Toxicology in Vitro 25 (2011) 286–293

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Toxicology in Vitro

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In vitro effect of aspartame in angiogenesis induction

Renata Alleva a,⇑, Battista Borghi a, Lory Santarelli b, Elisabetta Strafella b, Damiano Carbonari b,c,Massimo Bracci b, Marco Tomasetti b

a Department of Anesthesiology Research Unit, IRCCS Orthopaedic Institute Rizzoli, Bologna, Italyb Department of Molecular Pathology and Innovative Therapies, Clinic of Occupational Medicine, Polytechnic University of Marche, Ancona, Italyc Department of Occupational Hygiene, National Institute for Occupational Safety and Prevention, Rome, Italy

a r t i c l e i n f o

Article history:Received 25 January 2010Accepted 2 September 2010Available online 17 September 2010

Keywords:AspartameAngiogenesisGenotoxicityIL-6VEGFROS

0887-2333/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.tiv.2010.09.002

⇑ Corresponding author. Tel.: +39 051 6366844; faxE-mail address: rena.alleva@gmail.com (R. Alleva)

a b s t r a c t

Aspartame (APM) is the most widely used artificial sweetener and is added to a wide variety of foods,beverages, drugs, and hygiene products. In vitro and in vivo tests have reported contradictory data aboutAPM genotoxicity. We evaluated the angiogenic effect of APM in an in vitro model using blood vesseldevelopment assay (Angio-Kit), cultured endothelial cells and fibroblasts. The release of IL-6, VEGF-A,and their soluble receptors sIL-R6 and sVEGFR-2 were determined over time in the conditioned mediumof the Angio-Kit system, endothelial cells and cell lines with fibroblast properties after APM treatment.Reactive oxygen species (ROS) formation, cell viability, and stimulation of the extracellular signal-regu-lated kinases (erk1/2) and protein p38 were also evaluated. Exposure to APM induced blood vessel for-mation. ROS production was observed in endothelial cells after APM treatment, which was associatedwith a slight cell cytotoxicity. Neither intracellular ROS formation nor cell death was observed in fibro-blasts. APM increases the levels of inflammatory mediator IL-6, VEGF and their soluble receptors releasedfrom endothelial cells into the medium. APM treatment induces VEGF-pathway activation by erk1/2 andp38 phosphorylation. APM at low doses is an angiogenic agent that induces regenerative cytokine pro-duction leading to the activation of MAPKs and resulting in the formation of new blood vessels.

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1. Introduction

Aspartame (APM) is the most widely used artificial sweetenerand is added to a wide variety of foods, beverages, drugs, and hy-giene products. It was first approved by the US Food and DrugAdministration (FDA) for limited use in solid food in 1981 (FDA,1981). Later, its usage was extended to soft drinks (FDA, 1983),which currently account for >70% of the sale of APM. The accept-able daily intake (ADI) of APM is currently 50 mg/kg body weight(bw) in the United States and 40 mg/kg bw in the European Unionfor both children and adults. The estimated daily intakes of APM bychildren and adolescents are below the ADI (Butchko and Kotsonis,1991; Lino et al., 2008). In a Norwegian study, the consumers ofbeverages had APM intakes ranging from 6.1 to 10.2 mg/kg bw,which are below the ADI, but it has been reported that the contri-bution from other food categories might increase the estimated in-take to levels above ADI (Husøy et al., 2008).

The toxicity of APM is linked to the formation of methanol andformaldehyde. APM is metabolized in the gastric tract to its threemetabolites: aspartic acid, phenyalanine and methanol. Asparticacid is transformed into alanine and oxaloacetate, phenylalanine

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is transformed into tyrosine and, to a lesser extent, phenylethylala-nine, and methanol is converted into formaldehyde and then toformic acid (Ranney and Oppermann, 1979). Studies focusing onthe evaluation of APM toxicity showed controversial results. In vi-tro and in vivo tests have shown that APM is not genotoxic. DNArepair assay for the evaluation of genotoxicity did not show anyDNA-damaging properties for APM (Jeffrey and Williams, 2000).Recently, a genotoxic effect of APM has been observed. APM in-duces chromosome aberration, micronuclei formation, whereas itdoes not induce mutagenesis (Rencüzogullari et al., 2004). APM in-duces DNA strand breaks in bone marrow cells, as revealed by co-met assay. However, it could not act as a potential mutagen in theAmes/Salmonella/microsome test (Bandyopadhyay et al., 2008).Animal studies have showed that APM does not have any cancer-inducing effects, even in very high doses (Hagiwara et al., 1984;Ishii, 1981). Human studies have confirmed the safety of APM(Trefz et al., 1994; Tephly, 1999), although it has been suggestedthat APM consumption might constitute a hazard to humans be-cause of the formaldehyde adducts (Trocho et al., 1998). The firstevidence of a carcinogenetic effect of APM was described in ananimal model by Soffritti et al. (2006) (Belpoggi et al., 2006).Long-term carcinogenicity bioassay on APM demonstrates themultipotential carcinogenicity of APM at a dose close to the humanADI. Furthermore, the authors demonstrate that when life-span

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exposure to APM began during fetal life, its carcinogenic effects in-creased (Soffritti et al., 2007). However, an epidemiological studyreported the absence of an adverse effect of low-calorie sweetener(including aspartame) consumption on the risk of common neo-plasms such as gastric, pancreatic, and endometrial cancers inthe Italian population (Weihrauch and Diehl, 2004; Bosetti et al.,2009). Over the years, the toxic effects of APM have been mainlyfocused on its ability to induce cell transformation. Once a malig-nant cell is established, it requires factors to promote cell growth.Tumor growth depends on the formation of new blood vesselsfrom pre-existing capillaries (angiogenesis). Angiogenic signalingpathways play an important role in different pathologies such ascancers (Folkman, 1995; Petruzzelli, 2000; Maffei et al., 2010), car-diovascular diseases (Jude et al., 2010), diabetes (Waltenberger,2009), rheumatoid arthritis (Szekanecz et al., 2010) and retinopa-thies (Vedula and Krzystolikm, 2008), as well as vessel aging (Desaiet al., 2009; Tosetti et al., 2009). Evidence suggests that behavioralfactors such as life style, dietary habits and physical activity cansubstantially affect the risk of developing disease (Horton, 2009;Janiszewski and Ross, 2009; Byers et al., 2002; Baghurst et al.,1992).

In the present study, we evaluated the angiogenic effect of APMin an in vitro model using human endothelial cells in small islandwithin a culture matrix and the release of interleukin-6 (IL-6), vas-cular endothelial growth factor-A (VEGF-A), soluble interleukin-6receptor (sIL-R6) and soluble vascular endothelial growth factorreceptor-2 (sVEGFR-2) was evaluated over time in the conditionedmedium. Human endothelial cells and a human cell line (IMR-90)with fibroblast properties were separately treated with increaseddoses of APM ranging from 20 to 100 lM and intracellular reactiveoxygen species (ROS) formation and cell viability, release of IL-6and VEGF-A and their soluble receptors were analyzed in the cul-ture medium on days 1–3 of APM incubation.

2. Material and methods

2.1. In vitro angiogenesis assay

Blood vessel development was assessed by the Angio-Kit model(TCS Cell Works Ltd., Buckingham, UK/TEMA Research, BolognaItaly). Human endothelial cells were co-cultured with fibroblastsin a specially designed medium. The endothelial cells initiallyformed small islands within the culture matrix. They subsequentlybegan to proliferate and then entered a migratory phase duringwhich they moved through the matrix to form threadlike tubulestructures. These gradually joined up (9–14 days) to form a net-work of anastomosing tubules.

According to the manufacturer’s instructions, cultures weretreated with 20 and 100 lM APM and checked daily to monitorthe progress of tubule formation. To avoid medium changes,2.0 ml of fresh medium were added to the cell culture on day 1 in-stead of 0.5 ml as suggested by the manufacturer. On days 4–7–14,aliquots of conditioning medium (100 ll) were collected for cyto-kine analysis, and after 14 days of incubation the cultures werefixed in cold formalin 2% and subsequently stained by a tubulestaining kit containing anti-CD31 primary antibody (PECAM-1)conjugated with phosphatase, relevant secondary antibody andsubstrate (p-nitrophenol phosphate) to allow tubule visualization.The reliability of the Angio-Kit was assessed using a positive(Ctrl-pos) and negative (Ctrl-neg) controls performed by addingVEGF (2 lg/ml) and suramin (2 mM), respectively, to the culturemedia. Tubule formation was visualized by optical microscope andresults are shown as comparative images. Quantification was per-formed by adding a soluble substrate and after incubation for15 min at 37 �C, 100 ll aliquots were removed from each well

and absorbance read on a plate reader at 405 nm. The IL-6,VEGF-A and their soluble receptors sIL-R6 and sVEGFR-2 weredetermined in the conditioned medium of the Angio-Kit at the ba-sal level and after 4–7–14 days of incubation with APM at 20 and100 lM.

2.2. Cell culture and treatments

HUVEC, human umbilical vein endothelial cells (Lonza, Walk-ersville, MD) were grown by using Clonetics EGM Bullekit (Lonza,Walkersville, MD) containing endothelial cell basal medium(EBM) plus growth supplements (bovine brain extract BBE, hEGF,hydrocortisone, FBS, gentamicin–amphotericin B GA-1000). Cellswere harvested from monolayer cultures using One ReagentPack(Lonza, Walkersville, MD) containing trypsin/EDTA, trypsin neu-tralizing solution and HEPES buffered saline solution. IMR-90 hu-man cell line with fibroblast properties (ATCC, Rockville, MD)were grown in DMEM medium (Euroclone) supplemented with2 mM L-glutamine, 100 U/ml penicillin, 100 lg/ml streptomycinand 10% FBS. The cells were cultured at 37 �C and 5% CO2 in ahumidified incubator. Aspartame (Sigma, St. Louis, MO) was dis-solved in PBS and diluted in complete culture medium to a finalconcentration of 20–40–60–80–100 lM.

2.3. Cell viability assay

Cell viability was evaluated by the MTT method. HUVEC andIMR-90 cells were plated in 96-well flat-bottom tissue cultureplates at a density of 10 � 103 per well. The cells were allowed toattach overnight, and then treated with APM at various concentra-tions (20–100 lM) on days 1–3. After APM incubation, 200 ll ofthe conditioned medium was harvested for growth factor andinflammatory mediator determination (see below) and then200 ll of fresh cell culture medium was replaced. Ten microlitersof (3-4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT, Sigma, St. Louis, MO) was added to each well and then theplate was incubated in a humidified CO2 incubator at 37 �C for3 h. After removing the media, 200 ll of isopropanol was addedand mixed to dissolve crystals. Absorbance was read at 550 nmin an ELISA plate reader (Sunrise, Tecan) and the results were ex-pressed as percentage variation with respect to controls designedas 100%. The cell viability was repeated in triplicate with threeindependent experiments.

2.4. Inflammatory mediator and growth factor determination

The IL-6, VEGF-A and their soluble receptors sIL-R6, sVEGFR-2were analyzed by multiplex sandwich ELISA (SearchLight, PierceBiotechnology, Rockford, IL), according to the manufacturer’sinstructions. Each well of the microplate provided was pre-spottedwith target protein-specific antibodies. These antibodies capturethe specific target protein in the standard and plasma samplesadded to the plate. Unbound proteins were washed away and bio-tinylated detecting antibodies were added. After washing, antibodystreptavidin–horseradish peroxidase was used for detection. Eachsample was tested in duplicate and was repeated in triplicate withthree independent samples. The results are expressed both as pg/ml and fold change with respect to the control (untreated sample).

2.5. Intracellular ROS assay

Intracellular ROS were determined using fluorescent dye, 2070-dichlorofluorescein diacetate (DCFA). HUVEC and IMR-90 cellswere seeded in 24-well, flat-bottom plates and 20 lM of DCFA, acell-permeable, ROS-sensitive dye, was added to cell culture med-ia. After 30 min of incubation the fluorescent probe was removed

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and the cells were exposed to APM (20–100 lM) on days 1, 2 and 3.After APM incubation the cells were collected, washed and sus-pended in PBS and analyzed by flow cytometry (FACS Calibur,BD, Rutherford, NJ, USA). The amount of ROS was expressed as fluo-rescence intensity normalized for a blank without DCFA. The assaywas repeated in triplicate with three independent experiments.

2.6. Western blot analysis

HUVEC and IMR-90 cells were treated with increasing concen-trations of APM (range 20–100 lM), after 24 h of incubation thecells were lysed in a buffer containing 250 mM NaCl, 25 mMTris–HCl (pH 7.5), 5 mM EDTA (pH 8), 1% Nonidet P-40, and a

Fig. 1. Blood vessel formation in the Angio-Kit model. (A) Endothelial cells in small islan100 lM), and an untreated sample was used as the control (Ctrl). After 14 days of incubatstaining kit containing anti-CD31 primary antibody, relevant secondary antibody and suusing a positive (Ctrl-pos) and negative (Ctrl-neg) controls performed by adding Vformation was visualized by optical microscope and results are shown as comparativincubation for 15 min at 37 �C; 100 ll aliquots were removed from each well and ab(untreated sample) was determined. (B) The release of IL-6, VEGF-A and their soluble recbasal level and after 4–7–14 days of treatment with APM at 20 and 100 lM. Results are retime points of incubation; untreated cells vs APM-treated cells, p < 0.05.

cocktail of protease inhibitors (2 lg/ml aprotinin, 2 lg/ml leupep-tin, 1 mM phenylmethyl-sulfonyl fluoride and 2 lg/ml proteinin)and stored at �80 �C until analysis. The protein level was quanti-fied using the Bradford assay (Sigma). Protein samples (50 lg perlane) were boiled for 5 min and resolved using 12.5% SDS–PAGE,and transferred to a nitrocellulose membrane. The membranewas blocked (PBS containing 0.1% Tween and 5% skimmed milk)for 1 h, and incubated overnight with anti-VEGFR-2, anti-phosphop38 and anti-p38, anti-phospho erk1/2 and anti-erk1/2 IgG. Afterincubation with an HRP-conjugated secondary IgG (Amersham,London UK), the blots were developed using the ECL kit (Pierce,Rockford, IL, USA). Protein loading was corrected for by usinganti-b-actin IgG. The density of bands was quantified by Quantity

ds within the culture matrix were treated with two aspartame (APM) doses (20 andion the cultures were fixed in cold formalin 2% and subsequently stained by a tubulebstrate to allow tubule visualization. The reliability of the Angio-Kit was assessedEGF (2 lg/ml) and suramin (2 mM), respectively, to the culture media. Tubulee images. Quantification was performed by adding a soluble substrate and aftersorbance read on a plate reader at 405 nm. Fold change with respect to controlseptors sIL-6R, sVEGFR-2 were evaluated in the conditioning medium of Angio-Kit atpresentative of two independent experiments performed in duplicate. *Basal level vs

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One software (Bio-Rad Laboratories, MI, Italy) and the results ex-pressed as P-erk/erk and P-p38/p38 ratio.

2.7. Statistical analysis

All experiments were conducted at least two times, and data areshown as mean ± SD. Multiple comparisons were evaluated byKruskal–Wallis analysis, and the differences between groups wereperformed using the Mann–Whitney-U-test. Data were consideredstatistically significant at p < 0.05. All data in this study were ana-lyzed by the Statistical Package for Social Sciences version 15 (SPSSInc, Chicago, IL).

3. Results

3.1. Aspartame induces tubule formation

The capacity of APM to induce blood vessel formation was as-sessed in vitro by a model which uses human endothelial cells insmall islands co-cultured with other human cells. Treatment with20 lM of APM enhanced blood vessel formation which was ob-served after 14 days of incubation. The highest dose of APM(100 lM) did not increase the tubule density with respect to thelowest APM concentration (Fig. 1A). Both APM doses induced thesecretion of inflammatory cytokine such as IL-6 and its receptorsIL-6R and the growth factor VEGF-A and its receptor sVEGFR-2after 7 days of incubation (Fig. 1B).

Angiogenesis is a complex process which involves the activationof endothelial cell proliferation and migration. Endothelial cells areassembled in tubular structures around which blood vessel wallsare then formed. During vascular network maturation, the capillar-

Fig. 2. Effect of APM on ROS formation in HUVEC and IMR-90 cells. (A) HUVEC andIMR-90 cells were seeded in 24-well, flat-bottom plates and 20 lM of 2070-dichlorofluorescein diacetate (DCFA) was added to cell culture media. After 30 minof incubation the florescent probe was removed and the HUVEC and IMR-90 cellswere exposed to APM (range 20–100 lM) or (B) incubated with 20 lM of APM ondays 1, 2 and 3. After APM incubation the cells were collected, washed andsuspended in PBS and analyzed by flow cytometry. The amount of ROS wasexpressed as fluorescence intensity normalized for a blank without DCFA. The assaywas repeated in triplicate of three independent experiments.*Untreated cells vsAPM-treated cells; HUVEC vs IMR-90 cells, p < 0.05.

ies fuse into bigger vessels, arteries, and veins. Vessels consist oftwo main cell types: endothelial and mural cells, the latter com-prise a heterogeneous population of mesenchymal cells likesmooth muscle cells, osteoblasts, and fibroblasts. To evaluate theeffect of APM treatment on these cell types and their interactionwith each other during APM incubation, endothelial cells (HUVEC)and a human cell line with fibroblast properties (IMR-90), a com-ponent of mural cells, were separately treated with increasingAPM doses and the biological effects were evaluated.

3.2. Aspartame induces ROS formation

Incubation of APM (range 20–100 lM) induces intracellular ROSformation in HUVEC cells but not in IMR-90 cells (Fig. 2A). The low-est dose (20 lM) induces the maximal amount of ROS and no fur-ther ROS formation was observed by increasing the APM doses(Fig. 2A). ROS induction was observed after 1 day of APM incuba-tion, which decreased on the 2nd and 3rd day of APM treatment(Fig. 2B).

3.3. Cytotoxic effect of aspartame

To determine the cytotoxic effect of APM, HUVEC and IMR-90cells were incubated with increasing doses of APM on days 1–3and the cell viability checked by MTT assay. HUVEC were com-pletely resistant to APM at concentrations of up to 100 lM, show-ing a slight loss of cell viability in the presence of the agent atprolonged exposure times (Fig. 3A). IMR-90 cells were resistantto APM treatment, thus showing an induction of cell proliferationat the lowest APM doses (Fig. 3B).

Fig. 3. Cell proliferation and cytotoxicity of HUVEC and IMR-90 under APMincubation. Cells were seeded into 96-well tissue culture plates (10 � 103 per well)and treated with increasing concentrations of APM. The survival curves for HUVEC(A) and IMR-90 (B) are based on the results of MTT assays on days 1–3 of drugexposure. All data are expressed as percentage variation with respect to controlsdesigned as 100%. The experiments were repeated in triplicate with threeindependent experiments. *Untreated cells vs APM-treated cells, p < 0.05.

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3.4. Aspartame induces the release of IL-6 and VEGF and their solublereceptors

Tissue activity of angiogenesis depends on the balance of manystimulating or inhibiting factors. A number of cytokines andgrowth factors show angiogenic activity. To investigate the effectof APM in the induction of paracrine factors secreted by endothe-lial cells and surrounding cells, HUVEC and IMR-90 cells were trea-ted with increasing concentrations of APM and the cell culturemedium collected after days 1–3 of incubation. The conditionedmedium was analyzed for the inflammatory cytokine IL-6 and itssoluble receptor sIL-6R and growth factor VEGF-A and its solublereceptor sVEGFR-2 content.

In HUVEC, APM (20 lM) increases the secretion of the inflam-matory cytokine IL-6 into the growth medium 2-fold, with respectto untreated samples, which was found after 48 h (day 2) of incu-bation (Fig. 4A and B, left panel). The release of IL-6 was associatedwith a concomitant release of its soluble receptor sIL-6R, whichwas observed after 24 h (day 1) of APM (20 lM) exposure(Fig. 4B, right panel). The IL-6 and sIL-6R secretion was not dose-dependent, their content did not enhance by increasing the APMdose. As shown in Fig. 4A right panel, the fibroblasts were notresponsive to APM treatment.

Fig. 4. IL-6 and sIL-6R release from HUVEC and IMR-90 cells after exposure to APM. Hudensity of 10 � 103 per well. The cells were exposed to increasing concentrations of APMquantified in cell culture supernatants (n = 3) on days 1, 2 and 3 using the multiplexexperiments and the results are expressed in pg/ml. (B) IL-6 and sIL-6R fold change odetermined over time. *Untreated sample time points vs APM-treated sample time poin

No changes in VEGF-A induction were observed in IMR-90 afterAPM treatment, whereas APM induced in HUVEC a dose-depen-dent release of VEGF-A (10–20-fold with respect to controls) andsVEGFR-2 observed after APM treatment, as shown in Fig. 5A andB. Notably, VEGF-A was transiently induced by APM treatment,which peaked on day 2 and then returned to the basal levels. Atemporary induction was found also for sVEGFR-2 which was se-creted into the medium after day 1 of APM exposure.

3.5. Effect of APM exposure on mitogen-activated protein kinases(MAPKs) phosphorylation

It is assumed that the critical event in the regulation of angio-genesis is the signaling cascade involving VEGF. Therefore, experi-ments have been performed to elucidate the VEGF cellularsignaling pathway. We investigated the relevance of the MAPKpathway (Erk and p38) in APM-mediated VEGF expression. Bothcell types (HUVEC and IMR-90) expressed the VEGF receptor VEG-FR-2. The incubation of APM increased Erk1/2 and p38 activation inHUVEC after 24 h of incubation in a dose-dependent manner, asshown by immunoblots for phosphorylation (Fig. 6).

Activation of Erk1/2 was also observed in IMR-90 cells withoutp38 phosphorylation.

vec and IMR-90 cells were plated in 96-well flat-bottom tissue culture plates at a(range 20–100 lM) on days 1–3. (A) Secreted IL-6 and sIL-6R concentrations were

sandwich ELISA assay. Each sample was tested in duplicate of three independentf APM-treated (20 lM) cells with respect to the control (untreated sample) wasts; HUVEC vs IMR-90 cells, p < 0.05.

Fig. 5. VEGF-A and its soluble receptor sVEGFR-2 release from HUVEC and IMR-90 cells after exposure to APM. HUVEC (left panel) and IMR-90 (right panel) cells were platedin 96-well flat-bottom tissue culture plates at density of 10 � 103 per well. The cells were exposed to increasing concentrations of APM (range 20–100 lM) on days 1–3. (A)Secreted VEGF-A and sVEGFR-2 concentrations were quantified in cell culture supernatants on days 1, 2 and 3 using multiplex sandwich ELISA assay. Each sample was testedin duplicate of three independent experiments and the results are expressed in pg/ml. (B) The VEGF-A, sVEGFR-2-fold change of APM (20 lM) treated cells with respect tocontrol (untreated sample) were determined over time. *Untreated sample time points vs APM-treated sample time points; HUVEC vs IMR-90 cells, p < 0.05.

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4. Discussion

Since their discovery, the safety of artificial sweeteners hasbeen controversial. Scientists disagree about the safeness of theiruse due to the suggested relationships between sweeteners andlymphomas, leukemias, cancers of the bladder and brain, chronicfatigue syndrome, Parkinson’s disease, Alzheimer’s disease, multi-ple sclerosis, autism, and systemic lupus (Whitehouse et al.,2008). Population-based human epidemiological studies and pre-clinical studies in animal models show that factors affecting energybalance, such as the caloric intake, nutritional status and exercise,can influence cancer development and progression (Demark-Wahnefried et al., 2009; Brennan et al., 2010; Patel et al., 2004).Several studies have linked malignant transformation with angio-genesis (Folkman, 1986a,b; Petruzzelli, 2000; Kitadai, 2010).

Here, we hypothesized that the intake of APM, an artificialnutrient, might be involved in diseases associated with angiogene-sis. The ability of APM to induce angiogenesis was evaluated usingan in vitro model which uses human endothelial cells co-culturedin a matrix of human fibroblasts. A marked production of capil-lary-like structures associated with the formation of endothelial is-lands, appeared as a particular feature when endothelial cells wereincubated with APM (Fig. 1A). The primary vascular plexus signif-icantly expanded due to capillary branching and was transformedinto a highly-organized vascular network. The formation of bloodvessels was associated with a transient release of IL-6 andVEGF-A and their soluble receptors into the medium (Fig. 1B).

Angiogenesis requires many interactions that are tightly regulatedin a spatial and temporal manner. Such efficient control is medi-ated by the action of growth factors and cytokines that are tran-siently present during the angiogenetic phases (Liekens et al.,2001).

A transient increase in the medium of the inflammatory cyto-kine IL-6, the growth factor VEGF-A, and their cognate solublereceptors was also observed in cultured HUVEC following APMexposure (Figs. 4 and 5). The increase of cytokine and growth factorlevels in medium, peaking between days 1 and 2, coincided withthe temporary induction of ROS (Fig. 2B). APM induces the forma-tion of ROS in endothelial cells, but not in IMR-90, thus suggestingthat the genotoxic effect of APM is related to the target cell types(Fig. 2). The APM induced oxidative stress did not result in aremarkable cell cytotoxicity in HUVEC (Fig. 3).

ROS are conventionally considered cytotoxic and mutagenic andat high levels they induce cell death, apoptosis and senescence.However, ROS at low levels function as signaling molecules tomediate cell growth, migration, differentiation, and gene expres-sion. Of note, ROS play an important role in angiogenesis (Ushio-Fukai, 2006). Exogenous ROS stimulate the induction of a numberof cytokines and growth factors by various cell types and promotecell proliferation and migration (Stone and Collins, 2002). Amongthem, the IL-6/sIL-R6 complex showed angiogenic effects via theproduction of VEGF (Adachi et al., 2006). In the present study, weshowed that the lowest dose of APM (20 lM) induced HUVEC to se-crete IL-6 and its soluble receptor sIL-6R. The release of IL-6 into

Fig. 6. Effect of APM on the activation of MAPKs. HUVEC and IMR-90 cells were treated with increasing concentrations of APM (range 20–100 lM); after 24 h of incubationthe cells were lysed and stored at �80 �C until analysis. Protein samples (50 lg per lane) were resolved using 12.5% SDS–PAGE, and transferred to a nitrocellulose membrane.The membrane was blocked (PBS containing 0.1% Tween and 5% skimmed milk) for 1 h, and incubated overnight with anti-VEGFR-2, anti-phospho p38 and anti-p38, anti-phospho erk1/2 and anti-erk1/2 IgG. After incubation with an HRP-conjugated secondary IgG, the blots were developed using the ECL kit. Protein loading was corrected for byusing anti-b-actin. The results are representative of three independent experiments and expressed as P-erk/erk and P-p38/p38 ratio. *Untreated cells vs APM-treated cells,p < 0.05.

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the medium was associated with the increased secretion of VEGF-Aobserved on the days 1 and 2 of incubation and sVEGFR-2 immedi-ately released after day 1 of APM exposure. Although the IMR-90cells were shown to secrete VEGF-A into the medium at higher con-centrations with respect to endothelial cells, no significant changeswere observed in IMR-90 cells after APM exposure. It is well ac-cepted that angiogenesis is activated by IL-6 and amplified in thepresence of the soluble IL-6 receptor (Ara and Declerck, 2010; Nils-son et al., 2005) and the interaction of VEGF with VEGFR-2 is crit-ical for VEGF-induced biological responses (Liekens et al., 2001).Soluble VEGFRs have been reported to inhibit angiogenesis bothin vitro and in vivo (Wu et al., 2010). Even though the physiologicalrole of the soluble form of VEGFR-2 is not clear, sVEGFR-2 is likelyto be a negative regulator of VEGF availability (Kou et al., 2004).However, the balance between circulating levels of VEGF and itssoluble receptors reflects and/or affects VEGF signaling. VEGFR-2appears to mediate almost all of the known cellular responses to

VEGF. As a major mediator of VEGF, VEGFR-2 is activated throughligand-stimulated receptor dimerization and trans(auto) phos-phorylation at least six tyrosine residues (Neufeld et al., 1999;Zachary, 2003). The activation of VEGFR-2 induces an array of sig-nal transduction pathways and subsequent biological responses,including vascular permeability, migration and proliferation ofendothelial cells (Neufeld et al., 1999; Zachary, 2003; Takahashiand Shibuya, 2005). The finding that APM enhances VEGF produc-tion may favor an angiogenic micro-environment, thus inducingendothelial cells to proliferate through the activation of MAPKssuch as erk1/2 and p38 (Fig. 6). The activities of specific kinasesare essential for the VEGF-induced enhancement of vascular per-meability (Takahashi and Shibuya, 2005). Paracrine factors se-creted by stromal cells such as fibroblasts, inflammatory cells andvasculature-related cells (endothelial cells, perycites and smoothmuscle cells) play a critical role by stroma–epithelium crosstalkin carcinogenesis and progression of cancer (Yi-Nong and Shu-Jie,

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2009). Thus, a shift of the balance towards angiogenesis stimula-tion could play an important role in malignant transformationand its progression. Although APM has not been implicated in theinitiation of carcinomas, its uncontrolled intake might facilitatepathological condition characterized by inflammation.

In conclusion, APM at low doses is a potential angiogenic agentthat can induce regenerative cytokine production. In particular, itenhances IL-6 and VEGF and their soluble receptor release fromendothelial cells and leads to the activation of MAPKs (erk andp38) resulting in the formation of new blood vessels, thus creatingfavorable conditions that in vivo may result in the development ofpathological conditions like diabetic retinopathies, rheumatoidarthritis and tumor cell invasion and spreading of metastases.We are aware that our data were obtained from an in vitro model,but they indicate that more attention should be paid to the use ofAPM in inflammatory diseases characterized by the over-produc-tion of ROS.

5. Conflict of interest statement

The authors declare that they have no conflict of interest orcompeting financial interests.

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