Dr Costantino Salerno
email: costantino.salerno@uniroma1.it

E Capuozzo

C Crifò

W Siems

α-Tocopherol increases caspase-3 up-regulation and apoptosis by ß -carotene cleavage products in human neutrophils

Keywords: tocopherol, carotenoid, neutrophil, apoptosis, oxidative stress

It has been found that ß-carotene cleavage products (CarCP), besides having mutagenic and toxic effects on mitochondria due to their prooxidative properties, also initiate spontaneous apoptosis of human neutrophils. Therefore, it was expected that antioxidants such as a-tocopherol would inhibit the stimulation of apoptosis and caspase-3 activity by CarCP. However, we found that a-tocopherol increases caspase-3 up-regulation and stimulation of apoptosis of human neutrophils by CarCP. Ascorbic acid does not alter this caspase-3 up-regulating and proapoptotic effect exerted by a -tocopherol. Both a -tocopherol and ascorbic acid, in the absence of CarCP, decrease intracellular caspase-3 activity and spontaneous apoptosis of neutrophils. Uric acid alone or in combination with CarCP does not exert apparent effects on caspase-3 activity and apoptosis. Upregulating effect of a-tocopherol is not observed in the presence of retinol that markedly stimulates apoptosis by itself, whereas increase of caspase-3 activity is induced by concomitant addition of atocopherol and ß-ionone, a cyclohexenyl degradation product of ß-carotene with shorter aliphatic chain.

A C C E P T E D M A N U S C R I P T

Introduction Epidemiological studies indicated that high consumption of fruits and vegetables rich in carotenoids is associated with low risk for various forms of cancer, cardiovascular disease and some age-related degenerative diseases [START_REF] Mayne | Beta carotene, carotenoids, and disease prevention in humans[END_REF]. However intervention trials showed little, no or even detrimental effects of β-carotene supplementation on human health [START_REF] Hennekens | Lack of effect of long-term supplementation with beta carotene on the incidence of malignant neoplasms and cardiovascular disease[END_REF]. An increase in the risk of lung cancer among smokers and asbestos workers who were supplemented by high dosage of βcarotene was also reported [START_REF] Omenn | Risk factors for lung cancer and for intervention effects in CARET, the Beta-Carotene and Retinol Efficacy Trial[END_REF][START_REF] Bowen | Carotene and Retinol Efficacy Trial. Stopping the active intervention: CARET[END_REF][START_REF] Bendich | From 1989 to 2001: what have we learned about the "biological actions of betacarotene[END_REF].

Prooxidative effect of β-carotene cleavage products was discussed as possible reason for the procarcinogenic action in the case of malignant lesions [START_REF] Young | Antioxidant and pro-oxidant properties of carotenoids[END_REF][START_REF] Siems | Betacarotene cleavage products induce oxidative stress in vitro by impairing mitochondrial respiration[END_REF]. It was reported that carotenoids do behave as oxygen "carriers" from peroxyl radicals to cellular targets [START_REF] Edge | Prooxidant and antioxidant reaction mechanisms of carotene and radical interactions with vitamins E and C[END_REF]. A switch from prooxidant to antioxidant behaviour might be achieved at relatively low oxygen partial tension as well as by adding vitamin E and C [START_REF] Edge | Prooxidant and antioxidant reaction mechanisms of carotene and radical interactions with vitamins E and C[END_REF]. Synergistic protection against peroxynitrite and nitrogen dioxide radicals was observed in mixture of β-carotene, vitamin E and C, as compared to the individual anti-oxidants, and this was explained in terms of an electron transfer in which the β-carotene radical is repaired by vitamin C [START_REF] Böhm | Beta-carotene with vitamins E and C offers synergistic cell protection against NOx[END_REF].

We recently reported that carotenoid derivatives induce apoptosis of human neutrophils and suggested that premature neutrophil death may greatly increase the potential risk for host tissue injury [START_REF] Siems | Carotenoid cleavage products modify repiratory burst and induce apoptosis of human neutrophils[END_REF]. In this report, we studied the effect of α-tocopherol and ascorbic acid on the apoptosis of human neutrophils induced by carotenoid cleavage products. The scavenger effect of uric acid, an inhibitor of peroxynitrite-related chemical reactions [START_REF] Robinson | Triuret: a novel product of peroxynitrite-mediated oxidation of urate[END_REF], was also studied.

Materials and methods

All chemicals were from Sigma (St.Louis, MO) unless stated otherwise and were of the highest grade of purity available. Urate stock solution was prepared by dissolving uric acid in 1 M NaOH; the solution was neutralized by addition of HCl before use. Stock solutions of α-tocopherol and benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD; Calbiochem-Behring, San Diego, CA) were prepared in methanol (MetOH) and dimethyl sulfoxide (DMSO), respectively. Ascorbic acid was dissolved in water immediately before the experiments.

Mixtures of carotene cleavage products (CarCP) were prepared by mixing 40 µM methanolic solution of β-carotene (supplied by Cognis Pty Limited, Sydney, Australia) with 1 mM hypochlorous acid at room temperature as previously described [START_REF] Sommerburg | β-Carotene cleavage products after oxidation mediated by hypochlorous acid -A model for neutrophil-derived degradation[END_REF]. When the "bleaching" reaction was finished, as indicated by a stable colour in the reaction mixture, the cleavage products were extracted twice in hexane. The hexane phases were combined and then evaporated and dried with nitrogen. The residues were redissolved in aliquots of DMSO, which were stored at -80°C until used. Degradation of 10 µmol β-carotene yielded 5.5 mg of dried cleavage products.

Blank solutions without carotene cleavage products, α-tocopherol, ascorbic acid and uric acid were prepared under exactly the same conditions employed to dissolve these compounds in stock solutions. The solutions were used as blank controls for the experiments with the cells.

Leukocytes were purified from heparinized human blood freshly drawn from healthy donors. Leukocyte preparations containing 90% to 98% neutrophils and apparently free of contaminating erythrocytes were obtained by one-step procedure involving centrifugation of blood samples layered on Ficoll-Hypaque medium (Polymorphprep, Axis-Shield, Oslo, Norway) [START_REF] Ferrante | Optimal conditions for simultaneous purification of mononuclear and polymorphonuclear leukocytes from human peripheral blood by the Ficoll-Hypaque method[END_REF]. The cells were suspended in isotonic phosphate-buffered saline (PBS), pH 7.4, with 5 mM glucose and stored in ice. Each preparation produced cells with a viability higher than 90% up to 6 h after purification. The viability of the cells was measured by trypan blue exclusion test. The incubations were carried out at 37°C. Caspase-3 activity was tested in neutrophil lysates by measuring the release of the fluorescent 7-amino-4-methyl-coumarin (AMC) moiety from the synthetic substrate acetyl-Asp-Glu-Val-Asp-7-amido-4-methylcoumarin (AcDEVD-AMC). Neutrophils (5 x 10 6 cells) were collected by centrifugation and lysed in 0.5 ml volumes of 50 mM HEPES, 5 mM 3 -[3-(cholamidopropyl)dimethylammonio]-1-proanesulfonate (CHAPS), 5 mM dithiothreitol (DTT), pH 7.4. The reaction was started by adding 100 µl aliquots of the lysates in 2 ml solutions containing 16 µM AcDEVD-AMC, 20 mM HEPES, 0.1% CHAPS, 5 mM DTT, 2 mM EDTA, pH 7.4. The assay mixtures were incubated at 20°C in the dark for 1-1.5 h. The fluorescence increase was compared with solutions containing appropriate blank controls, 10 µM acetyl-Asp-Glu-Val-Asp-al, a specific caspase-3 inhibitor [START_REF] Nicholson | Identification and inhibition of the ICE/CED-3 proteased necessary for mammalian apoptosis[END_REF], or standard preparations of recombinant caspase-3. A calibration curve obtained with standard AMC solutions was employed for quantitative analysis. The excitation and emission wavelengths of AMC were 360 and 460 nm, respectively.

Fragmentation of neutrophil DNA was tested by agarose gel electrophoresis. Neutrophils (5 x 10 6 cells) were incubated for 20 min in 400 µl of ice-cold hypotonic buffer (10 mM Tris-HCl, 10 mM EDTA, 0.2% Triton X-100, pH 8.0). After addition of 100 µl NaCl (5 M) and 500 µl isopropanol, the samples were held at -20°C for 60 min and then centrifuged at 14,000 rpm for 10 min. The dried pellet was resuspended in 30 µl of a buffer containing 90 mM Tris-borate, 25 mM EDTA, pH 8.3. The fragments of DNA (1 µg per lane) were separated by electrophoresis in 1% agarose gel and stained with ethidium bromide.

Specific differences among treatments were examined using Student's t test that assumed unequal variations.

Results

Apoptosis was assessed by measuring caspase-3 activity in lysates of neutrophils (5 x 10 6 cells/ml) that had been preincubated for 3.5 h at 37°C. If the cells were suspended in phosphate buffered saline with 5 mM glucose, caspase-3 activity in the cell extracts was roughly equivalent to the production of 2 pmol AMC/(min x 10 6 cells).

Addition of carotenoid cleavage products (13 µg/ml, corresponding to 24 µM β-carotene) in 0.28 M DMSO caused a two-fold increase of intracellular caspase-3 activity in the neutrophil suspension (Table I), in good agreement with previously reported results [START_REF] Siems | Carotenoid cleavage products modify repiratory burst and induce apoptosis of human neutrophils[END_REF]. The proapoptotic effect exerted by the carotenoid cleavage products was less pronounced but still evident in the presence of 0.25 M MetOH. Both DMSO and MetOH did not significantly affect intracellular caspase-3 activity in the absence of carotenoid cleavage products (Table I).

α-Tocopherol alone (10, 20, 50, or 100 µM in 0.25 M MetOH) caused a slight but statistically significant decrease of intracellular caspase-3 activity (P < 0.05), while a pronounced up-regulation of intracellular caspase-3 activity was observed for combinations of various concentrations of α-tocopherol and carotenoid cleavage products (13 µg/ml). The effect of the combined treatment (see Fig. 1, second to fifth column pairs) was considerably higher than that observed in the presence of carotenoid derivatives alone, which is presented by the first column pair from the left side of Fig. 1.

Uric acid (50, 100, 250, or 500 µM), used alone or in combination with carotenoid cleavage products, did not exert apparent effect on the intracellular caspase-3 activity (Fig. 2). Ascorbic acid (100 µM) slightly decreased intracellular caspase-3 activity (P < 0.05), if used alone, but did not alter significantly the caspase-3 up-regulating and proapoptotic effect exerted by α-tocopherol in combination with carotenoid cleavage products (Fig. 3).

Retinol (10-20 µM) and β-ionone (20 µM), two degradation products of β-carotene that still retain cyclohexenyl ring, exerted different effect on neutrophil caspase-3 activity (Fig. 4). Retinol stimulated caspase-3 activity in a concentration-dependent manner, but its proapoptotic effect was mitigated by a-tocopherol. β-Ionone did not significantly affect the enzyme activity, if used alone, but stimulated caspase-3 activity in combination with a-tocopherol (P < 0.05).

Chromatin fragmentation was examined as a qualitative marker for neutrophil apoptosis (Fig. 5). If neutrophils were preincubated for 5 h at 37°C in PBS, gel electrophoresis demonstrated a slightly increased amount of low-molecular-weight DNA. In the presence of 13 µg/ml carotenoid cleavage products, neutrophils exhibited low-molecular-weight DNA in larger quantities, which were electrophoresed in a dense ladder pattern. This effect was more evident by using 13 µg/ml carotenoid cleavage products in combination with a-tocopherol, in particular with 100 µM αtocopherol. Similar results were obtained by incubating neutrophils in the presence of 0.5 mM cycloheximide. Control experiments showed, furthermore, that chromatin fragmentation by carotenoid cleavage products and α-tocopherol was greatly reduced in the presence of z-VAD, a broad-spectrum caspase inhibitor [START_REF] Chandler | Different subcellular distribution of caspase-3 and caspase-7 following Fas-induced apoptosis in mouse liver[END_REF].

Discussion

This report shows that carotenoid cleavage products induce apoptosis in human neutrophils and that the concomitant addition of α-tocopherol enhances the apoptotic effect of the carotenoid cleavage products including caspase-3 up-regulation. The proapoptotic activity of carotenoid cleavage products was affected neither by uric acid, an efficient antioxidant that has recently emerged as peroxynitrite scavenger [START_REF] Robinson | Triuret: a novel product of peroxynitrite-mediated oxidation of urate[END_REF][START_REF] Spitsin | Comparison of uric acid and ascorbic acid in protection against EAE[END_REF], nor by ascorbic acid that has been reported to exert, in the presence of β-carotene and α-tocopherol, synergistic protective effect from oxidative stress in model systems [START_REF] Böhm | Beta-carotene with vitamins E and C offers synergistic cell protection against NOx[END_REF]. By the contrast, in the absence of carotenoid cleavage products, α-tocopherol and ascorbic acid slightly decreased caspase-3 activity and neutrophil apoptosis, while uric acid did not exert significant effect. Previous works [START_REF] Siems | Carotenoid cleavage products modify repiratory burst and induce apoptosis of human neutrophils[END_REF] indicated that superoxide production was very low under the employed experimental conditions, since we used fresh preparation of neutrophils in resting state and a relatively high carotenoid derivative concentration (corresponding to 24 µM βcarotene), which inhibits the already very low production of oxygen radicals by unstimulated cells.

The results are in agreement with recent findings of [START_REF] Miyoshi | a-Tocopherolmediated caspase-3 up-regulation enhances susceptibility to apoptotic stimuli[END_REF] who demonstrated an a-tocopherol-mediated caspase-3 up-regulation enhancing the susceptibility to apoptotic stimuli such as Fas, hydrogen peroxide, or etoposide in several human cell lines [START_REF] Miyoshi | a-Tocopherolmediated caspase-3 up-regulation enhances susceptibility to apoptotic stimuli[END_REF]. In those experiments, a-tocopherol also elevated the pro-caspase-3 protein level and mRNA expression in a time-and dose-dependent manner. a -Tocopherol promoted caspase-3 expression without changing the specific activity of the enzyme.

The results question the role of carotenoid breakdown products, which are known to exert toxic effect on mitochondria and even mutagenic effects [START_REF] Siems | Betacarotene cleavage products induce oxidative stress in vitro by impairing mitochondrial respiration[END_REF][START_REF] Alija | Cytotoxic and genotoxic effects of beta-carotene breakdown products on primary rat hepatocytes[END_REF][START_REF] Alija | Beta-carotene breakdown products enhance genotoxic effectrs of oxidative stress in primary rat hepatocytes[END_REF], as intermediates in redox reactions in human neutrophil apoptosis. The findings are somewhat unexpected since several independent observations support a role for oxidative mechanisms in this process [START_REF] Kane | Bel-2 inhibition of neural death: decreased generation of reactive oxygen species[END_REF][START_REF] Jacobson | Reactive oxygen species and programmed cell death[END_REF]. It has been suggested that the detrimental effect of carotenoids is due to the prooxidative properties of βcarotene and of its oxidation products [START_REF] Edge | Prooxidant and antioxidant reaction mechanisms of carotene and radical interactions with vitamins E and C[END_REF][START_REF] Böhm | Beta-carotene with vitamins E and C offers synergistic cell protection against NOx[END_REF]. The formation of carotenoid radical cations has been regarded as an intermediate step in the reaction cascade triggered by peroxyl radicals that are produced by the cells and damage the cell structures [START_REF] Truscott | The interaction of carotenoids with reactive oxy-species[END_REF][START_REF] Cantrell | Singlet oxygen quenching by dietary carotenoids in a model membrane environment[END_REF]. A relationship between apoptosis induction and changes of intracellular redox potential has been observed in human adenocarcinoma cells [START_REF] Palozza | β-Carotene at high concentrations induces apoptosis by enhancing oxy-radical production in human adenocarcinoma cells[END_REF]. Indeed, it has been reported that, unlike human neutrophils, adenocarcinoma cells increase oxygen radical production in the presence of high concentrations of β-carotene. β-Carotene induced apoptosis in adenocarcinoma cells and both the prooxidant and the growth-inhibitory effects of β-carotene were reversed by addition of the antioxidant α-tocopherol in these cells.

Although the prooxidative and proapoptotic effects of carotenoids are well documented, the pathways for induction of apoptosis are still controversial. It has recently reported [START_REF] Müller | Caroteoids induce apoptosis in the T-lymphoblast cell line Jurkat E6.1[END_REF] that αtocopherol did not protect T -lymphoblast cell line from apoptosis induced by carotenoids. Moreover, the antioxidant BO-653 led to a slight but statistically significant increase of cell apoptosis when it was used alone, and to a dramatic increase of apoptosis when it was used in combination with β-carotene. It was suggested that a second pathway by which carotenoids might induce apoptosis is via the formation of retinoic acid or retinoic acid analogues [START_REF] Konta | Selective roles of retinoic acid receptor and retinoid X receptor in the suppression of apoptosis by all-trans-retinoic acid[END_REF][START_REF] Lowe | Lycopene and β-carotene protect against oxidative damage in HT29 cells at low concentrations but rapidly lose this capacity at higher doses[END_REF]. Other mechanisms, which were taken into account to explain cell apoptosis, include direct DNA damage [START_REF] Lowe | Lycopene and β-carotene protect against oxidative damage in HT29 cells at low concentrations but rapidly lose this capacity at higher doses[END_REF] and down-regulation of cyclooxigenase-2 activity [START_REF] Cacciola | Lycopene inhibits proliferation and regulates cyclooxygenase-2 gene expression in neoplastic rat mammari epithelial cells[END_REF] and epidermal growth-factor receptors [START_REF] Muto | Growth-retardation in human cervical dysplasia-derived cell lines by beta-caotene through down-regulation of epidermal growth-factor receptor[END_REF]. The cooperative apoptotic effect of the antioxidant BO-653 in combination with β-carotene is still unexplained. Here we have, with a-tocopherol, an antioxidant, which is able to stimulate caspase-3 up-regulation and apoptosis of human neutrophils induced by a mixture of ß-carotene cleavage products. Up-regulating effect of a-tocopherol was not observed in the presence of retinol that markedly stimulated apoptosis by itself, whereas increase of caspase-3 activity was induced by concomitant addition of a-tocopherol and ß-ionone, a cyclohexenyl breakdown product of ßcarotene with shorter aliphatic chain. It is noteworthy to mention that the effect of carotenoid cleavage products on neutrophil viability was partially influenced by the addition of methanol. This suggests that solvents, which may influence the repartition of lipophilic vitamins between different compartments in the cells, should be considered to explain, at least in part, the effects exerted by carotenoid derivatives in combination with other compounds.

Because of the obvious difficulty to compare in vitro with in vivo models, caution must be employed in discussing the physiological role of cooperative proapoptotic effect of α-tocopherol and carotenoid cleavage products. Anyhow, it must be stressed that the concentrations of αtocopherol and carotenoid derivatives employed in our work are comparable with those present in human tissues. It is well known [START_REF] Ricciarelli | Vitamin E: protective role of a Janus molecule[END_REF] that α-tocopherol, independently of its antioxidant scavenging ability, affects signal transduction and gene expression in different tissues and inhibits cell proliferation. The recommended optimal plasma concentration of α-tocopherol is 30 µM or greater, which corresponds to a daily intake of about 15-30 mg of vitamin E [START_REF] Traber | Vitamin E in humans: demand and delivery[END_REF][START_REF] Hu | α-Tocopherol dietary supplement decreases titers of antibody against 5hydroxymethyl-2'-deoxyuridine[END_REF]. Plasma concentration of β-carotene is highly variable and depends on the biological availability of this compound in food [START_REF] Thurmann | Plasma concentration response to drinks containing beta-carotene as carrot juice or formulated as a water dispersed powder[END_REF]. β-Carotene levels in patient's serum were 3 µM after the intake of 24 mg β-carotene per day in an observation of Stahl et al. [START_REF] Stahl | Dietary tomato paste protects against ultraviolet light-induced erythema in humans[END_REF] and 4 µM in the CARET study [START_REF] Omenn | Risk factors for lung cancer and for intervention effects in CARET, the Beta-Carotene and Retinol Efficacy Trial[END_REF]. High concentrations of carotenoid cleavage products in combination with excessively high number of neutrophils are present very likely in situations of heavy oxidative stress in various organs such as lung (smoking, environmental pollution, ozone, inflammation) [START_REF] Chelchowska | Level of retinol and beta-carotene in plasma of smoking and non-smoking women[END_REF][START_REF] Papi | Pathophysiology of exacerbations of chronic obstructive pulmonary disease[END_REF][START_REF] Battaglia | Differential distribution of inflammatory cells in large and small airways in smokers[END_REF] and in sun-or photoirradiated skin after local application of carotenoid containing cosmetics [START_REF] Tolleson | Photodecomposition and phototoxicity of natural retinoids[END_REF][START_REF] Stanfield | Sun protection of a hydroquinone 4%/retinol 0.3% preparation containing sunscreens[END_REF].

A C C E P T E D

M A N U S C R I P T Chromatin fragmentation in neutrophils exposed to carotenoid cleavage products, tocopherol and z-VAD. Agarose gel electrophoresis patterns observed with molecular size markers (line 7), neutrophils incubated at 0°C for 5 h in absence of effectors (line 1), neutrophils incubated at 37°C for 5 h in absence of effectors (line 2), neutrophils incubated at 37°C for 5 h in presence of 13 µg/ml carotenoid cleavage products (line 3), 13 µg/ml carotenoid cleavage products and 100 µM αtocopherol (line 4), 13 µg/ml carotenoid cleavage products, 100 µM α-tocopherol and 20 µM z-VAD (line 5), 0.5 mM cycloheximide (line 6). Other experimental conditions are reported in the text.

Figure 1 .

 1 Figure 1.Combined effect of tocopherol and carotenoid cleavage products on neutrophil caspase-3 activity. The purified cells were suspended in PBS containing 5 mM glucose, 0.28 M DMSO and 0.25 M MetOH in the absence (closed bars) and presence (open bars) of 13 µg/ml carotenoid cleavage products and 0 -100 µM α-tocopherol. Cell suspensions were incubated at 37°C for 3.5 h. Intracellular caspase-3 activity was determined as described in the text. Values are given as mean + SD (n = 4). Significant difference (P < 0.05) is observed versus controls without α-tocopherol.

Figure 2 .

 2 Figure 2.Combined effect of uric acid and carotenoid cleavage products on neutrophil caspase-3 activity. The purified cells were suspended in PBS containing 5 mM glucose and 0.28 M DMSO in the absence (closed bars) and presence (open bars) of 13 µg/ml carotenoid cleavage products and 0 -500 µM uric acid. Cells suspensions were incubated at 37°C for 3.5 h. Intracellular caspase-3 activity was determined as described in the text. Values are given as mean + SD (n = 4).

Figure 3 .

 3 Figure 3.Combined effect of α-tocopherol, ascorbic acid and caroteinoid cleavage products on neutrophil caspase-3 activity. The purified cells were suspended in PBS containing 5 mM glucose, 0.28 mM DMSO, 0.25 M MetOH and 100 µM ascorbic acid in the absence (closed bars) and presence (open bars) of 13 µg/ml carotenoid cleavage products and 0 -100 µM α-tocopherol. Shaded bar refers to an experiment performed in absence of vitamin derivatives. Cell suspensions were incubated at 37°C for 3.5 h. Intracellular caspase-3 activity was determined as described in the text. Values are given as mean + SD (n = 4). Significant difference (P < 0.05) is observed versus controls without αtocopherol.

Figure 4 .

 4 Figure 4. Combined effect of tocopherol, retinol and β-ionone on neutrophil caspase-3 activity. The purified cells were suspended in PBS containing 5 mM glucose and 0.25 M MetOH in the presence of retinol (open bars:10 µM; shaded bars: 20 µM), 20 µM β-ionone (closed bars) and 0 -100 µM αtocopherol. Cell suspensions were incubated at 37°C for 3.5 h. Intracellular caspase-3 activity was determined as described in the text. Values are given as mean + SD (n = 4). Significant difference (P < 0.05) is observed versus controls without α-tocopherol.

Figure 5 .

 5 Figure 5.

  of carotenoid cleavage product and solvents on the intracellular caspase-3 activity. Intracellular caspase-3 activity was determined as described in the text. Values are given as mean + SD (n = 4). Significant difference (* P < 0.05) is observed versus solvent alone. M DMSO + 0.25 M MetOH 2.2 ± 0.1 3.2 ± 0.1 (*) A C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T

A C C E P T E D

M A N U S C R I P T


Bingmei Yang

Andrea Hodgkinson

Peter J Oates

Beverley A Millward

Professor Andrew G Demaine
email: andy.demaine@pms.ac.uk

High glucose induction of DNA binding activity of the transcription factor NFκB in patients with diabetic nephropathy

Keywords: Nuclear factor kappa B, Diabetic nephropathy, Aldose reductase, Aldose reductase inhibitor, siRNA

The aim of this study was to investigate whether high glucose induces aldose reductase (AKR1B1) expression through NFkB, which may contribute to the pathogenesis of diabetic nephropathy. 34 Caucasoid patients with type 1 diabetes were recruited; 20 nephropaths and 14 long-term uncomplicated subjects. Peripheral blood mononuclear cells (PBMCs) were cultured under normal or high glucose (25 mmol/l of D-glucose) with or without an aldose reductase inhibitor (ARI). High glucose increased NFκB binding activities in the PBMCs from nephropaths compared to the uncomplicated subjects (1.77+0.22 vs. 1.16+0.04, p=0.02). ARI induced a substantially greater decrease of NFκB binding activities in the nephropaths compared to the uncomplicated subjects (0.58+0.06 vs. 0.79+0.06, p=0.032). AKR1B1 protein levels in the nephropaths were increased under high glucose conditions and decreased in the presence of an ARI, whilst the silencing of the NFκB p65 gene in-vitro reduced the transcriptional activities of AKR1B1 in luciferase assays. These results show that NFκB induces AKR1B1expression under high glucose conditions, and the pattern of expression differs between nephropaths and the uncomplicated subjects.

A C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T INTRODUCTION

A large number of patients with type 1 and type 2 diabetes mellitus will develop one or more chronic microvascular complications during the course of their diabetes. The precise mechanisms that initiate and promote diabetic microvascular complications are still being elucidated. It is well established that high glucose stimulates increased flux through the polyol pathway, and this is linked to abnormalities such as osmotic and oxidative stresses that have been cited as promoters of diabetic microvascular disease [START_REF] Brownlee | The Pathobiology of Diabetic Complications: A unifying mechanism[END_REF][START_REF] Hansen | The role of taurine in diabetes and the development of diabetic complications[END_REF][START_REF] Dan | Interaction between the polyol pathway and non-enzymatic glycation on mesangial cell gene expression[END_REF].

Aldose reductase (AKR1B1) is the first and rate-limiting enzyme of the polyol pathway and its expression is tightly regulated by intracellular osmolality at the transcriptional level [START_REF] Smardo | Kidney aldose reductase gene transcription is osmotically regulated[END_REF]. This is mediated through the osmotic response elements (OREs) located in the 5' flanking sequences of the AKR1B1 gene [START_REF] Ko | Identification and characterisation of multiple osmotic response sequences in the human aldose reductase gene[END_REF]. There are three OREs, (OREA, OREB and OREC), that act as specific binding sites for the transcription factor, nuclear factor of activated T cells 5 (NFAT5) or the TonE binding protein (TonEBP) [START_REF] Ko | Identification and characterisation of multiple osmotic response sequences in the human aldose reductase gene[END_REF].

Our previous study has demonstrated that high glucose significantly increased the DNAbinding activity of NFAT5 to the OREs, in particular to the OREC [START_REF] Yang | Elevated activity of transcription factor nuclear factor of activated T-cells 5 (NFAT5) and diabetic nephropathy[END_REF]. Furthermore, this increase was significantly higher in patients with type 1 diabetes and nephropathy compared with those patients without microvascular complications. These results indicate that AKR1B1 is up-regulated by the transcriptional factor NFAT5 under high glucose conditions.

Nuclear factor kappa B (NFκB) is a redox-sensitive transcriptional factor which shares the binding site with OREC in the promoter region of AKR1B1 gene with NFAT5 [START_REF] Iwata | Osmotic response element is regulated for the induction of aldose reductase by tumor necrosis factor-α[END_REF]. NFκB is a widely expressed and is an inducible transcriptional factor with an inactive cytoplasmic and active nuclear DNA-binding form. Canonically, upon stimulation, NFκB is activated through a rapid phosphorylation of IκBα, which is dissociated from NFκB and

A C C E P T E D M A N U S C R I P T

is rapidly translocated to the nucleus where it binds to a κB site and activates its target genes. High glucose-increased flux through the polyol pathway induces redox change and oxidative stress, which activates NFκB. As a result, increased NFκB activity may induce the expression of AKR1B1 by binding to the κB motif in the promoter region of AKR1B1 gene under high glucose conditions. At the same time, NFκB binds to other target genes to increase transcription of these genes, such as macrophage chemotactic protein (MCP-1)

and metallo-matrix-proteinases (MMPs) which are involved in the accumulation of extracellular matrix and contributes to the development of diabetic complications [START_REF] Rovin | Activation of nuclear factor κB correlates with MCP-1 expression by human mesangial cells[END_REF][START_REF] Wahab | Modulation of neutral protease expression in human mesangial cells by hyperglycaemic culture[END_REF][START_REF] Maxwell | Peripheral blood level alterations of TIMP-1, MMP-2 and MMP-9 in patients with type 1 diabetes[END_REF].

Increased activity of NFκB in patients with type 1 as well as type 2 diabetes with microvascular complications have been observed by several groups [START_REF] Mohamed | The role of oxidative stress and NF-kappa B activation in late diabetic complications[END_REF][START_REF] Hofmann | Insufficient glycaemic control increases nuclear factor -κB binding activity in peripheral blood mononuclear cells isolated from patients with type 1 diabetes[END_REF][START_REF] Hofmann | Peripheral blood mononuclear cells isolated from patients with diabetic nephropathy show increased activation of the oxidative-stress sensitive transcription factor NF-κB[END_REF][START_REF] Bierhaus | Diabetes-associated sustained activation of the transcription factor nuclear factor-κB[END_REF]. Ramana et al. [START_REF] Ramana | Activation of nuclear factor -κB by hyperglycemia in vascular smooth muscle cells is regulated by aldose reductase[END_REF] demonstrated that inhibition of AKR1B1 prevented hyperglycemia-induced NFκB activation in vascular smooth muscle cells. They observed that aldose reductase inhibitors (ARI) inhibited the translocation of NFκB and decreased NFκB DNA binding activities.

The aims of the present study were to determine whether the AKR1B1 gene expression is regulated by NFκB under high glucose conditions, and whether this expression is different between those patients with or without nephropathy. Finally, we investigated the effects of ARI on the NFκB binding, and the effects of the silencing of the NFkB p65 subunit on the expression of AKR1B1 gene in vitro.

MATERIALS AND METHODS

Subjects

The following Caucasoid subjects were included in this study: 34 patients with type 1 diabetes. All patients with type 1 as defined by The Expert Committee On The Diagnosis And Classification Of Diabetes Mellitus [START_REF]The Expert Committee on the diagnosis and classification of diabetes mellitus, Report of the Expert Committee on the diagnosis and classification of diabetes mellitus[END_REF] had attended the Diabetes Clinic,

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 5
Derriford Hospital, Plymouth. The study was approved by the Local Research Ethical Committee and informed consent was obtained from all subjects. The criteria for diabetic microvascular complications have been published previously [START_REF] Heesom | Polymorphism in the 5'end of the aldose reductase gene is strongly associated with the development of diabetic nephropathy in type 1 diabetes[END_REF].

Uncomplicated: (Uncompls: n=14) have been diagnosed with type 1 for at least 20 years but remain free of retinopathy (fewer than five dots or blots per fundus), proteinuria (urine Albustix negative on at least three consecutive occasions over 12 months) and neuropathy (overt neuropathy was defined if there was any clinical evidence of peripheral or autonomic neuropathy).

Diabetic nephropathy: (Nephropaths: n=20) have had type 1 for at least 8 years with persistent proteinuria (urine Albustix positive on at least three consecutive occasions over 12 months or three consecutive total urinary protein excretion rates >0.5g/24h) in the absence of hematuria or infection on midstream urine samples. Diabetic nephropathy was always associated with retinopathy. Retinopathy was defined as more than five dots or blots per eye; hard or soft exudates, new vessels, or fluorescein angiographic evidence of maculopathy or previous laser treatment for pre-proliferative or proliferative retinopathy;

and maculopathy or vitreous haemorrhage. Fundoscope was performed by both a diabetologist and an ophthalmologist.

Cell isolation and cultures

Peripheral venous blood samples (20ml) were collected into 5% EDTA Vacutainers (Becton Dickinson, UK). The PBMC were separated by using Histopaque (Sigma, Dorset, UK) and grown in RPMI 1640 supplemented with D-glucose at a 5.5 mmol/l of concentration, 10% calf serum and 2 mmol/l L-glutamine, 100units/ml penicillin G sodium and 100mg/ml streptomycin sulphate with PHA-P at 5μg/ml of concentration in a 37 0 C incubator with a controlled, humidified atmosphere of 95% air/5%

CO2. These cells were divided into three groups from each individual in 200ml-flasks.

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Group 1 (NG): cells were cultured in the above medium. Group 2 (HG): 19.5mmol/l extra D-glucose was added into above-mentioned medium. Group 3 (ARI+HG): sorbinil (10 µmol/l) which is an ARI was added into above-mentioned medium and 3 hours later [START_REF] Hodgkinson | Aldose reductase expression is induced by hyperglycemia in diabetic nephropathy[END_REF].5mmol/l extra D-glucose was added to the culture medium [START_REF] Ramana | Activation of nuclear factor -κB by hyperglycemia in vascular smooth muscle cells is regulated by aldose reductase[END_REF]. All cells were incubated for 5 days. At the end of the incubation time, cells were harvested and nuclear and cytoplasmic proteins were extracted from PBMC as below. A portion of the PBMCs in some samples were cultured with mannitol for 5 days (a final concentration at 25mM) as positive controls. HEK 293 were cultured in EMEM medium supplemented with Dglucose at a 5.5 mmol/l of concentration, 10% calf serum and 2 mmol/l L-glutamine, 100units/ml penicillin G sodium and 100mg/ml streptomycin sulphate for 5 days at NG, HG and ARI+HG conditions.

Extraction of nuclear protein and cytoplasmic proteins

Cells were collected and re-suspended in 100μl of buffer A (10 μmol/l, HEPES, pH 7.9, 1.5 mmol/l MgCl 2 , 0.5 mmol/l dithiothreitol (DTT), 0.2% NP-40, 100 mmol/l 4-

(2-aminoethyl)-bezenesulfonyl fluoride (AEBSF), 18.4 mg/ml sodium orthovanadate, 42 mg/ml sodium flouride and 2.2 mg/ml aprotonin) and held on ice 15 minutes. The resulting cell lysate was then centrifuged at 13000rpm for 10 minutes. The supernatant containing cytoplasmic proteins was transferred into a fresh tube for Western blotting and the nuclear pellets were re-suspended in 50μl of buffer C (20 mmol/l HEPES pH 7.9, 25% glycerol, 0.42 mol/l NaCl, 1.5 mmol/l, MgCl 2 , 0.5 mmol/l DTT, 0.2 mmol/l EDTA, 100 mmol/l AEBSF, 18.4 mg/ml sodium orthovanadate, 42 mg/ml sodium fluoride, 2.2 mg/ml aprotonin), and incubated on ice for 10 minutes. After centrifugation at 13000 rpm for 10 minutes the supernatant containing the nuclear protein was transferred into a fresh tube and stored at -70 0 C until use. The concentrations of both nuclear and cytoplasmic proteins

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were determined using a Coomassie® Plus Protein Assay kit (Peribo Science Ltd., Chest, UK).

Electrophoretic mobility shift assay (EMSA)

The NFκB probe with consensus sequence to κB motif AAATGGGAAATCACCGGC [START_REF] Iwata | Osmotic response element is regulated for the induction of aldose reductase by tumor necrosis factor-α[END_REF] (Fig. 1A) of AKR1B1 gene was labelled with [α- 32 P] deoxy-ATP by T4 polynucleotide kinase (Amersham Pharmacia Biotech, Buckinghamshire, UK). The labelled probe along with a gel binding buffer was incubated with 25 μg of nuclear proteins at room temperature for 20 minutes. The binding mixtures were resolved by eletrophoresis on a 4% non-denaturing polyacrylamide gel at 100V for 3 to 4 hours. The gel was exposed to X-Omax photographic paper. The specificity of the DNA binding protein for the putative binding sites was established by specific competitors and antibodies against human NFκB p65 (Insight Biotechnology Ltd, Wembley, UK), respectively.

Western blotting

Briefly, a total 50μg of cytoplasmic proteins were loaded onto a 7.5 or 10% precast SDS-PAGE (BIO-RAD Laboratory Ltd, Hempstead, UK), electrophoresed for 2-3 hours at 100 volts, transferred to nitrocellular membrane (Amersham, USA) overnight. Next, the membrane was blocked with 5% non-fat milk and 0.05% Tween 20 -PBS for one hour at room temperature. Immunoblotting was performed with primary rabbit antibodies against human AKR1B1 and IκB-α (Abcam Ltd, Cambridge, UK) as well as with primary mouse antibodies against NFκB p65, phosphorylated IκB-α (Insight Biotechnology Ltd, Wembley, UK) in appropriate dilutions, respectively. Secondary antibodies against rabbit or mouse IgG of horse-radish peroxidase-conjugated were used in a 1:10,000 dilution (Sigma, UK). A chemiluminescence kit (Pierce, UK) and Kodak X-Omat film (Amersham, UK) were used to detect the amount of proteins. Meantime, in order to have

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 8
an equal amount of proteins in all wells, β-actin levels were measured by using a mouse antibody against human β-actin in 1:10,000 dilution (Sigma, UK) and a secondary antibody against mouse IgG of horse-radish peroxidase-conjugated in 1:10,000 dilution (Sigma, UK).

DNA binding activities of NFκB and protein levels of AKR1B1, NFκB p65, phosphorylated IκB-α and IκB-α were analysed and quantified by using a phosphoimager (BIO-RAD, UK) with multi-analyst software. All results were expressed as means of fold increases due to high glucose treatment, calculated by dividing the amount of density in high glucose-treated cells by the amount of density in untreated cells or as means of fold decreases due to ARI treatment, calculated by dividing the amount of density in high glucose with ARI treated cells by the amount of density in high glucose-treated cells.

SiRNA assay

In order to investigate effects of NFκB on the transcription of AKR1B1 gene under high glucose conditions, we silenced the NFκB gene by RNA interference. Three siRNA duplex sequences were pre-designed from Ambion (Ambion Limited, UK), targeting to exon 7, 8 and 9 of the NFκB p65 gene, respectively. A negative control siRNA was purchased from Ambion as well.

Transfection of siRNA was carried out by using siFECT siRNA transfection reagent (Promega, Southampton, UK) and transfection of plasmids was performed by using Tfx-20 reagent (Promega, Southampton UK). The day before transfection, HEK 293 cells (which express NFκB p65 identified by RT-PCR, data not shown) were seeded at 30% to 50% confluence in 96-well plates and each transfection was performed in duplicates. siRNA at a concentration range from 10 nmol/l to 1000 nmol/l was tested and a 500 nmol/l of siRNA gave a detectable silencing effect. Among three siRNAs, the one targeting exon 9 gave the highest silencing. Therefore, siRNA targeting at exon 9 and conditions (ARI was added 3 hours earlier before D-glucose was added). All cells were cultured for another 30 hours. At the end of culture, cells were lysed using 20 μl of lysis buffer (Promega, Southampton, UK). AKR1B1 transcription activity was measured using a Dual-luciferase reporter assay system (Promega, Southampton, UK) in a MLX luminometer (Dynex Technologies Inc., USA). For each transfection, the relative activity was defined as firefly luciferase activity normalised by the activity of the internal control (Renilla luciferase) as described previously [START_REF] Yang | Elevated activity of transcription factor nuclear factor of activated T-cells 5 (NFAT5) and diabetic nephropathy[END_REF][START_REF] Yang | Functional differences between the susceptibility Z-2/C-106 and protective Z+2/T-106 promoter region polymorphisms of the aldose reductase gene may account for the association with diabetic microvascular complications[END_REF].

Statistical Analysis

All data were expressed as the means + standard error of the means (SE). Student t-test was used to compare differences between two groups and ANOVA was used to compare differences between more than two groups. A p value of less than 0.05 was considered to be significant.

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10

RESULTS

Clinical characteristics of patients with type 1 diabetes with or without nephropathy are

shown in Table 1. There were no differences in age, gender, age at onset of diabetes, duration of diabetes, Hb1Ac and glucose levels between two groups.

High glucose significantly increased NFκB binding activities to the κB motif site in

PBMCs

We observed that two binding bands were formed in the gel shit with NFκB probe, which were consistent with Ramana et al. study [START_REF] Ramana | Activation of nuclear factor -κB by hyperglycemia in vascular smooth muscle cells is regulated by aldose reductase[END_REF]. In the presence of cold (unlabelled)

NFκB probe, the band was abolished (Figure 1B). The super shift analysis of NFκB showed that following the incubation with antibodies against human NFκB p65, the binding complex with the probe to κB motif were shifted upwards as indicated in Figure 1B.

The DNA-binding activities of NFκB to the κB motif in PBMCs exposed to either normal glucose, high glucose or, ARI with high glucose conditions, from a nephropath and an uncomplicated subject are shown in Figure 1C. Following the incubation with high glucose there was a significantly increased DNA binding activities of NFκB to κB motif in PBMCs in both the nephropaths and uncomplicated subjects. These increases were particularly significant in the nephropaths compared with uncomplicated group (1.77+0.22 vs. 1.16+0.04, p=0.02) (Table 2). ARI treatment significantly decreased the DNA-binding activities of NFκB to κB motif under high glucose conditions in both the nephropaths by 67% (1.77+0.22 vs. 0.58+0.06, p=0.0001) and uncomplicated by 32% (1.16+0.04 vs. 0.79+0.06, p=0.0002) groups, respectively (Table 2). The ARI-linked decrease in NFκB binding activity was significantly greater in the nephropaths compared to the uncomplicated (0.58+0.06 vs. 0.79+0.06, p=0.032) (Table 2).

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High glucose significantly increased protein level of the AKR1B1

Western blots were used to measure the AKR1B1, NFκB p65, IκB-α and phosphorylated IκB-α protein levels (Figure 2). In the presence of high glucose there was a significant increase in AKR1B1 protein in PBMCs from the nephropaths compared to the uncomplicated (1.26+0.08 vs. 0.93+0.07, p=0.02). This is consistent with our earlier study of elevated AKR1B1 mRNA and proteins by high glucose in the nephropaths [START_REF] Yang | Elevated activity of transcription factor nuclear factor of activated T-cells 5 (NFAT5) and diabetic nephropathy[END_REF][START_REF] Hodgkinson | Aldose reductase expression is induced by hyperglycemia in diabetic nephropathy[END_REF]. In the presence of the ARI, the elevation of AKR1B1 protein by high glucose was significantly suppressed in PBMCs from the nephropaths (1.26+0.08 vs. 0.76+0.07, p=0.01), but not from uncomplicated subjects (0.93+0.07 vs. 0.81+0.06, p=0.24) (Table 3). High glucose slightly increased the expression of NFκB p65 and phosphorylated IκB-α and IκB, respectively, in PBMC from the nephropaths, but those increases were not significantly different compared to PBMC from the uncomplicated subjects. In the presence of ARI, those protein levels were decreased by various degrees from 10 to 20% compared with that under high glucose conditions from the nephropaths, respectively.

Again, these changes were not statistically significant. All results are summarised in Table 3.

Silencing of NFκB reduces AKR1B1 gene transcription

These results were consistent with our previous studies showing [START_REF] Yang | Elevated activity of transcription factor nuclear factor of activated T-cells 5 (NFAT5) and diabetic nephropathy[END_REF][START_REF] Yang | Functional differences between the susceptibility Z-2/C-106 and protective Z+2/T-106 promoter region polymorphisms of the aldose reductase gene may account for the association with diabetic microvascular complications[END_REF] that the recombinants with the Z-2/C-106 AKR1B1 haplotype had the highest transcriptional activity among all haplotypes. The Z/C-106 AKR1B1 haplotype had the second highest transcriptional activity in transfected HEK 293 cells in luciferase assays. High glucose significantly increased AKR1B1 transcription by 1.2 fold in the HEK293 cells transfected with both Z-2/C-106 and Z/C-106 AKR1B1 haplotypes, respectively (Figure 3A andB). In the presence of the siRNA targeting NFκB p65, transcriptional activities of AKR1B1 in the Z-2/C-106 recombinants were reduced by 50%, 56%, 60% and 50% under NG, HG, AKR1B1 with Z/C-106 recombinants were decreased by 50%, 57%, 56% and 37% under NG, HG, ARI and ARI with HG, respectively (Figure 3B). The silencing of NFκB p65 gene was confirmed by Western blotting by using an antibody against human NFκB p65 (Figure 3C). However, the ARI did not decrease transcriptional activities in those cells transfected with either Z-2/C-106 or Z/C-106 AKR1B1 haplotypes without siRNA transfection. There were no significant changes in transcriptional activities in transfected cells with the control siRNA compared to cells without siRNA between either normal glucose, high glucose or, high glucose and ARI for both haplotypes (data not shown).

DISCUSSION

Chronic hyperglycaemia is a major initiator of microvascular complications in diabetes. Accelerated flux through the polyol pathway may be a key to the development of diabetic nephropathy and other complications [START_REF] Dan | Interaction between the polyol pathway and non-enzymatic glycation on mesangial cell gene expression[END_REF]. There is strong evidence that those patients with diabetic nephropathy have increased expression and activity of AKR1B1.

We previously demonstrated that the increased expression of AKR1B1 gene is accompanied by elevated AKR1B1 protein level, is ORE-dependent, and regulated by NFAT5 under high glucose conditions [START_REF] Yang | Elevated activity of transcription factor nuclear factor of activated T-cells 5 (NFAT5) and diabetic nephropathy[END_REF]. High glucose induced NFAT5 binding activities to the OREs has been also observed in the myo-inositol oxygenase (MIOX) gene [START_REF] Nayak | Modulation of renal-specific oxidoreductase myo-inositol oxygenase by high-glucose ambience[END_REF][START_REF] Prabhu | Up-regulation of human myoinositol oxygenase by hyperosmotic stress in renal proximal tubular epithelial cells[END_REF], which is closely linked to the polyol pathway and has been suggested to play an important role in the development of diabetic nephropathy [START_REF] Arner | Expression of myoinositol oxygenase in tissues susceptible to diabetic complications[END_REF].

NFκB is activated by a large variety of stimuli including cytokines, viruses, oxidative stress and genotoxic drugs. There is growing evidence that high glucose as well as diabetes can activate NFκB expression [START_REF] Bierhaus | Diabetes-associated sustained activation of the transcription factor nuclear factor-κB[END_REF][START_REF] Yerneni | Hyperglycaemia induced activation of nuclear transcription factor k B in vascular smooth muscle cells[END_REF][START_REF] Guijarro | Transcription factor-kB (NFkB) and renal disease[END_REF][START_REF] Hattori | High-glucose-induced nuclear factor κB activation in vascular smooth muscle cells[END_REF]. Previous studies have shown that

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NFκB-DNA binding activity was increased in response to elevated glucose in cultured porcine vascular smooth muscle cells [START_REF] Yerneni | Hyperglycaemia induced activation of nuclear transcription factor k B in vascular smooth muscle cells[END_REF] and PBMCs from patients with type 1 diabetes [START_REF] Bierhaus | Diabetes-associated sustained activation of the transcription factor nuclear factor-κB[END_REF] and the activity correlated with blood glucose levels. In present study, we demonstrated that the binding activity of NFκB to the κB motif of the AKR1B1 gene was significantly increased under high glucose conditions in nephropaths. In contrast, there was no increase in NFκB-DNA binding activity in patients without microvascular complications. The exact mechanisms have still to be elucidated and explored, but genetic factors or an imbalance of redox probably make important contributions. Our previous report [START_REF] Hodgkinson | The response of antioxidant genes to hyperglycemia is abnormal in patients with type 1 diabetes and diabetic nephropathy[END_REF] indicated there was a defect of anti-oxidant gene expression in patients with nephropathy. Since high glucose causes excess free radical generation [START_REF] Obrosova | How does glucose generate oxidative stress in peripheral nerve?[END_REF][START_REF] Gupta | Hyperglycemia increases endothelial superoxide that impairs smooth muscle cell Na+-K+-ATPase activity[END_REF], this implies that the increase in NFκB binding activity may be due to a generalized response to oxidative stress. This is supported by our previous report that PBMCs of nephropaths have decreased expression of cytoplasmic anti-oxidant genes in response to excess glucose, whilst those from patients with no complications have a marked increase in expression of these genes [START_REF] Hodgkinson | The response of antioxidant genes to hyperglycemia is abnormal in patients with type 1 diabetes and diabetic nephropathy[END_REF]. Since NFκB activation is redox dependent [START_REF] Baeuerle | NF-κB: ten years after[END_REF][START_REF] Barnes | Nuclear factor-κB a pivotal transcription factor in chronic inflammatory diseases[END_REF], and increased metabolic flux through AKR1B1 is strongly linked to a change in redox potential within the cell [START_REF] Chung | Contribution of polyol pathway to diabetes-induced oxidative stress[END_REF], it is possible that NFκB could provide positive feedback to regulate the level of AKR1B1 expression through κB motif. This notion is supported by our results that ARI decreased the DNA binding activities of NFκB in both the nephropaths and uncomplicated subjects. It is also supported by Ramana et al. study [START_REF] Ramana | Activation of nuclear factor -κB by hyperglycemia in vascular smooth muscle cells is regulated by aldose reductase[END_REF] that ablation of AKR1B1 by siRNA prevented high glucose induced NFκB activation in vitro. Furthermore, silencing of NFκB p65 significantly decreased the transcriptional activity of AKR1B1 in vitro in this study, which provided solid evidence that high glucose regulates the expression of AKR1B1 gene through the transcriptional factor NFκB. The susceptible haplotype (Z-2/C-106) to the diabetic nephropathy showed the highest transcriptional activity and the

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common haplotype (Z/C-106) in population showed the second higher transcriptional activity in luciferase assays, which are consistent with our previous results [START_REF] Yang | Elevated activity of transcription factor nuclear factor of activated T-cells 5 (NFAT5) and diabetic nephropathy[END_REF][START_REF] Yang | Functional differences between the susceptibility Z-2/C-106 and protective Z+2/T-106 promoter region polymorphisms of the aldose reductase gene may account for the association with diabetic microvascular complications[END_REF]. The different haplotypes may change the binding activity of NFκB p65 to the κB motif in the promoter region of the AKR1B1 gene as we have seen in nephropaths. Taken together, reduced luciferase activities by silencing of both NFAT5 [START_REF] Yang | Elevated activity of transcription factor nuclear factor of activated T-cells 5 (NFAT5) and diabetic nephropathy[END_REF] and NFκB p65 expression in HEK 293 cells transfected with those recombinants supports the notion that interaction between NFAT5 and NFκB is required for transcription of AKR1B1 gene although we believe that there are more accessory proteins involved in the binding complex because our supershift analysis could not completely abolish the bands by using antibodies against NFκB and NFAT5 (data not shown).

We have demonstrated that increases in NFκB binding activities in response to high glucose were prevented by treatment with an ARI in PBMCs, particularly in the nephropaths. These findings together with those reported in cell lines and other species [START_REF] Ramana | Activation of nuclear factor -κB by hyperglycemia in vascular smooth muscle cells is regulated by aldose reductase[END_REF][START_REF] Obrosova | How does glucose generate oxidative stress in peripheral nerve?[END_REF][START_REF] Ramana | Aldose reductase mediates cytotoxic signals of hyperglycaemia and TNF-in human lens epithelial cells[END_REF][START_REF] Williamson | Hyperglycemic pseudohypoxia and diabetic complications[END_REF][START_REF] Ramana | Aldose reductase mediates mitogenic signalling in vascular smooth muscle cells[END_REF] strongly suggest that increased expression and activity of AKR1B1 is intricately linked with the activation of the pro-inflammatory transcription factor NFκB.

The precise mechanism underlying these observations has still to be determined. Recently it has been shown that inhibition of aldose reductase either by ARIs or SiRNA can abolish many of the pro-inflammatory effects of NFκB in animal models of systemic inflammation. The inhibition of aldose reductase can modify a number of NFκB related molecular and cellular pathways within the cell including mitogenic signalling and the accumulation of products of lipid peroxidation [START_REF] Ramana | Endotoxin-induced cardiomyopathy and systemic inflammation in mice is prevented by aldose reductase inhibition[END_REF]. A recent study from the same group showed that high glucose induced NFκB activation could be prevented by ARI through inhibition of TNF-α synthesis and secretion in vascular smooth muscle cell [START_REF] Ramana | Aldose reductase-regulated tumour necrosis factor-alpha production is essential for high glucoseinduced vascular smooth muscle cell growth[END_REF]. All those results indicate that ARI could be modulating the response by decreasing the action of AKR1B1 through its action as a pro-inflammatory mediator [START_REF] Ramana | Aldose reductase mediates cytotoxic signals of hyperglycaemia and TNF-in human lens epithelial cells[END_REF].

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The absence of any significant detectable changes in protein levels of cytoplasmic NFκB, IkB-α and P-IkB-α in the patient groups may be due to a number of reasons including higher DNA binding affinity of NFκB in the diabetic nephropaths compared with diabetic controls. This might be influenced by genetic variation in the promoter region [START_REF] Yang | Functional differences between the susceptibility Z-2/C-106 and protective Z+2/T-106 promoter region polymorphisms of the aldose reductase gene may account for the association with diabetic microvascular complications[END_REF]. In addition, previous reports suggest that the reservoir of individual nuclear proteins is far greater than the total amount of NFκB protein actually required for binding to the DNA binding motifs [START_REF] Natoli | Interaction of NF-κB with chromatin: the art of being at the right place at the right time[END_REF]. Finally, it is likely that access to these motifs differs between patients with or without nephropathy [START_REF] Yang | Functional differences between the susceptibility Z-2/C-106 and protective Z+2/T-106 promoter region polymorphisms of the aldose reductase gene may account for the association with diabetic microvascular complications[END_REF][START_REF] Saccani | Two waves of nuclear factor κB recruitment to target promoters[END_REF]. Consequently, in our study there may be reduced requirement for NFkB to translocate from the cytoplasm to the nucleus.

We cannot fully explain patterns of phosphorylated IκB-α and IκB-α protein levels under various conditions in both groups. Theoretically, an increase in phosphorylated IκBshould be accompanied by a decrease in total IκB due to phosphorylation. A recent review [START_REF] Janssens | Signals from within: the DNA-damage-induced NFκB response[END_REF] suggested that there are three major NFκB activating pathways responding to various stimuli: the canonical, the alternative and atypical pathways. Oxidative stress may lead to the atypical pathway with a weak and slow NFκB signal (with peak activities reached after 2-4 hours) [START_REF] Li | Is NFκB the sensor of oxidative stress?[END_REF] and long-term high glucose may generate oxidative and genotoxic stresses, which may partly induce NFκB activities in an IKK-independent way. Therefore, the absence of significant decreases in IκB-α expression in PBMCs may reflect this fact, cells exposed to chronic high glucose. Our observation is consistent with previous reports showing sustained NFκB activation in the absence of significant decreases in IκB-α expression in PBMCs exposed to chronic high glucose [START_REF] Bierhaus | Diabetes-associated sustained activation of the transcription factor nuclear factor-κB[END_REF].

In conclusion, the exposure of human PBMCs to high glucose in vitro caused increased binding of NFκB to the κB motif. These increases were significantly higher in patients with the nephropathy compared to the uncomplicated subjects and were accompanied with an increase in protein level of AKR1B1. ARI decreased the binding Data are means+SE (ranges) in years. Levels of plasma and Hemoglobin A1c (HbA1c) are expressed as means+SE in mmol/l and %, respectively. Uncomplicated: patients have been diagnosed with type 1 for at least 20 years but remain free of retinopathy (fewer than five dots or blots per fundus), proteinuria (urine Albustix negative on at least three consecutive occasions over 12 months) and neuropathy (overt neuropathy was defined if there was any clinical evidence of peripheral or autonomic neuropathy). Diabetic nephropaths: patients have had type 1 for at least 8 years with persistent proteinuria (urine Albustix positive on at least three consecutive occasions over 12 months or three consecutive total urinary protein excretion rates >0.5g/24h) in the absence of hematuria or infection on midstream urine samples.

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  A C C E P T E D M A N U S C R I P T negative control at a 500 nmol/l were used in our study. For analysing the effect of silencing of NFκB p65 on the transcription of AKR1B1 gene, recombinants pGL3 reporter plasmids at 98 ng which contain the promoter region with OREC/κB motif and haplotypes Z-2/C-106 or Z/C-106 of the AKR1B1 gene [6, 18] and firefly luciferase gene downstream were co-transfected with pRL-TK control plasmids which contain the Renilla reniformis luciferase gene into the cells at 2 ng after 24 hours of siRNA transfection. Next day, cells were divided into four groups: (a) cells were maintained in original media; (b) cells were switched to HG (a final concentration at 25 mmol/l of D-glucose) and (c) cells were switched to ARI (sorbinil at 10 μmol/l); (d) cells were switched to HG and ARI

ALEGENDSFigure 1 .

 1 Figure 1. A: A sequence scheme of the promoter region of AKR1B1 gene. (AC)n is the

Figure 2 .

 2 Figure 2. Western blotting results with antibodies against human AKR1B1, NFκB p65,

Figure 3 .

 3 Figure 3. Data are means+SE (5 experiments were performed). Results are relative

  Figure 2

Table 1 . Clinical characteristics of patients with type 1 diabetes

 1

		Nephropaths	Uncomplicated
		(n=20)	(n=14)
	Male:Female	6:14	4:10
	Age	49.6+13.9	48.1+14.9
	(years)	(30-82)	(19-74)
	Age at onset of	12.3+8.8	15.2+9.4)
	diabetes	(1-35)	(2-38)
	(years)
	Duration of diabetes	34.9+8.1	34.3+9.4)
	(years)	(21-49)	(20-53)
	Plasma Glucose	9.8+0.8	10.1+1.5
	(mmol/L)
	HbA1c	8.2+0.3	8.3+0.3
	(%)

A C C E P T E D M A N U S C R I P T


Rosalinda Madonna

Marika Massaro

M.D Raffaele De Caterina

Marika Massaro°

Insulin potentiates cytokine-induced VCAM-1 expression in human endothelial cells

Keywords: Insulin, atherosclerosis, vascular cell adhesion molecule-1, cytokines, type 2 diabetes, insulin resistance

Hyperinsulinemia is an independent risk factor for cardiovascular events and may contribute to cardiovascular disease. Low grade, chronic inflammation has been implicated in the pathogenesis of atherosclerosis. We aimed at determining the impact of pathophysiologically high insulin concentrations on cytokine-induced endothelial activation in human umbilical vein endothelial cells (HUVEC). HUVEC were incubated with insulin (0-24 h) ± tumor necrosis factor(TNF)-α or lipopolysaccharide (LPS). At pathophysiological/pharmacological concentrations (10 -9 -10 -7 mol/L), insulin selectively induced VCAM-1 expression and potentiated the effects of TNF-α and LPS, effects reverted by the proteasome inhibitor lactacystin. Compared with TNF-α alone, insulin+TNFα doubled U937 cell adhesion. Insulin markedly increased TNF-α-induced NF-κB activation and induced phosphorylated IκBα accumulation. Therefore, hyperinsulinemia enhances cytokineinduced VCAM-1 expression in endothelial cells, thus potentially contributing to detrimental effects of other inflammatory stimuli on atherogenesis.

Introduction

Increased circulating levels of pro-inflammatory cytokines, such as interleukin(IL)-6 [START_REF] Fernandez-Real | Circulating interleukin 6 levels, blood pressure, and insulin sensitivity in apparently healthy men and women[END_REF]and tumor necrosis factor (TNF)-α [START_REF] Mishima | Relationship between serum tumor necrosis factor-alpha and insulin resistance in obese men with Type 2 diabetes mellitus[END_REF], as well as high levels of acute phase reactants [START_REF] Mcmillan | Increased levels of acute-phase serum proteins in diabetes[END_REF], have been found in patients with insulin resistance, obesity and type 2 diabetes. Low-grade, chronic inflammation may play a role in the pathogenesis of insulin resistance in obesity and type 2 diabetes. Indeed, inflammatory cytokines inhibit insulin signaling by impairing the tyrosine kinase activity of both the insulin receptor (IR) and insulin receptor substrate(IRS)-1, thus acting as mediators of insulin resistance [4; 5]. Furthermore, mice lacking TNF receptor function have been shown to develop protection from obesity-induced insulin resistance [START_REF] Uysal | Protection from obesityinduced insulin resistance in mice lacking TNF-alpha function[END_REF]. Insulin resistance and compensatory hyperinsulinemia are probably causally associated with elevated levels of inflammatory markers and increased risk of cardiovascular disease [START_REF] Despres | Hyperinsulinemia as an independent risk factor for ischemic heart disease[END_REF]. Thus, by determining insulin resistance, systemic low-grade chronic inflammation would act as a key pathogenetic factor for cardiovascular disease in type 2 diabetes and the metabolic syndrome [START_REF] Festa | Chronic subclinical inflammation as part of the insulin resistance syndrome: the Insulin Resistance Atherosclerosis Study (IRAS)[END_REF].

A growing body of evidence now suggests the existence of a bidirectional relationship between hyperinsulinemia and low-grade chronic inflammation, by which hyperinsulinemia might contribute to vascular inflammation, and vascular inflammation leads to insulin resistance and consequent compensatory hyperinsulinemia. Indeed, in humans, plasma concentrations of the proinflammatory cytokines IL-6 and TNF-α increase when insulin is administered together with bacterial lipopolysaccharide (LPS) [START_REF] Soop | Euglycemic hyperinsulinemia augments the cytokine and endocrine responses to endotoxin in humans[END_REF]. In human smooth muscle cells [START_REF] Golovchenko | Hyperinsulinemia enhances transcriptional activity of nuclear factor-kappaB induced by angiotensin II, hyperglycemia, and advanced glycosylation end products in vascular smooth muscle cells[END_REF], in murine glomerular vascular endothelial cells [START_REF] Hasan | Differential regulation of early growth response gene-1 expression by insulin and glucose in vascular endothelial cells[END_REF] and in human adipose tissue [START_REF] Krogh-Madsen | Insulin stimulates interleukin-6 and tumor necrosis factor-alpha gene expression in human subcutaneous adipose tissue[END_REF] hyperinsulinemia exacerbates inflammation and increases levels of markers of oxidative stress, while on the contrary insulin exerts prevailing anti-inflammatory effect at low, physiological, concentrations [13; 14; 15; 16].

Finally, in macrophages, insulin, at pathophysiologically relevant concentrations, stimulates TNF-α expression and production [START_REF] Iida | Insulin up-regulates tumor necrosis factor-alpha production in macrophages A C C E P T[END_REF].

A C C E P T E D M A N U S C R I P T

ACCEPTED MANUSCRIPT Madonna R et al.,page 4 We have previously shown that in human umbilical endothelial cells (HUVEC) insulin per se increases the expression of vascular cell adhesion molecule-1 (VCAM-1) [18; 19], probably the adhesion molecule most relevant to the development of atherosclerosis [START_REF] Cybulsky | A major role for VCAM-1, but not ICAM-1, in early atherosclerosis[END_REF]. However, the impact of high insulin on the inflammatory response to cytokines is still poorly characterized. In this study we Cell cultures -HUVEC were harvested enzymatically from umbilical cords with Type-II collagenase at 0.1 mg/mL, and cultured as described [START_REF] Carluccio | Olive oil and red wine antioxidant polyphenols inhibit endothelial activation: antiatherogenic properties of Mediterranean diet phytochemicals[END_REF]. Cell viability after treatments was assessed by evaluating several parameters, including cell morphology at phase-contrast microscopy, Trypan blue exclusion, determination of total proteins and of the surface expression of a constitutive and non-cytokine-inducible endothelial cell antigen (E1/1) [START_REF] Caterina | Nitric oxide decreases cytokine-induced endothelial activation. Nitric oxide selectively reduces endothelial expression of adhesion molecules and proinflammatory cytokines[END_REF].

Materials and Methods

Materials

Cell surface immunoassays -Assays of cell surface molecules were carried out by cell surface enzyme immunoassays (EIA), using mouse anti-human monoclonal antibodies against VCAM-1 (Ab E1/6), E-Selectin (Ab H18/7), intercellular adhesion molecule(ICAM)-1 (Ab HU5/3), or a constitutive and non-cytokine-inducible endothelial cell antigen (E1/1) [START_REF] Caterina | Nitric oxide decreases cytokine-induced endothelial activation. Nitric oxide selectively reduces endothelial expression of adhesion molecules and proinflammatory cytokines[END_REF]. Details of the EIA have been previously reported [START_REF] Caterina | Nitric oxide decreases cytokine-induced endothelial activation. Nitric oxide selectively reduces endothelial expression of adhesion molecules and proinflammatory cytokines[END_REF].

Flow cytometry -Flow cytometric analysis of adhesion molecule expression was performed by incubating HUVEC with the mouse anti-human monoclonal antibody E1/6 against VCAM-1 for 60 min at 4° C. After washing, cells were exposed to goat anti-mouse F(ab') Measurement of TNF-α release -HUVEC seeded into 24-well culture plates (5 x 10 5 cells/well) were stimulated with insulin (10 -9 -10 -7 mol/L) for 24 h with or without LPS (0.1 ng/mL).

TNF-α release in the supernatants was measured by an enzyme-linked immunosorbent assay (ELISA), using a commercial kit (Bender MedSystems, Vienna, Austria) and following the manufacturer's protocols. A standard curve was prepared from seven TNF-α dilutions. The lower limit of sensitivity was 7.8 pg/mL. The overall inter-assay and intra-assay coefficients of variation were <10%. In each well, residual cells that had been deprived of medium were lysed with ice-cold RadioImmuno Precipitation Assay (RIPA) buffer, and total cellular proteins were measured with a BCA assay (Pierce, Rockford, IL). The TNF-α content in the medium was normalized to cell protein values.

Immunoblotting -Total proteins were isolated from HUVEC in ice-cold RIPA buffer.

Nuclear proteins were purified as previously described [START_REF] Carluccio | Olive oil and red wine antioxidant polyphenols inhibit endothelial activation: antiatherogenic properties of Mediterranean diet phytochemicals[END_REF]. Proteins were separated under reducing

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conditions and electroblotted onto polyvinylidene fluoride membranes (Immobilon-P; Millipore, Bedford, MA). After blocking, the membranes were incubated overnight at 4° C with primary antibodies against VCAM-1 (Santa Cruz Biotechnologies, Santa Cruz, CA), and with primary antibodies against the constitutive and Ser 32 -phosphorylated inhibitor IκBα (Santa Cruz Biotechnology). The blots were developed using a SuperSignal West Pico Chemiluminescent Substrate kit (Pierce). The intensity of each immunoreactive protein band was measured by densitometry.

Adhesion assays -HUVEC grown at confluence in 6-well plates were incubated with 1 mL of 1 × 10 6 cells/mL U937 cell suspension (American Tissue Culture Collection, Rockville, MD) , under rotating conditions (63 r.p.m.) at room temperature. To test the VCAM-1-dependence of adhesion, some monolayers were treated with the mouse anti-human monoclonal antibody E1/6, directed against VCAM-1. After 10 min, non-adhering cells were removed and monolayers fixed in 1% paraformaldehyde. The number of adhering cells was determined by counting 8 different highpower fields (h.p.f.) using an ocular grid and a 20× objective (0.09 mm 2 /field). Fields for counting adherent leukocytes were randomly located at half-radius distance from the center of the monolayers.

Northern Analysis -Total RNA was isolated by a single extraction using an acid guanidinium thiocyanate-phenol-chloroform method [START_REF] Chomczynski | Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction[END_REF]. Details of Northern analysis have been previously reported [START_REF] Carluccio | Olive oil and red wine antioxidant polyphenols inhibit endothelial activation: antiatherogenic properties of Mediterranean diet phytochemicals[END_REF].

Electrophoretic mobility (gel) shift assay -Nuclear proteins from stimulated or unstimulated HUVEC were purified as previously described [START_REF] Carluccio | Olive oil and red wine antioxidant polyphenols inhibit endothelial activation: antiatherogenic properties of Mediterranean diet phytochemicals[END_REF]. Protein concentration was determined by the Bio-Rad Laboratories Protein Assay (Bio-Rad Laboratories Inc, Hercules, CA). The doublestranded synthetic oligonucleotides NF-κB motif (5'-AGT TGA GGG GAC TTT CCC AGG C-3'

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and 5'-CCT GGG AAA GTC CCC TCA ACT-3') was labeled with [γ-32 P]-ATP. Binding reactions containing 10 µg of crude nuclear extract were performed using the electrophoretic mobility gel shift assay core system (Promega, Madison, WI) according to the manufacturer's protocol. Insulin, in a concentration-dependent manner, per se induced VCAM-1 surface expression (Fig. 1,A and Table 1), but also potentiated the effects of TNF-α and LPS. Quiescent endothelial cells co-incubated with pathophysiologically relevant concentrations of insulin (10 -9 -10 -8 mol/L) and low concentrations of TNF-α (0.1 ng/mL) had an average 3.9-fold increase in VCAM-1 expression (p<0.001 vs untreated cells), which was greater than the stimulation by insulin alone (2 fold) or TNF-α alone (2.4-fold, * p<0.001 vs untreated cells ) (Fig. 1,A). In the absence of any cytotoxic effect (Online Supplement Figure ), the addition of insulin to TNF-α-treated cells produced a maximum of 1.4-fold increase in VCAM-1 surface expression (** p<0.05, insulin + TNF-α-vs TNF-α-treated cells). Similar at-least-additive and concentration-dependent effects of insulin were obtained when a higher TNF-α concentration (1 ng/mL) was used (Fig. 1,B).

Statistical Analysis

The co-stimulatory effect of insulin and TNF-α seen at cell surface EIA was paralleled by an increase of the total cellular content of VCAM-1, as assessed by immunoblotting (Fig. 1,E).

Consistently, flow cytometry demonstrated that HUVEC treated with insulin for 16 h showed not only a significant increase in mean fluorescent intensity for VCAM-1 compared with untreated cells, but also an expression greater than that obtained after co-incubation with TNF-α 1 ng/mL (Table 1). We also observed an at-least-additive and concentration-dependent effect on VCAM-1 expression by adding increasing concentrations of insulin (10 -9 -10 -8 mol/L) to low concentrations of LPS (0.1 ng/mL). The maximum induction of VCAM-1 expression with insulin and LPS coadministration was 2.3-fold vs basal untreated cells, whereas it was 1.7-fold vs LPS-treated cells (# p<0.05), and 1.4-fold with LPS alone vs untreated cells (* P<0.05) (Fig. 1,C).

Immunocytochemistry on endothelial cell monolayers further confirmed the effect of insulin + TNF-α on VCAM-1 surface expression. (Fig. 1,D). Since a robust costimulatory effect of insulin

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT Madonna R et al., page 10
was seen at a concentration of TNF-α of 1 ng/mL, further experiments were performed with this concentration of TNF-α.

Ruling out possible cytolytic or cytostatic effects of TNF-α and LPS [START_REF] Old | Tumor necrosis factor[END_REF], phase-contrast microscopy revealed no morphologic signs of injury to human endothelial cells due to TNF-α (1 ng/ml, 24 h) in these experiments in the presence or absence of insulin (10 -9 -10 -8 mol/L, 24 hours) (Online Supplement Figure ). Similarly, LPS (0.1 ng/mL given for 24 h) or TNF-α (1 ng/ml given for 24 h) or insulin alone (10 -9 -10 -8 mol/L given for 24 h) did not reduce cell number or protein content of endothelial cultures, or expression of the constitutive surface endothelial protein E1/1

(Online Supplement Table).
To rule-out that endotoxin contamination contributed to the effect of insulin on VCAM-1 expression, the LPS inactivator polymixin-B sulfate (PMBS) was added in a parallel set of experiments. PMBS completely abrogated LPS-induced VCAM-1 expression, but had no effect on insulin ± TNF-α-induced VCAM-1 expression (data not shown).

Inflammatory mediators such as interferon-γ and LPS are known to increase the secretion of TNF-α from several cell types, including macrophages [START_REF] Gifford | Requirement for the continual presence of lipopolysaccharide for production of tumor necrosis factor by thioglycollate-induced peritoneal murine macrophages[END_REF], monocytes [START_REF] Libby | Endotoxin and Tumor Necrosis Factor Induce Interleukin-1 Gene Expression in Adult Human Vascular Endothelial Cells[END_REF] and smooth muscle cells [START_REF] Warner | Human smooth muscle cells. Target for and source of tumor necrosis factor[END_REF].

In HUVEC, LPS has been demonstrated however not to induce TNF secretion [START_REF] Libby | Endotoxin and Tumor Necrosis Factor Induce Interleukin-1 Gene Expression in Adult Human Vascular Endothelial Cells[END_REF]. In macrophages, insulin by itself has been shown to increase gene and protein expression of TNF-α [START_REF] Iida | Insulin up-regulates tumor necrosis factor-alpha production in macrophages A C C E P T[END_REF]. To test whether insulin -alone or in combination with LPS -induces TNF-α secretion in HUVEC, which might account, in an autocrine manner, for the potentiation by insulin of the effects of other agents, we measured TNF-α protein mass by ELISA in the cell medium of HUVEC treated with insulin (10 -9 -10 -7 mol/L) in the presence or absence of LPS (0.1 ng/mL). Treatment of HUVEC with insulin alone or in the presence of LPS for 16 h did not end up in any detectable production of TNF-α (TNF-α concentrations always below the detection limit of our assay (7.8 pg/mL)).

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Madonna R et al., page 11

Insulin, at pathophysiological concentrations, increases cytokine-induced monocytoid cell adhesion to human umbilical endothelial cells.

Because the induction of VCAM-1 on the endothelium is known to support monocyte adhesion, we examined the functional consequences of endothelial treatment with insulin in terms of monocytoid (U937) adhesion to endothelial cells, using a rotational adhesion assay. In agreement with data on VCAM-1 surface expression, treatment of quiescent endothelial cells with insulin (10 -8 mol/L) alone for 16 h produced a 2.8-fold increase in U937 cell adhesion compared with control, unstimulated endothelial cells (Fig. 2, panels A and C, conditions a and b). Stimulation of endothelial cells with TNF-α (1 ng/mL) substantially increased U937 cell adhesion, and this effect was further potentiated by the addition of insulin (2-fold increase in U937 cell adhesion compared with TNF-α alone, Fig. 2, panels A and C, conditions d and e). This insulin-and cytokine-induced adhesion was markedly suppressed (by about 70%) by a 30 min pre-treatment of endothelial cells with the blocking anti-VCAM-1 monoclonal antibody E1/6, at saturating concentrations (Fig. 2, panels A and C, conditions c and f).

Insulin, at pathophysiological concentrations, induces and co-induces VCAM-1 mRNA at

Northern analysis.

We observed increased mRNA levels in cells treated with 10 -8 mol/L insulin ± TNF-α (1 ng/mL), consistent with the observed increase of VCAM-1 protein expression (Fig. 2, panels B and D). In untreated cells, VCAM-1 mRNA was not detectable, while in cells treated with insulin alone substantial levels of mRNA were detected (albeit, compared with stimulations in the presence of TNF-α, with longer exposure times and a double intensifying screen) (p<0.05). Compared with TNF-α alone, the addition of insulin to TNF-α increased levels of VCAM-1 mRNA by 1.6 fold (p<0.05), as determined by densitometry of Northern blots at 4-6 h in two different experiments.

Overall, these results indicate that increased VCAM-1 surface levels after insulin ± cytokine stimulation occurs at a pre-traslational levels, while the absence of VCAM-1 mRNA levels in

Discussion

We had previously demonstrated that insulin, at pathophysiologically relevant concentrations, exerts pro-inflammatory, pro-atherogenic effects on endothelial cells by enhancing VCAM-1 expression and monocyte-endothelial interactions [18; 19]. The present findings now (a)

demonstrate the ability of insulin to act in concert with other pro-inflammatory stimuli in enhancing the surface expression of VCAM-1 on endothelial cells; (b) provide a mechanistic explanation of the observed effects. This is -to the best of our knowledge -the first report showing the additive effects of insulin and pro-inflammatory cytokines on endothelial activation.

We used insulin concentrations between 10 -9 and 10 -7 mol/L, in the range of binding and activation of insulin receptors in endothelial cells [START_REF] Lee | The insulin receptor: structure, function, and signaling[END_REF]. Such concentrations, equivalent to 139-13,900 µU/mL, span from pathophysiological to pharmacological levels of insulinemia.

Concentrations of 10 -9 and 10 -8 mol/L are indeed attainable in fasting and post-prandial states of individuals with insulin resistance [START_REF] Saad | Sequential changes in serum insulin concentration during development of non-insulin-dependent diabetes[END_REF], indicating the plausibility, with all the limitations of extrapolating from in vitro findings, that these observations are applicable to in vivo settings where hyperinsulinemia occurs.

TNF-α and LPS are potent inflammatory mediators, known to synergize in the upregulation of endothelial adhesion molecule expression during immune and inflammatory processes [START_REF] Jersmann | Bacterial lipopolysaccharide and tumor necrosis factor alpha synergistically increase expression of human endothelial adhesion molecules through activation of NF-kappaB and p38 mitogen-activated protein kinase signaling pathways[END_REF]. TNFα has been shown to be a mediator of insulin resistance in infection, tumor cachexia and obesity [4; 5]. Indeed TNF-α is known to impair tyrosine kinase activity of both insulin receptor (IR) and IR substrate-1 (IRS-1), with subsequent blocking of the IRS-1/phosphatidyl inositol(PI)-3 kinase association, through multi-site phosphorylations of serine/threonine residues as a consequence of the activation of multiple serine/threonine protein kinases or the inactivation of serine/threonine phosphatases [START_REF] Hotamisligil | IRS-1mediated inhibition of insulin receptor tyrosine kinase activity in TNF-alpha-and obesityinduced insulin resistance[END_REF]. In addition, TNF-α, through serine/threonine or tyrosine phosphorylations, has been shown to enhance the activity of mitogen-activated protein (MAP) kinases [START_REF] Vietor | Tumor necrosis factor-induced activation and increased tyrosine phosphorylation of mitogen-activated protein (MAP) kinase in human fibroblasts[END_REF], particularly p38MAP kinase [START_REF] Kyriakis | The stress-activated protein kinase subfamily of c-Jun kinases[END_REF]. Furthermore, insulin has been recently shown to stimulate TNF-α production

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in macrophages, by regulating the expression of TNF-α mRNA through the ERK signalling pathway [START_REF] Iida | Insulin up-regulates tumor necrosis factor-alpha production in macrophages A C C E P T[END_REF]. All these interactions, together with the known ability of these primary cytokines to activate the gene expression of adhesion molecules through NF-κB activation, provide ample biological rationale for the observed at least additive interaction between insulin and inflammatory cytokines or LPS, here for the first time formally demonstrated. The ability of insulin, alone or in combination with low concentrations of TNF-α, to promote VCAM-1 expression in our system appears related, at least in part, to the ability of insulin to increase steady-state levels of mRNA, in turn due to insulin ability to activate transcription factors traditionally implicated in VCAM-1 transactivation, such as NF-κB.

In conclusion, mimicking hyperinsulinemia and the simultaneous exposure to inflammatory mediators in vitro we have shown here the promotion of pro-atherogenic actions of insulin in endothelial cells. Favoring the pro-atherogenic properties of inflammatory cytokines on the endothelium may represent a major pathogenetic mechanism involved in increased atherogenesis and the promotion of vascular disease in patients with type 2 diabetes and the metabolic syndrome.

  therefore investigated (a) whether insulin promotes endothelial activation in human endothelial cells exposed to pro-inflammatory cytokines; (b) the relevance of nuclear factor(NF)-κB in insulinmediated signaling and (c) the functional consequences of the selective modulation of insulin signaling on monocytoid cell adhesion to endothelial cells. A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT Madonna R et al., page 5

-

  Human recombinant insulin was purchased from Novo-Nordisk Farmaceutici S.p.A., Rome, Italy. TNF-α, endotoxin, polymixin B and lactacystin were purchased from Sigma, St. Louis, MO. The monoclonal antibodies against VCAM-1 (Ab E1/6), E-selectin (Ab H18/7), ICAM-1 (Ab Hu 5/3), and a constitutive and non-cytokine-inducible endothelial antigen (Ab E1/1) were kindly provided by M.A. Gimbrone, Jr., Brigham & Women's Hospital, Boston, MA.

  -Two-group comparisons were performed by the Student's t-test for unpaired values. Comparisons of means of ≥3 groups were performed by analysis of variance (ANOVA), and the existence of individual differences, in case of significant F values at ANOVA, tested by Scheffé's multiple contrasts. Comparisons of distribution of fluorescence intensities at flow cytometry were performed by the Kolgomorov-Smirnov statistics with the aid of the CellQuest TM software package from Becton Dickinson, 1997. A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT Madonna R et al., page 9ResultsInsulin, at pathophysiological concentrations, potentiates cytokine-induced VCAM-1 expression in human umbilical endothelial cells.

3 A, lanes 3 and 4 )Fig. 3 ,

 3343 al., page 12 control conditions and their presence after stimulation with insulin alone indirectly argues for an induction of transcription. Insulin, at pathophysiological concentrations, increases cytokine-induced NF-κB activation and the phosphorylated form of IκB-α in the cytosol of human umbilical endothelial cells Insulin treatment markedly increased TNF-α-induced activation of NF-κB (Fig. . The specificity of the NF-κB DNA-protein complex was verified by successful competition with unlabeled (cold) NF-κB oligonucleotide and by the lack of any binding reaction with a mutated oligonucleotide (not shown). In addition, there was no significant and specific binding with the unlabeled (cold) oligonucleotide (Fig. 3, lane 5), or by omitting the nuclear protein extracts or the oligonucleotide probe (not shown), as well as by using nuclear protein extracts from unstimulated HUVEC (lane 1). Insulin treatment further increased TNF-α-induced cellular accumulation of Ser 32 -phosphorylated IκB-α in a concentration-dependent manner (Fig. 4 B). Treatment with insulin was thus shown to promote IκB-α phosphorylation, thus accounting for NF-κB translocation into the nucleus.We next examined the effect of the proteasome inhibitor lactacystin (10 µmol/L) on insulininduced VCAM-1 expression in the presence of TNF-α. In the absence of any cytotoxicity, treatment of HUVEC with lactacystin significantly decreased TNF-α-induced VCAM-1 expression (* = p<0.05 vs TNF-treated cells, Fig.4 A), and this effect was further potentiated in HUVEC cotreated with insulin and TNF-α (# = p<0.001, Fig.4 A). Taken together, these results demonstrate that NF-κB plays a key role in mediating the pro-inflammatory effects of high insulin in cytokineinduced human endothelial cells.A C C E P T E DM A N U S C R I P T ACCEPTED MANUSCRIPTMadonna R et al., page 13

Fig. 1 .

 1 Fig. 1. Costimulatory effects of insulin and TNF-α or LPS on VCAM-1 surface expression in

Fig. 2 . 21 Fig. 3 Fig. 4 .

 22134 Fig. 2. Effect of insulin and TNF-α on monocytoid cell adhesion to endothelial cells and on

  3 154.5 + 18.1 Subconfluent serum-starved HUVEC were treated with TNF-α and/or insulin (10 -9 -10 -8 mol/L) for 16 h. Cells were then immunostained for VCAM-1 or FITC-conjugated IgG isotyepe control and submitted to flowcytometry. Values (a) are mean ± S.D. of % cells expressing VCAM-1; values (b) are mean ± S.D. of units of fluorescence intensity. Results are representative of three independendent experiments. * = p<0.05 vs unstimulated control; ** = p<0.05 vsTNF-α.

Table 1 :

 1 The co-induction of VCAM-1 expression by insulin and TNF-α at flow cytometry

	Treatment	% gated (a)	fluorescence intensity (b)
	untreated	1.07 + 0.05	5.11 + 1.53
	insulin 10 -9 mol/L *	2.08 + 0.7	9.22 + 2.18
	insulin 10 -8 mol/L *	4.22 + 1.2	15.6 + 3.2
	TNF-α 1 ng/mL *	20.27 + 4.2	115.8 + 11
	insulin 10 -9 mol/L + TNF-α 1 ng/mL **	32.87 + 5.3	148.7 + 14.3
	insulin 10 -8 mol/L + TNF-α 1 ng/mL **	38.97 + 8.

Acknowledgements

This work has been supported through funding from the Center of Excellence on Aging (C.E.A.) Project, through a PRIN grant from the Italian Ministry of the University and Scientific Research, and through a grant from the Consorzio Italiano Ricerche Cardiovascolari (C.I.R.C.), to the Chair of Cardiology at "G. d'Annunzio" University, Chieti. We thank Michael A. Gimbrone Jr., Brigham & Women's Hospital, Boston, MA, for providing antibodies against endothelial antigens, and Dr. Elena Toniato and Dr. Raffaella Faricelli for help in the TNF-α ELISA.