] Begleiter | Factors influencing the induction of DT-diaphorase activity by 1,2-dithiole-3-thione in human tumor cell lines[END_REF].

Each of these cell lines have been genetically engineered to stably overexpress functional NQO1 [35]. In order to evaluate any contribution of p53, the HCT116 p53 +/+ and -/-pair of cell lines were used [START_REF] Bunz | Requirement for p53 and p21 to sustain G2 arrest after DNA damage[END_REF]. The values for enzyme activity given in Table 2 vary by a factor of 250. Endogenous values of enzyme activity range from 20 to 3011 (nmoles of cytochrome c reduced / min / mg protein) in the MDA-MB-468 and A549 cells respectively. The maximum activity is 5284 in the overexpressing T47D DTD cells. Where measurements have been made

previously, the values reported here compare extremely well [START_REF] Robertson | The sensitivity of human tumour cells to quinine bioreductive drugs: what role for DT-diaphorase?[END_REF]. It has been suggested [START_REF] Cullen | Dicumarol inhibition of NADPH:quinone oxidoreductase induces growth inhibition of pancreatic cancer via a superoxide-mediated mechanism[END_REF] that the toxicity of dicoumarol is mediated by its ability to inhibit NQO1 thereby increasing cellular exposure to reactive oxygen species. Therefore, the toxicity of dicoumarol (S1) and analogues from the S and AS series were evaluated in all the cell lines. Values of IC 50 , the concentration required to reduce proliferation by 50%, are given in Table 3. Dicoumarol is consistently one of the most toxic compounds in all of the cell lines and it is clear that neither NQO1 nor p53-status plays a role in determining toxicity. There also appears to be no relationship between the toxicity induced by the compounds and their ability to inhibit

NQO1enzymatic activity (Table 1).

A c c e p t e d M a n u s c r i p t 12

Production of intracellular superoxide

Dicoumarol has been shown to dose-dependently increase the production of superoxide in MIA PaCa-2 pancreatic cancer cells [START_REF] Du | Mitochondrial production of reactive oxygen species mediates dicumarol-induced cytotoxicity in cancer cells[END_REF]. This result was recapitulated here using flow cytometry to measure the superoxide-mediated oxidation of dihydroethidium to 2hydroxyethidium. Figure 2 shows fluorescence intensity histograms acquired following treatment of MIA PaCa-2 cells with increasing concentrations of dicoumarol (S1) for 4 hours.

In all experiments, 50μM menadione was used as a positive control for superoxide production [START_REF] Kappus | Toxic drug effects associated with oxygen metabolism: redox cycling and lipid peroxidation[END_REF]. From the fluorescence intensity profile obtained for untreated control cells, the R3 region is defined as that containing cells with increased fluorescence. Clearly, treatment with dicoumarol results in a dose-dependent increase in the number of fluorescent cells, which is consistent with previous data [START_REF] Du | Mitochondrial production of reactive oxygen species mediates dicumarol-induced cytotoxicity in cancer cells[END_REF] suggesting it was dicoumarol-induced superoxide formation that caused the increased intracellular fluorescence. To confirm this, the superoxide dismutase mimetic, ambroxol, was added to the cells exposed to dicoumarol. Figure 2, panel F, shows the fluorescence profile obtained when cells were exposed to the combination of 200μM dicoumarol plus 500μM ambroxol. This non-toxic combination shows little, if any, formation of the fluorescent product 2-hydroxyethidium, which suggests that any superoxide formed by a dicoumarol-mediated process is scavenged by ambroxol, thereby preventing reduction of dihydroethidium.

The S and AS compounds were then evaluated for their ability to generate superoxide in MIA PaCa-2 cells. Figure 3 shows the percentage of cells with increased fluorescence relative to untreated controls, following exposure of cells to varying concentrations of the compounds.

There is substantial variation in the ability of the compounds to generate superoxide and there appears to be no relationship with the ability of the different compounds to inhibit NQO1.

A c c e p t e d

M a n u s c r i p t 13 However, interestingly, the compounds in the S series that contain a methoxy group in the coumarin ring (S3, S4, S5 and S11) and those in the AS series that contain the simpler Xsubstituent (AS3, AS6 and AS14) show substantially lower induction of superoxide than dicoumarol (S1) and the other compounds. This is reflected in an apparent correlation between superoxide production and the ability of the compounds to inhibit the proliferation of MIA PaCa-2 cells as measured by the MTT assay (Figure 4).

In order to confirm whether the phenomenon of intracellular superoxide production is not mediated by NQO1, measurements were made of changes in fluorescence intensity in the MDA-MB-231 WT and DTD cells, treated with a variety of compounds from the S and AS series. The cells were incubated for 4 hours with 100µM of each compound and the percentage of cells showing increased fluorescence relative to control is shown in Figure 5.

Clearly, there is no significant difference in the generation of intracellular superoxide between the two cell lines for any of the compounds. It is also apparent that the same lack of dependence on NQO1 for superoxide production is seen for menadione (also shown in Figure 5).

Effects on cell cycle progression and induction of apoptosis

It has been shown previously that dicoumarol can promote the growth of HL-60 cancer cells via cell cycle perturbation [START_REF] Bello | Dicoumarol relieves serum withdrawal-induced G0/1 blockade in HL-60 cells through a superoxide-dependent mechanism[END_REF]. In order to determine the effects of dicoumarol and the S and AS analogues on cell cycle progression, MIA PaCa-2 cells were exposed to 200µM of each of the compounds for 24 hours before staining with propidium iodide for cell cycle analysis.

Figure 6 shows typical DNA histograms of untreated cells and cells treated with dicoumarol (S1), S3 and AS2. Inspection of these profiles suggests that there is no substantive change in A c c e p t e d M a n u s c r i p t 14 the percentage of cells in each of the phases of the cell cycle. However, for both dicoumarol and AS2 there is an indication of an increased number of cells in the sub-G1 region. The percentage of cells in each phase of the cell cycle following treatment with each compound is given in the Supplementary Data Table 1. The percentage of cells in the sub-G1 region following exposure to a 200µM concentration of each of the S and AS compounds is shown in the top panel in Figure 7. Clearly, there are significant differences, with compounds such as S3, S4, S5 and S11 (those showing low superoxide production) providing little if any increase in the sub-G1 population compared to controls. In contrast, the remaining compounds, including dicoumarol (S1) show significantly higher numbers of cells in the sub-G1 region than in untreated populations. In order to determine whether the cells in the sub-G1 region are undergoing apoptosis [START_REF] Ormerod | Apoptosis in interleukin-3-dependent haemopoietic cells. Quantification by two flow cytometric methods[END_REF], cells were collected after 24 hour exposure to 200µM of dicoumarol and each of the S and AS compounds, cell lysates prepared and Western blots probed for cleaved PARP. These are shown in the bottom panel of Figure 7 and a clear concordance exists between expression of cleaved PARP and the percentage of cells in the sub-G1 area, which strongly suggests these cells are undergoing apoptosis. A comparison of the proportion of cells in the sub-G1 region and the generation of superoxide in MIA PaCa-2 cells treated with of each of the putative NQO1 inhibitors is given in the Supplementary Data Figure 1. While there is no correlation between these end-points, it is quite clear that the compounds generating high levels of superoxide are those that also seem to cause the greatest amount of apoptosis. However, importantly, there is no clear relationship between induction of apoptosis or generation of superoxide and the ability of the compounds to inhibit the enzymatic activity of NQO1 (Table 1). Finally, to confirm that there was no link between induction of apoptosis and cellular levels of NQO1, cell cycle analysis of the MDA-MB-231 WT and DTD cells was made following treatment for 24 hours with 200µM of dicoumarol (S1). The proportion of cells in the sub-G1 region following treatment is given in the

Discussion

The purpose of this work has been to identify compounds that are potent inhibitors of NQO1 but with superior pharmacological acceptability than the conventional inhibitor, dicoumarol.

A series of substituted coumarins has been synthesized (Figure 1 and Table 1) and many of these have equivalent or greater potency for inhibiting NQO1 than dicoumarol [START_REF] Nolan | Synthesis and biological evaluation of coumarin-based inhibitors of NAD(P)H: quinone oxidoreductase-1 (NQO1)[END_REF][START_REF] Nolan | Phamacological inhibitors of NAD(P)H quinone oxidoreductase, NQO1: Structure/activity relationships and functional activity in tumour cells[END_REF].

Further, these agents can also inhibit the functional activity of NQO1 in intact HT29 colon cancer, A549 non small cell lung cancer and MIA PaCa-2 pancreatic cancer cell lines [START_REF] Nolan | Phamacological inhibitors of NAD(P)H quinone oxidoreductase, NQO1: Structure/activity relationships and functional activity in tumour cells[END_REF]A number of these compounds (S3, S10, S13, AS2 and AS14) are consistently more potent than dicoumarol, which suggests they may have general applicability as NQO1 inhibitors in cells.

It is known that dicoumarol has a variety of "off-target" effects that can confound interpretation of its action as an inhibitor of NQO1 in cells. Therefore, to establish the potential pharmacological usefulness of the substituted coumarins, experiments have been carried out to determine their toxicity in a large panel of cell lines, their ability to generate superoxide and their ability to cause apoptosis.

In the toxicity studies reported here, there is no relationship between the ability of the compounds to inhibit NQO1 and the values of IC 50 in any of the cell lines (irrespective of the endogenous level of NQO1). Furthermore, there appears to be no dependence on p53 (HCT116 cells). Interestingly, dicoumarol is the most toxic agent in each cell line, irrespective of NQO1 content. For the other compounds there is no systematic relationship between structure and toxicity. In contrast, there are clear differences between the compounds A c c e p t e d M a n u s c r i p t 16 for their ability to generate superoxide. Experiments were initially done in MIA PaCa-2 cells as these cells were used by Du, Cullen and colleagues [START_REF] Du | Mitochondrial production of reactive oxygen species mediates dicumarol-induced cytotoxicity in cancer cells[END_REF][START_REF] Lewis | Treatment of pancreatic cancer cells with dicumarol induces cytotoxicity and oxidative stress[END_REF][START_REF] Cullen | Dicumarol inhibition of NADPH:quinone oxidoreductase induces growth inhibition of pancreatic cancer via a superoxide-mediated mechanism[END_REF][START_REF] Bello | Dicoumarol relieves serum withdrawal-induced G0/1 blockade in HL-60 cells through a superoxide-dependent mechanism[END_REF] who initially proposed that dicoumarol generated superoxide via an NQO1-mediated mechanism. In the S series, compounds such as S10 and S13 are as efficient, if not more so, than dicoumarol for generating superoxide. These compounds are effective for inhibiting NQO1 in cells, but so is S3, and this compound generates little if any superoxide. A molecular feature of the compounds in the S series that do not generate superoxide is that they all contain a methoxy group in the coumarin ring. For the AS series, these compounds generally cause formation of superoxide. However, there are exceptions, with AS3 and AS6 generating substantially less superoxide than similar concentrations of dicoumarol; additionally, AS14, which is a more efficient inhibitor of NQO1 in cells than dicoumarol, generates little if any superoxide. Taken together with results showing similar superoxide production in isogenic cells expressing differing levels of NQO1 (MDA-MB-231 cells) and those in previous work reported by our group [START_REF] Nolan | Coumarinbased inhibitors of human NAD(P)H:quinone oxidoreductase-1. Identification, structure-activity, off-target effects and in vitro human pancreatic cancer toxicity[END_REF], and others [START_REF] Reigan | Development of indolequinone mechanism-based inhibitors of NAD(P)H:quinone oxidoreductase 1 (NQO1), NQO1 inhibition and growth inhibitory activity in human pancreatic MIA PaCa-2 cancer cells[END_REF][START_REF] Dehn | 5-Methoxy-1,2dimethyl-3-[(4-nitrophenoxy)methyl]-indole-4,7-dione, a mechanism-based inhibitor of NAD(P)H:quinine oxidoreductase 1, exhibits activity against human pancreatic cancer in vitro and in vivo[END_REF], these data strongly suggest that cellular NQO1 plays little if any role in the generation of superoxide in cells following treatment with dicoumarol and its analogues. However, the generation of superoxide may be the basis for the toxicity of the coumarin analogues as is suggested by the correlation shown in Figure 4. Interestingly, the relationship between superoxide production and apoptosis is less apparent, although it is quite clear that those compounds generating the greater amount of superoxide are those that produce most apoptosis.

Other off-target effects associated with dicoumarol include extensive protein binding and mitochondrial uncoupling. Protein binding has previously been shown [START_REF] Nolan | Synthesis and biological evaluation of coumarin-based inhibitors of NAD(P)H: quinone oxidoreductase-1 (NQO1)[END_REF] to be much reduced with some of the analogues, including S3. Further, preliminary experiments carried out to measure effects on oxygen respiration have shown that S3 shows a much reduced It is not the purpose of this work to ascertain the underlying mechanism by which the coumarin analogues elicit their off-target effects. Instead, we are seeking to identify molecular features of compounds related to dicoumarol that would help facilitate their development as pharmacological inhibitors of NQO1. It is particularly obvious that the 5-methoxy substituted analogue of dicoumarol, S3, is the most potent inhibitor of NQO1 in cells, while being consistently less toxic in the nine cell lines used here. S3 also shows much reduced generation of superoxide and does not induce apoptosis. From the AS series, AS3 and AS14 stand out, as they also show comparable NQO1-inhibitory potency to dicoumarol in cells with generally less generation of superoxide, less apoptosis and reduced toxicity. A potential advantage of the AS compounds compared to their S analogues is their improved water solubility [START_REF] Nolan | Synthesis and biological evaluation of coumarin-based inhibitors of NAD(P)H: quinone oxidoreductase-1 (NQO1)[END_REF]. Thus, we have provided a firm basis for development of compounds to evaluate the impact of NQO1 inhibition in cells.

Using gene knockout technology it has been shown that loss of NQO1 is associated with reduced p53 stability [START_REF] Gong | NRH:quinone oxidoreductase 2 and NAD(P)H:quinone oxidoreductase 1 protect tumor suppressor p53 against 20s proteasomal degradation leading to stabilization and activation of p53[END_REF][START_REF] Iskander | Lower induction of p53 and decreased apoptosis in NQO1-null mice lead to increased sensitivity to chemical-induced skin carcinogenesis[END_REF] and changes in NFκB signalling [START_REF] Ahn | Genetic deletion of NAD(P)H:quinone oxidoreductase 1 abrogates activation of nuclear factor-kappaB, IkappaBalpha kinase, c-Jun N-terminal kinase, Akt, p38, and p44/42 mitogenactivated protein kinases and potentiates apoptosis[END_REF]. Treatment of cells with dicoumarol can also cause degradation of p53 and other onco-proteins in a process that is mediated by NQO1. There is good evidence to show that transient disruption of p53 during and after irradiation can protect against radiation damage [START_REF] Strom | Small-molecule inhibitor of p53 binding to mitochondria protects mice from gamma radiation[END_REF][START_REF] Ghosh | Radiation protection by a new chemical entity, Ex-Rad: efficacy and mechanisms[END_REF]. Thus, there may well be substantial scope for developing NQO1 inhibitors for therapeutic use [START_REF] Nolan | Phamacological inhibitors of NAD(P)H quinone oxidoreductase, NQO1: Structure/activity relationships and functional activity in tumour cells[END_REF]. In summary, it has been shown that there are a number of compounds that act as potent inhibitors of NQO1 and, beneficially, they lack the off-target effects associated with dicoumarol.

*Graphical Abstract

A c c e p t e d M a n u s c r i p t Table 1. Substituents in the S and AS series (Figure 1) together with the concentration of each compound that inhibits the activity of recombinant human NQO1 by 50% (IC 50 ) in the presence of 0.14% (w/v) BSA [START_REF] Plumb | DT-diaphorase protects cells from the hypoxic cytotoxicity of indoloquinone EO9[END_REF]. Also given are concentrations of each inhibitor that causes 50% toxicity in cells in the presence of 1µM EO9. This concentration of EO9 alone will kill ≥90% of the HT29, A549 or MIA PaCa-2 cells; hence the values quoted give a measure of the efficiency of protection against EO9 toxicity [START_REF] Asher | P53 hot-spot mutants are resistant to ubiquitin-independent degradation by increased binding to NAD(P)H:quinone oxidoreductase 1[END_REF]. This reflects the ability of the S and AS compounds to inhibit NQO1 activity in the cancer cells. Each experiment was repeated on at least three separate occasions and mean values are given ± standard deviation. dicoumarol (S series). Substituents R 5 -R 8 and X are given in Table 1.

CO 2 .

 2 All cell lines were grown in RPMI-1640 medium (Invitrogen, Paisley, UK) supplemented with 10% (v/v) heat-inactivated foetal calf serum (FCS) (Biosera, East Sussex, UK) and 2mM L-glutamine (Invitrogen, Paisley, UK), except MIA PaCa-2 cells which were grown in Dulbecco's Modified Eagle Medium (DMEM) (Invitrogen, Paisley, UK)

5 x 10 5 cells

 5 per well and left overnight to adhere. Cells were treated with 200µM of dicoumarol (S1) or the S or AS compounds, incubated for 24 hours and then harvested by scraping into lysis buffer (50mM Tris-HCl (pH 7.4) (Fisher Scientific, Leicestershire, UK); 120mM NaCl; 5mM EDTA; 0.5% (v/v) Nonidet P-40; 1mM dithiothreitol; 1mM phenylmethanesulphonyl fluoride; 2mM sodium orthovanadate; 2mM sodium fluoride; 20mM B-glycerolphosphate; 5mM A c c e p t e d M a n u s c r i p t 10 sodium pyrophosphate; and 1mg/ml Protease Inhibitor Cocktail tablet (Roche, Hertfordshire, UK

  2 and these results clearly show that the cellular NQO1 status has no influence on the number of cells in sub-G1.

A

  c c e p t e d M a n u s c r i p t ability to cause mitochondrial uncoupling when compared to dicoumarol (Scott KA unpublished data).

  Differential effects of dicoumarol analogues as inhibitors of NQO1, production of superoxide and induction of apoptosis and toxicity in cancer cells.

A c c e p t e d M a n u s c r i p t Figure captions Figure 1 .

 captions1 Figure captions

Figure 2 .

 2 Figure 2. Dose-dependent increase of intracellular superoxide following treatment of

Figure 3 .

 3 Figure 3. Superoxide production in MIA PaCa-2 cells treated for 4 hours with

Figure 4 .

 4 Figure 4. The relationship between superoxide production and toxicity in the MIA

Figure 5 .

 5 Figure 5. Intracellular superoxide production is independent of the activity of NQO1

  Figure captions

Figure 1 .

 1 Figure 1.

Figure A c c e p t e d M a n u s c r i p t

  Figure

Figure 2 .

 2 Figure 2.

Figure 3 .

 3 Figure 3.

Figure 4

 4 Figure 4.

Figure 5 .

 5 Figure 5.

Figure 6 .

 6 Figure 6.

Table 3 .

 3 Toxicity of compounds in a panel of cell lines with varying NQO1 activity and p53 functionality. Values represent the means and standard deviations of at least three independent experiments.

					96 hour IC 50 (µM)
	Compound	MIA	HCT116	HCT116	MDA-MB-231	MDA-MB-468	T47D
		PaCa-2	WT	p53 -/-	WT	DTD	WT	DTD	WT	DTD
	S1	52 ± 26	19 ± 13	22 ± 13	50 ± 37	67 ± 31	30 ± 12	47 ± 23	51 ± 6.2	41 ± 8.3
	S3	350 ± 142	80 ± 27	58 ± 33	133 ±16	165 ± 37 174 ± 45 125 ± 5.2	98 ± 52	190 ± 77
	S4	405 ± 22	48 ± 1.4	43 ± 13	77 ± 7.1	105 ± 60	58 ± 22	38 ± 4.2	283 ± 46 103 ± 84
	S5	303 ± 32	187 ± 17	180 ± 7.1 202 ± 82 162 ± 78 148 ± 16 155 ± 35 109 ± 56	68 ± 31
	S10	106 ± 33	35 ± 1.4	33 ± 5.6	66 ± 51	41 ± 2.6	51 ± 26	56 ± 30	66 ± 47	45 ± 16
	S11	> 400	>400	>400	> 400	> 400	> 400	> 400	> 400	> 400
	S13	119 ± 42	35 ± 16	41 ± 13	114 ± 36 173 ± 15 123 ± 31 141 ± 22 111 ± 38 103 ± 32
	AS1	188 ± 2.9	56 ± 4.9	76 ± 48	32 ± 9.0	51 ± 64	107 ± 17	45 ± 29	124 ± 67 116 ± 71
	AS2	162 ± 28	180 ± 21	165 ± 44	119 ± 48 115 ± 59 142 ± 68	75 ± 51	143 ± 57	95 ± 74
	AS3	267 ± 31	178 ± 2.8	122 ± 65	102 ± 70 185 ± 18 231 ± 70	99 ± 46	145 ± 42 117 ± 69
	AS4	163 ± 20	102 ± 18	111 ± 21	41 ± 5.2	60 ± 58	100 ± 21	67 ± 41	94 ± 22	88 ± 29
	AS5	152 ± 16	39 ± 2.8	31 ± 4.7	41 ± 15	58 ± 15	102 ± 22	58 ± 34	58 ± 36	51 ± 17
	AS6	222 ± 60	40 ± 45	68 ± 48	124 ± 40	94 ± 21	173 ± 27 100 ± 36	13 ± 61	89 ± 16
	AS14	407 ± 15	176 ± 6.4	172 ± 43	152 ± 36 164 ± 25 248 ± 37 154 ± 21 170 ± 7.0 138 ± 3.5

Acknowledgements

This work was funded by a MRC programme grant to IJS (G0500366) and a project grant from the Association for International Cancer Research to KAN, RCW and IJS. The HCT116 cells were kindly provided by Prof B Vogelstein (The Howard Hughes Medical Institute and The Johns Hopkins Oncology Center, 424 North Bond Street, Baltimore, MD 21231, USA. )

A c c e p t e d M a n u s c r i p t Supplementary data, Table 1. Cell cycle profiles were generated on at least three independent occasions.


Adrian J Stanley
email: adrian.stanley@ggc.scot.nhs.uk

Harry R Dalton

Oliver Blatchford

+C Dawn Ashley

Craig Mowat

Aidan Cahill

Daniel R Gaya

Emma Thompson

Usama Warshow

Nikki Hare

Adrian ; Stanley

A J Stanley

¶o Blatchford

A Mowat

D R Cahill

Gaya

+m Groome

G Benson

Dr A J Stanley

Consultant Gastroenterologist

Multicentre comparison of the Glasgow Blatchford and Rockall scores in the

Keywords: Outcomes research < Topics, Endoscopy < Topics, Non-variceal bleeding < Topics, Symptom score or index < outcomes, endoscopy, scoring systems, gastrointestinal bleeding

The Glasgow Blatchford Score (GBS) is increasingly being used to predict intervention and outcome following upper gastrointestinal haemorrhage (UGIH).

Aims:

To compare the GBS with both the admission and full Rockall scores in predicting specific clinical end-points following UGIH.

Patients and methods:

Data on consecutive patients presenting to four UK hospitals were collected.

Admission history, clinical and laboratory data, endoscopic findings, treatment and clinical follow-up were recorded. Using ROC curves, we compared the three scores in the prediction of death, endoscopic or surgical intervention, and transfusion.

Results:

1555 patients (mean age 56.7 years) presented with UGIH during the study period. 74 (4.8%) died, 223 (14.3%) had endoscopic or surgical intervention and 363 (23.3%) required transfusion. The GBS was similar at predicting death compared with both the admission Rockall (area under ROC curve 0.804 vs 0.801) and full Rockall score (AUROC 0.741 vs 0.790). In predicting endo-surgical intervention, the GBS was superior to the admission Rockall (AUROC 0.858 vs 0.705; p<0.00005) and similar to the full Rockall score (AUROC 0.822 vs 0.797). The GBS was superior to both admission Rockall (AUROC 0.944 vs 0.756; p<0.00005) and full Rockall scores (AUROC 0.935 vs 0.792; p<0.00005) in predicting need for transfusion.

Conclusions:

Despite not incorporating age, the GBS is as effective as the admission and full Rockall scores in predicting death after UGIH. It is superior to both the admission and [START_REF] Rockall | Risk assessment after acute upper gastrointestinal haemorrhage[END_REF][START_REF] Blatchford | A risk score to predict need for treatment for upper gastrointestinal haemorrhage[END_REF][START_REF] Cipolletta | Outpatient management for low-risk nonvariceal upper GI bleeding: a randomized controlled trial[END_REF][START_REF] Longstreth | Successful outpatient management of acute upper gastrointestinal hemorrhage: Use of practice guidelines in a large patient series[END_REF][START_REF] Saeed | A scoring system to predict rebleeding after endoscopic therapy of non-variceal upper gastrointestinal hemorrhage[END_REF][START_REF] Hay | Prospective evaluation of a clinical guideline recommending hospital length of stay in upper gastrointestinal tract hemorrhage[END_REF][START_REF] Cameron | Three year prospective validation of a pre-endoscopic risk stratification in patients with acute upper-gastrointestinal haemorrhage[END_REF][START_REF] Romagnuolo | Simple clinical predictors may obviate urgent endoscopy in selected patients with nonvariceal upper gastrointestinal tract bleeding[END_REF]. Most scoring systems require endoscopy including the commonly used Rockall score, which was introduced to assess risk of death following UGIH [START_REF] Rockall | Risk assessment after acute upper gastrointestinal haemorrhage[END_REF]. An abbreviated admission Rockall score which excludes the endoscopic parameters is sometimes used, however this has not been fully validated. The Glasgow Blatchford Score (GBS) appears to be accurate in identifying patients' risk of requiring hospital based intervention or death following UGIH (2, 9; see table 1). This score does not require endoscopy and is based on simple clinical and laboratory parameters which are available soon after the patient presents to the Emergency department.

Our recent publications have identified a low-risk cohort using the GBS, who could be safely managed without admission or in-patient endoscopy and suggested the GBS may be superior to the Rockall scores in predicting the combined outcome of intervention or death [START_REF] Stanley | Outpatient management of patients with low-risk upper gastrointestinal haemorrhage: multicentre validation and prospective evaluation[END_REF][START_REF] Stephens | Management of minor upper gastrointestinal haemorrhage in the community using the Glasgow Blatchford Score[END_REF]. However it is not clear how the scores compare with regard to specific clinical end-points. The aim of this study was to compare the GBS with both the full and admission Rockall scores in the separate prediction of death, endoscopic or surgical intervention and blood transfusion in a multicentre cohort of UK patients presenting with UGIH. GBS. For the last 12 months at Truro, patients were not admitted if the GBS was ≤2 and the patient <70 years old. However all patients not admitted in Glasgow and Truro were offered out-patient endoscopy, as were those >50yrs old in Stockton (and younger patients at the discretion of the clinician). Any patient who failed to attend for endoscopy was followed up at clinic or via discussion with their general practitioners a minimum of six months later, to ensure they had not died or been readmitted to require any intervention as described above.

Statistical analysis:

We compared the GBS with both admission and full Rockall scores in the separate prediction of need for transfusion, endoscopic therapy or surgery, and death. We used the STATA statistical package for data analysis (version 9). Data are shown as median with IQR unless otherwise specified. Comparisons between the scores for separate outcome of death, endoscopic or surgical therapy, and transfusion were made by calculation of the areas under the receiver-operator characteristic (ROC) curves with 95% CI's. CHI squared tests were used to compare the differences between ROC curves. The GBS and admission Rockall scores were compared using all patients with full data on presentation, but comparisons with the full Rockall score was made using the data on those patients who had also undergone endoscopy which allowed calculation of the full score. All described analyses were pre-specified. [START_REF] Rockall | Incidence of and mortality from acute upper gastrointestinal haemorrhage in the UK[END_REF][START_REF] Blatchford | Acute upper gastrointestinal haemorrhage in west of Scotland: case ascertainment study[END_REF]. Recent UK data have reported either an increased or stable incidence over time [START_REF] Taha | Upepr gastrointestinal haemorrhage associated with low-dose aspirin and anti-thrombotic drugs -a 6-year analysis and comparison with non-steroidal anti-inflammatory drugs[END_REF][START_REF] Button | Hospitalized incidence and case fatality for upper gastrointestinal bleeding from 1999 to 2007: a record linkage study[END_REF], with Spanish data suggesting a decreased incidence of UGIH there (16). This study has shown that the simple, non-endoscopic GBS has a high accuracy in predicting specific clinical end points after presentation with UGIH. Whilst the Rockall score was designed to predict death and the GBS designed to predict need for clinical intervention after UGIH, we have found the GBS equivalent to both the full and admission Rockall scores in predicting death, and superior to both in predicting need for blood transfusion. In addition, the GBS is superior to the admission and equivalent to the full Rockall score in predicting endoscopic therapy or surgery.

Advancing age is an obvious risk factor for death and most risk scoring systems for UGIH include age, including both the full and admission Rockall scores. However on initial formulation of the GBS by stepwise logistic regression, age was not found to be an independent risk factor when the other parameters were taken into account and therefore is not part of the GBS (2). Other scores have also been developed for predicting rebleeding or death which do not include age as a component variable [START_REF] Hay | Prospective evaluation of a clinical guideline recommending hospital length of stay in upper gastrointestinal tract hemorrhage[END_REF][START_REF] Romagnuolo | Simple clinical predictors may obviate urgent endoscopy in selected patients with nonvariceal upper gastrointestinal tract bleeding[END_REF]. It is interesting that in this study, despite not including age, the GBS was equivalent to both Rockall scores in predicting death. Recent preliminary data from centres in Europe and Asia have reported similar findings (17,18). The significant superiority of the GBS over both Rockall scores in predicting need for transfusion is interesting. This presumably relates to the development of the GBS to Hb was not found to be an independent predictor of mortality, whereas it was shown to be an independent predictor of intervention during development of the GBS. For many years, there has been a belief that serum urea may be a good biochemical marker of UGIH (19,20). This may be due to the fact that Hb is a low grade protein, which is deficient in isoleucine. Following UGIH, the Hb in the gastrointestinal tract is digested. However, because of its isoleucine deficiency, catabolism results in a rapid increase in serum urea (21). Serum urea values were was not collected in the 1993 national UK audit whose data was used in the development of the Rockall score.

Whilst there is some debate about the optimum Hb threshold for blood transfusion in patients with UGIH (18,23, 24), all centres in our study followed the BSG guidelines (11) suggesting transfusion if Hb was <10g/dL. Our study reflected "real-life" in that many A&E and general physicians were involved in initial care which was however guided by local gastroenterologists and surgeons. Clearly a different local transfusion threshold would lead to different transfusion outcomes. Therefore these results may not be applicable to all centres.

It is perhaps surprising that the GBS and the full Rockall scores are equivalent in predicting need for endoscopic therapy or surgery, considering the Rockall score includes the endoscopic diagnosis and presence of major stigmata of recent haemorrhage. These stigmata are recognised risk factors for re-bleeding, surgery and death and are indications for endoscopic therapy which improves outcome [START_REF] Rockall | Risk assessment after acute upper gastrointestinal haemorrhage[END_REF]23,25). Limitatons of our study include the retrospective collection of a small number of patients within the Stockton group and varying practices on out-patients endoscopy for the low risk patients at Glasgow and Stockton during the later data collection periods. This could have been a source of bias, however all patients were followed up for relevant outcomes and we believe this does not take away from the main comparisons in this paper. Whilst a score for optimal sensitivity and specificity could be calculated, in practice the clinician will err towards sensitivity at the cost of specificity. Therefore the scores are best used to identify low risk patients for non- admission or early discharge. As previously reported by our group, a GBS score of zero had 100% sensitivity for intervntion or death, but there were interventions and a death amongst those with an admission Rockall score of zero [START_REF] Stanley | Outpatient management of patients with low-risk upper gastrointestinal haemorrhage: multicentre validation and prospective evaluation[END_REF].

In conclusion, we have shown that the GBS is equivalent to both the full and admission Rockall scores in predicting death and superior to both in predicting need for blood transfusion. The GBS is similar to the full Rockall score and superior to the admission Rockall score in predicting need for endoscopic therapy or surgery. The GBS, which can be easily calculated in Emergency departments and medical receiving units, may be the best method of early risk assessment following presentation with UGIH.

full

  Rockall scores in predicting need for transfusion and superior to the admission Rockall score in predicting endoscopic or surgical intervention. systems exist to assess patients presenting with upper gastrointestinal haemorrhage (UGIH)

  from consecutive patients presenting with UGIH over a two year period at Royal Cornwall Hospital Truro, eighteen months at Glasgow Royal Infirmary (GRI), six months at University Hospital of North-Tees Stockton and three months at Ninewells Hospital Dundee. This was a follow-up study from our earlier publication[START_REF] Stanley | Outpatient management of patients with low-risk upper gastrointestinal haemorrhage: multicentre validation and prospective evaluation[END_REF], and includes analysis from all patients from that study in addition to a further 12 months data from Truro. All data were collected prospectively apart from the first 3 months at Stockton which was collected retrospectively. UGIH was defined as haematemesis, coffee ground vomit or melaena. Patients with an in-patient UGIH were excluded.At each hospital, an identified research nurse or junior doctor undertook data collection using a standard pro-forma. The data included patient characteristics, any history of melaena, syncope, cardiac failure or liver disease, haemodynamic and laboratory parameters and outcome data in the form of interventions (blood transfusion, endoscopic therapy or surgery) or death (in-patient mortality). These data were used to calculate the GBS and the admission Rockall score for each patient and the full Rockall score in those who had endoscopy. During the study, patients were admitted under general physicians, although both emergency endoscopy and surgery were available out-of hours. The British Society of Gastroenterology guidelines were followed regarding general management including transfusion (11).During the last 12 months at Glasgow and the last three months at Stockton, a local protocol advised non-admission for those patients who scored zero on the presentation

  1555 patients presented to the four centres with UGIH during the study period; 701 at Glasgow, 540 at Truro, 204 at Stockton and 110 at Dundee. Patient demographics and outcome are shown in table 2. Full data allowing area under the ROC curve comparisons between the GBS and admission Rockall scores were available in 1517 patients. 1054 patients underwent endoscopy, allowing calculation of the full Rockall score. These patients were used for area under the ROC curve comparisons between the full Rockall, GBS and admission Rockall scores.For the prediction of mortality, the GBS was similar to both the admission Rockall score: area under the curve 0.804 (CI 0.763-0.844) vs 0.801 (0.751-0.850) p=0.91, and the full Rockall score: 0.741 (0.679-0.804) vs 0.790 (0.725-0.856) p=0.20. There was also no difference between the admission and full Rockall scores in predicting death: 0.762 (0.697-0.828) vs 0.790 (0.724-0.855) p=0.32. The ROC curves for the three scores combined in predicting death are shown in figure1.In predicting the need for endoscopic therapy or surgical intervention, the GBS was superior to the admission Rockall score: 0.834 (0.810-0.858) vs 0.667 (0.629-0.705) p<0.00005 and similar to the full Rockall score: 0.793 (0.763-0.822) vs 0.764 (0.731-0.797) p=0.14. The full Rockall score was superior to the admission Rockall score for predicting endoscopic or surgical intervention: 0.762 (0.729-0.795) vs 0.628 (0.586-0.670) p<0.00005. The ROC curves for the three scores combined in predicting endoscopic or surgical intervention are shown in figure2.

  For predicting transfusion, the GBS was superior to both the admission Rockall score: 0.932 (0.919-0.944) vs 0.727 (0.698-0.756) p<0.00005, and the full Rockall score: 0.919 (0.903-0.935) vs 0.752 (0.722-0.783) p<0.00005. The full Rockall score was superior to the admission Rockall score for predicting transfusion: 0.751 (0.721-0.781) vs 0.688 (0.654-0.721) p<0.00005. The ROC curves for the three scores combined in predicting transfusion are shown in figure3.

  common medical emergency with a UK incidence of 103-172/100 000

  clinical intervention whilst the Rockall score primarily predicts mortality. In addition, the GBS incorporates haemoglobin (Hb) and serum urea, both of which are absent from the Rockall score. During development of the Rockall score,

  Many hospitals use the admission Rockall score to identify low-risk patients for nonadmission or early discharge. Therefore, our finding of superiority of the GBS over the admission Rockall score in predicting a patient's need for endoscopic therapy, surgery or transfusion is clearly relevant. Recent data from Hong Kong and (retrospectively from) the UK have shown the GBS to be superior to the admission Rockall score in predicting need for therapeutic endoscopy or combined intervention respectively, following UGIH (26, 27). There are ongoing problems in provision of out-of-hours endoscopic services at many UK hospitals with the recent national bleeding audit revealing that only 50% patients with UGIH had an endoscopy within 24 hours (28). Therefore accurate pre-endoscopic risk stratification of patients is of critical importance. A recent study has suggested the GBS accurately predicts outcomes including need for high dependency or intensive care (29). It would be helpful if further data confirm the role of GBS in predicting the level of care required and urgency of in-patient endoscopy following UGIH. However this issue is complicated by the variability of relevant resources in hospitals across the UK.

FormattedFormatted

  A, Garcia-Rodriguez LA, Polo-Tomas M et al. The changing face of hospitalisation due to gastroinetstinal bleeding and perforation. Aliment Pharmacol Ther 2011;33:585-91 17) Lim LG, Ho KY, Chan HY, Khor CJ, Rajnakova A, Yeoh KG. Glasgow Blatchford score as an effective triage tool for identifying patients with severe upper gastrointestinal haemorrhage. Gastroenterology 2010: suppl; S1096 18) Almela P, Bolos AG, Anton R, Pascual I, Pena A, Mas P, Minguez M, Benages A. Relationship between Rockall, Blatchford and Almela scores in non-variceal gastrointestinal bleeding. Gastroenterology 2010: suppl:S1102 19) Mailer C, Goldberg A, Harden RM, Grey-Thomas I, Burnett W. Diagnosis of upper gastro-intestinal bleeding. BMJ 1965:2:784-89 20) Silverstein FE, Gilbert DA, Tedesco FJ, Buenger FJ, Persing J. The national ASGE survey on upper gastrointestinal bleeding, II: clinical prognostic factors. Gastrointestinal Endosc 1981;27:80-93 21) Olde Damink SW, Dejong CH, Deutz NE, van Berlo CL, Soeters PB. Upper gastrointestinal bleeding: an ammoniagenic and catabolic event due to the total absence of isoleucine in the haemoglobin molecule. Med Hypotheses 1999; 52(6): 515-9 22) Jairath V, Hearnshaw S, Brunskill S, Doree C,Hopewell S, Hyde C, Travis S, Murphy MF. Red cell transfusion for the management of upper gastrointestinal haemorrhage. Cochrane database of systemic reviews (2):CD006613, 2009 23) Barkun AN, Bardou M, Kuipers EJ, Sung J, Hunt RH, Martel M, Sinclair P. International consensus recommendations on the management of patients with Formatted: French (France) Formatted: English (U.K.) bleeding. Ann Intern Med 2010:152:101-113 24) Hearnshaw SA, Logan RFA, Palmer KR, Card TR, Travis SPL, Murphy MF. Outcomes following early red blood cell transfusion in acute upper gastrointestinal bleeding. Aliment Pharmacol Ther 2010;32:215-224 25) Management of acute upper and lower GI bleeding. SIGN Guideline No. 105, ISBN 978 1 905813 37 7, September 2008 26) Pang SH, Ching JC, Lau JY, Sung JJ, Graham DY, Chan FKL. Comparing the Blatchford and pre-endoscopic Rockall score in predicting the need for endoscopy therapy in patients with upper gastrointestinal haemorrhage. Gastrointest Endosc 2010;71:1134-40 27) Chan JCH, Ayaru L. Analysis of risk scoring for the outpatient management of acute upper gastrointestinal bleeding. Frontline Gastroenterol 2011;2:19-25 28) Hearnshaw SA, Lowe D, Logan RF, Lowe D, Travis SPL, Murphy MF Palmer KR. Use of endoscopy for the management of acute upper gastrointestinal bleeding in the UK: results of a nationwide audit. Gut 2010;59:1022-1029 29) Srirajaskanthan R, Conn R, Bulwer C, Irving P. The Glasgow Blatchford scoring system enables accurate risk stratification of patients with upper gastrointestinal haemorrhage. Int J Clin Practice 2010; 64:868-874

Figure 2 .Figure 3 .

 23 Figure 2. Comparison of the GBS, full Rockall and admission Rockall scores with AUROC figures for the prediction of endoscopic or surgical intervention (GBS=0.793, admission Rockall=0.630, full Rockall=0.764)

Table 1 .

 1 Glasgow Blatchford score:

Full Glasgow-Blatchford bleeding score (GBS)

	Admission risk marker	Score value
	Blood urea (mmol/l)
	6.5-8	2
	8-10	3
	10-25	4
	>25	6
	Hb (g/l) for men
	120-130	1
	100-120	3
	<100	6
	Hb (g/l) for women 100-120 <100 Systolic blood pressure (mmHg) 100-109 F o r 1 6 1
	90-99 <90	2 3 P
	Other markers Pulse≥100/min Presentation with melaena Presentation with syncope Hepatic disease*	1 1 2 2	e e r
	Cardiac failure #	2	R
	* known history of or clinical/laboratory evidence of chronic or acute liver disease e
	# known history of or clinical/echocardiographic evidence of cardiac failure v i e
			w

Table 2 .

 2 Patient demographics and outcome (all four centres combined; n=1555) Comparision of the GBS, full Rockall and admission Rockall scores with AUROC figures for the prediction of death (GBS=0.741, admission Rockall=0.764, full

	Mean age:	56.7 years
	Gender:	965 (62%) male; 591 (38%) female
	In-patient endoscopy undertaken:	1054 (68%)
	In-patient death:	74 (4.8%)
	Endoscopic therapy or surgery required:	223 (14.3%)
	Transfusion required:	363 (23.3%)