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Biochimica et Biophysica Acta 1740 (2005) 222–239 http://www.elsevier.com/locate/bba Review Proangiogenic activity of beta-carotene is coupled with the activation of endothelial cell chemotaxis

A. Dembinska-Kieca,*, A. Polusa, B. Kiec-Wilka, J. Grzybowskaa, M. Mikolajczyka, J. Hartwicha, U. Raznya, K. Szumilasa, A. Banasa, M. Bodziocha, J. Stachurab, G. Dyduchb, P. Laidlerc, J. Zagajewskic, T. Langmand, G. Schmitzd

aDepartment of Clinical Biochemistry, The Jagiellonian University Medical College, Kopernika 15a, 31-501 Krako´w, Poland bDepartment of Pathology, The Jagiellonian University Medical College, Krako´w, Poland cDepartment of Medical Biochemistry, The Jagiellonian University Medical College, Krako´w, Poland dDepartment of Clinical Chemistry, Laboratory Medicine and Transfusiology, University of Regensburg, Regensburg, Germany

Received 27 September 2004; received in revised form 5 November 2004; accepted 22 November 2004 Available online 28 December 2004

Abstract

Endothelial cells play an important role in angiogenesis (formation of new vessels from preexisting ones), which is essential for organogenesis, tissue remodeling but also inflammatory response, carcinogenesis in all periods of our life. Beta-carotene (BC) in non-toxic concentrations (up to 3 AM) had no detectable effect on HUVECs (human umbilical vein endothelial cells) proliferation or apoptosis, despite significant changes of the expression patterns of pro- and anti-apoptotic . However beta-carotene did not change the tubulogenic activity of HUVEC in the in vitro angiogenesis model, it potently accelerated the bFGF-induced development of microcapillaries, as well as the migration of endothelial cells, in matrigel plug injected subcutaneously to mice. Potent activation of endothelial cell migration in the in vitro model of chemotaxis was also observed. According to the microarray data, genes involved in cell/ cell and cell/matrix adhesion, matrix reorganization, activation of chemotaxis, the G- regulated intracellular signaling as well as genes involved in the rapid remodeling of protein cytoskeleton were the most affected by BC in HUVEC. We conclude that beta-carotene in the physiological concentration range stimulates early steps of angiogenesis by the activation of cellular migration as well as matrix reorganization and decrease of cell adhesion. D 2004 Published by Elsevier B.V.

Keywords: Beta-carotene; Angiogenesis; Endothelium; Microarray; Chemotaxis

1. Introduction in new observations suggesting the influence of the new regulators. A number of growth factors (such as VEGF, Angiogenesis, the formation of new blood vessels from bFGF, TGFb, PDGF, IGF and others), cell/matrix (integ- the preexisting ones, plays a central role in a number of rins) and cell/cell (VE-cadherins, catenins, endoglins, physiologic and pathologic events including vascular ephrins, their receptors or Jagged/Notch pathway) inter- remodeling of growing ischemic tissue, inflammation, actions, and environmental factors (such as shear stress, diabetic retinopathy, as well as in progression of solid nutrients, oxygen supply and others) regulate the most tumor growth and invasiveness [1,2]. important steps in angiogenesis, which include detachment, The knowledge of the formation of new tubular proliferation, migration, homing and differentiation of the structures in angiogenesis is being continuously enriched vascular wall cells, mainly endothelial or their progenitors [3,4]. * Corresponding author. Tel.: +48 12 421 40 06; fax: +48 12 421 40 Carotenoids are lipid soluble hydrocarbon pigments 73. (C40H56) containing 11 conjugated double bonds. The main E-mail address: [email protected] (A. Dembinska-Kiec). plant carotenoids detected in human blood are: beta-carotene

0925-4439/$ - see front matter D 2004 Published by Elsevier B.V. doi:10.1016/j.bbadis.2004.11.017 A. Dembinska-Kiec et al. / Biochimica et Biophysica Acta 1740 (2005) 222–239 223

(BC), alpha-carotene, gamma-carotene and lycopene. They actin, vWF [16]. Experiments were performed on 70% are isomers varying in the position of double bonds and the confluent cell cultures (up to 5th passage). presence of acyclic ring. Carotenoids present in human For cell culture, 4 mM beta-carotene (BC) stock solution tissues function as free radical scavengers, immunomodula- in tetrahydrofurane (THF) (Sigma) was further diluted in tors, or some being the substrate for the synthesis of retinol ethanol(1:1v/vTHF/EtOHratio)toobtain2mM (Vitamin A) and retinoic acid (RA) are regulators of cell fate concentration of BC. The further dilutions were made with and differentiation [5]. RA, derived from beta-carotene, is a the tissue culture medium (EBM). The final concentration of well known activator of nuclear receptors such as retinoid/ THF/EtOH in the cell culture medium was 0.075%. rexinoid receptors (RXR/RAR). RA in cooperation with fatty HUVECs were incubated with the solvent (0.075% THF/ acids and fatty acid metabolites regulates the activity of the EtOH) containing medium (as the control) and beta-carotene PPAR/RXR heterodimer (peroxisome proliferator-activated (BC) (0.3–10 AM), arachidonic acid (AA) (3 AM) (Sigma), receptors, PPARs) [6]. As regulators of transcription they or both BC (3 AM) and AA (3 AM) for 24 h at 37 8Cin5% influence cell differentiation, maturation of tissues and body CO2 and 95% humidity (Jouan IG 150). organization in both fetal and adult periods of life [5–7]. Epidemiological studies suggested that the intake of 2.2. Beta-carotene uptake by HUVECs (measured by HPLC) fruit and vegetables rich in carotenoids results in the reduction of the cancer incidence, decreased risk of heart All solvents used for HPLC (THF, ethanol, tBME diseases, and enhanced immunity due to its free radical (tetra-butylmethylether) (Sigma) and BHT (2,6-Di-tert- scavenging properties, anti-inflammatory and increased butyl-p-cresol) (Sigma)) were analytical grade. THF and activity of NK cells [8–11]. Surprisingly, some of the large ethanol were additionally filtered through the Aluminum clinical studies, undertaken to prove the efficacy of beta- Oxide column (Sigma) before use. Following 24 h carotene supplementation in the prevention of coronary incubation of HUVECs with the studied compounds, cells heart disease and cancer, showed that administration of were detached by trypsinization and washed three times beta-carotene or vitamin A might increase the risk of lung with Ca+2 and Mg+2 free PBS (centrifugation: 400Âg for cancer, especially in smokers and patients afflicted with 10 min, at the room temperature). Cells were counted asbestosis (Alpha-Tocopherol Beta-Carotene Cancer Pre- using the Burker chamber and spinned down (400Âg,10 vention Study (ATBC), [12], Beta-Carotene Retinol Effi- min, at room temperature). The resulted pellet was frozen cacy Trial (CARET) [13], Physician’s Health Study (PHS), (at À80 8C) for further analysis. BC concentration in the [14]). cells and BC stability in culture medium (lack of Since 15% of beta-carotene ingested reach human target spontaneous degradation products), were assessed with cells in the non-metabolized form [15] and angiogenesis is the HPLC micromethod developed by Roche Vitamins AG an important part of remodeling of an ischemic tissue as (Kaiseraugust, Switzerland). Briefly, for extraction, 200 Al well as solid tumor malignancy [1], this study was under- of acetone (with 0.025% BHT f.c.) was added to a frozen taken to define the direct effects of beta-carotene on cell pellet, mixed for 1 min and dried in a vacuum endothelial cells in terms of angiogenic activity and centrifuge under argon (30 min at 50 mbar). 200 Al of the regulation of expression. BC extraction solvent (ethanol/THF/tBME 9:1:5, 0.025% BHT f.c.) was applied to the dried pellet, vigorously mixed and centrifuged (3 min, 8000Âg at 4 8C). The supernatant 2. Methods was used for HPLC. For the measurement of BC content in the tissue culture The HPLC grade beta-carotene (1 mg) filled with medium, 25 Al of the medium was mixed with 225 Al of the nitrogen in vials made of dark glass were kindly provided BC extraction solvent described above, mixed for 1 min and for the project by the Roche Vitamins AG, Kaiseraugust, centrifuged for 3 min at 8000Âg at 4 8C. The HPLC sample Switzerland. analysis was isocratically performed on a Vydac 218TP54 column C-18 (Roche) (4.6Â250 mm) at a constant column 2.1. The cell culture temperature 20 8C. The sample solvent consisted of ethanol/ THF/tBME 9:1:5, 0.025% BHT f.c. The mobile phase was: Primary endothelial cells (HUVECs) were isolated from acetonitryl/tert-butylmethylether/ammonium acetate (80 human umbilical veins using digestion. mM)/triethylamine (73:20:7:0.05) at a constant flow of HUVECs were cultured in EBM (EGM Bullet Kit, butylmethylether/1.5 ml/min. Shimadzu SCL-10AVP instru- Clonetics) with supplements: hEGF (10 ng/ml), hydro- ment (Shimadzu, Kyoto, Japan) with the SPD-10AV cortisone (1 ng/ml), Bovine Brain Extract (12 Ag/ml), detector was set at 450 nm for the carotenoids estimation. antibiotics: gentamicin (50 Ag/ml) as well as amphoter- In this HPLC system, 12V-apocarotenal, 8V-apocarotenal, 4V- icin-B (50 ng/ml), and 10% Fetal Bovine Serum (Clonetics). h-apocarotenal, all-trans-h-carotene and (Z)-h-carotene The resulting cell lines were characterized by morphological could be detected. The amount of BC was expressed as and immunohistochemical criteria such as desmin, alpha- pmol of BC/106 cells. 224 A. Dembinska-Kiec et al. / Biochimica et Biophysica Acta 1740 (2005) 222–239

2.3. Detection of cellular toxicity and apoptosis through the pores to the bottom side of the inserts, was measured with a fluorescence plate reader (Synergy HT, The possible toxic effects of used compounds and solvents BIO-TEK). The changes in intensity of fluorescence in a on HUVECs were assessed by the cellular lactic dehydrogen- sample with agents versus control (cells migrated against the ase (LDH) presence in the medium after 24-h incubation (Cy- EBM with solvents only) corresponded to the amount of toTox 96 Non-Radioactive Cytotoxicity Assay, Promega). migrating cells. The chemotactic activity of the endothelial For the estimation of apoptosis the ApoFluorR Green cells was expressed as the chemotaxis index (CHI) which Caspase Activity Assay (ICN Biomedicals Inc.) was used. represents the ratio of migration stimulated by investigated This test allows for the measurement of global cellular compound versus random migration of unstimulated caspase activity. Proapoptotic staurosporine (at 1 nM) was HUVECs in THF/EtOH control sample. used as the positive control for apoptosis. Following incubation with the fluorescent dye (1 h) cells were 2.6. The three dimensional [3D] model of tubulogenesis in harvested, washed to remove unbound dye with the Ca+2 matrigel in vitro [19] and Mg+2 free PBS and 1Â106 cells were placed in black microtiter plates (NUNC A/S, Roskilde). The fluorescence HUVECs harvested from 3–4 passages were resuspended was detected using a plate reader (Synergy HT, BIO-TEK) in Matrigel (containing , IV, entactin, and quantitated at 520 nm emission wavelength following heparan sulfate proteoglycans) (Becton Dickinson) to the the excitation with the 490 nm light. Results are expressed final concentration of 1Â106 cells/ml on ice. The cell– as the percentage of activity detected in the control matrigel mixture (50 Al) was placed into the cell culture (incubated with the solvent only) HUVECs. dishes and incubated in a humidified CO2 incubator (Jouan) at 37 8C for 30 min. Subsequently the EBM medium (100 2.4. Assay of HUVEC proliferation (The bromo- Al) supplemented with solvent (control probe), or BC (3 deoxyuridyne (BrdU) incorporation assay) AM), or AA (3 AM), or BC/AA mixture was applied on top of the cells immersed in matrigel and was incubated in a In order to determine the effect of BC and AA on the humidified CO2 incubator for 24 h at 37 8C. The number of HUVEC proliferation, the rate of DNA synthesis was tubules formed in the presence of VEGF (0.2 nM) or bFGF measured by the incorporation of the thymidine analog, (0.5 nM) served as the positive control. The formation of bromo-deoxyuridine (BrdU) to DNA [17]. HUVECs tubules by HUVECs suspended in matrigel was assessed (5Â104 cells) were incubated with the compounds for 24 under the light microscope (at magnitude Â10) and photo- h, as described above, and additionally with BrdU for the graphed. The lengths of the analyzed tubule-like structures last 3 h. Following incubation, cells were fixed and stained were calculated and expressed as the average sum of total with the anti-BrdU kit (Roche) according to the manufac- length of tubules visible under the light microscope. turer’s recommendations. HUVECs proliferation induced by VEGF (Vascular Endothelial Growth Factor) (0.2 nM) 2.7. Angiogenesis in vivo (the mouse model) (Sigma) or bFGF (basic fibroblast growth factor) (0.5 nM) (Sigma) for 24 h was served as the positive control. Results Protocol was accepted by the local University Ethic are given as the percent of the control BrdU incorporation to Committee. All animal experiments were performed accord- the HUVEC incubated in the medium with solvents only. ing to Polish laws and approved by the Polish Animal Inspectorate and Institutional Animal Care. Female Balb/c 2.5. Migration of endothelial cells (The Boyden’s Chamber mice (n=6) received sterile injections of 2Â500 Al matrigel assay) (Becton Dickinson) s.c. (dorsally). The matrigel plug con- tained solvent or BC (3 AM), bFGF (50 nM), or both BC and HUVECs harvested from 3–4 passages were suspended bFGF at the given concentrations. Six days later the animals (105 cells/100 Al) in the EBM medium with 0.5% FBS and were sacrificed by overdosing urethane anesthesia and the seeded into BD Falconk FluoroBlokk Inserts (3 Am pore, matrigel plug were removed, fixed and immersed in paraffin. Becton Dickinson). The inserts containing HUVECs were Immunohistochemistry was performed using routine proto- placed into a 24-well plate with 600 Al of the EBM medium col. Primary anti-CD31 antibodies (anti-PECAM-1, BD and 0.5% FBS, and incubated for 24 h at 37 8C. Media with Pharmingen) at 1:300 dilution were used. The slides were the addition of BC (3 AM) or AA (3 AM) as well as 1- rehydrated and incubated in 3% peroxide solution for 10 min phospho-sphingosine (S1P) (500 nM) (Sigma) as the to block endogenous peroxidase activity. The Streptavidin– positive control [18] were used as chemoattractant in the Biotin (BD Pharmingen) detection system was used. DAB lower chamber. After 24 h, HUVECs were stained with anti- was used as chromogen. The slides were contra-stained with CD31 antibody conjugated with phycoerythrin (PE) (10 Ag/ Mayer hematoxylin (DAKO, Denmark). The amount of ml) (Becton Dickinson) for 30 min at 37 8C. Subsequently, capillaries was counted under the microscope in five different cells in the inserts were washed with the Ca+2 and Mg+2 free fields in each of the three slices taken from different parts of PBS and the fluorescence of the cells, which had migrated each plugs. The number of capillaries detected in slices from A. Dembinska-Kiec et al. / Biochimica et Biophysica Acta 1740 (2005) 222–239 225 the plugs containing the studied compounds was compared to hybridization, RNA was reverse transcribed into cDNA with the plugs with solvents and expressed as the number of a primer containing the T7 promoter using Superscript II vessels with or without the lumen. Additionally, the number (Invitrogen Life Technologies). cDNA was used as a of separately detected endothelial PECAM-positive single template in a biotin-labeled transcription reaction (Enzo cells, not connected with capillaries, migrating to the matrigel BioArray, Affymetrix). The resulting target cRNA was was calculated. purified on RNeasy columns (QIAGEN) and fragmented for hybridization to Affymetrix HG-U133A GeneChips. 2.8. The protein synthesis (Western Blot) Hybridization was done overnight at 45 8C for 16 h in the GeneChip Hybridization Oven 640 (Affymetrix). The Western-blot was used to detect the synthesis of GeneChips were subsequently processed on the Affymetrix interleukin -8 (IL-8) by HUVECs incubated with inves- GeneChip Fluidics Workstation 400 according to the tigated compounds. EukGE-WS2v4 protocol. The GeneChips were scanned with The cells were rinsed with the Ca+2 and Mg+2 free PBS the Hewlett Packard GeneArray Scanner, and the results and lysed in CelLyticTM-M Mammalian Cell Lysis/Extrac- analyzed using Affymetrix Microarray Analysis Suit 4.0. tion Reagent (Sigma) containing inhibitors (Protease Inhibitor Cocktail Tablets complete TM, Roche). Protein 2.11. Analysis of the microarray data content was determined by Bradford’s method [20]. were electrophoretically separated on an SDS-polyacryla- Changes in relative gene expression were calculated mide gel under reducing conditions and transferred to PVDF versus the control (THF/EtOH solvent). Only spots with (polyvinylidine difluoride) membranes (BIORAD). Transfer significant differences in signal intensity (more than 1.4-fold was performed in 100 V for 2 h. Blots, stained with 2% and only when the P value was at least 0.05) were included Ponceau S (Sigma), in 3% trichloroacetic acid to visualize in the analysis. proteins, were saturated with 1% blocking solution (Lumi- In order to identify genes belonging to pathways regulated LightPLUS Western Blotting Kit Mouse/Rabbit, Amersham by beta-carotene and/or arachidonic acid in HUVECs we Pharmacia Biotech) and incubated for 1 h with mouse anti-IL- searched promotor sequences of the identified genes to find 8 antibody (Mouse monoclonal anti-IL-8 antibody [B-2]). similar transcription factors binding sites. The 4-kb upstream Following washing in TBST (Lumi-LightPLUS Western sequences relative to the transcription start site were retrieved Blotting Kit Mouse/Rabbit), membranes were incubated for from the database of the Transcriptional Start Sites DBTSS 30 min, subsequently with anti-mouse Ig-POD Fab fragments (http://dbtss.hgc.jp/index.html). Searches of transcription (Lumi-LightPLUS Western Blotting Kit Mouse/Rabbit, factor binding sites were done using the TRANSFAC Roche) or monoclonal anti-goat/sheep IgG Monoclonal database at a default threshold setting of 90 (http:// Anti-Goat/Sheep IgG Clone GT-34 peroxidase conjugated molsun1.cbrc.aist.go.jp/research/db/TFSEARCH.html). (Sigma). Monoclonal antibodies against actin were used as a control of protein loading (mouse monoclonal anti-actin 2.12. cDNA synthesis and quantitative real-time PCR antibodies—Actin [C-2], Santa Cruz Biotechnology, Inc.). Enhanced chemiluminescence, performed according to In order to confirm the regulation of the expression of manufacturer’s instructions (Amersham), was used to genes important for demonstration of the proangiogenic demonstrate positive bands that were visualized after activity of BC in HUVECs, which were identified by the exposure on a transparent medical X-ray film. microarray experiments, the quantitative expression analysis IL-8, CXCR4, VCAM-1, EGR-1, MAD1L1, BIRC5 and 2.9. Isolation of total RNA MEOX2 was performed by real-time PCR using GAPDH as the reference gene. For the cDNA synthesis 1 Ag of total RNA Following 24-h incubation with the studied compounds, was reverse transcribed at 42 8C for 50 min in a total volume total RNA was isolated from HUVECs by the guanidine of 40 Al reaction buffer containing 5Â First Strand Buffer, thiocyanate–caesium chloride method [21] using Trizol DTT, oligo(dT) (Sigma), deoxy-NTPs (Promega), and 200 (Invitrogen Life Technologies) and was purified using the units of SUPERSCRIPT II reverse transcriptase (Invitrogen SV total RNA Isolation System Kit (Promega). The quality Life Technologies). The reaction mixture was heated to 70 8C of RNA was confirmed by denaturing gel electrophoresis for 15 min and immediately chilled on ice. Subsequently, and an analysis on the Agilent 2100 Bioanalyser (Agilent cDNA was subjected to real-time PCR in a reaction mixture Technologies). containing QuantiTect SYBR Green PCR (Qiagen) mix and primers. The sequences of the primers used in this study are 2.10. Microarray affymetrix HG-U133A hybridization presented in Table 1.The primers were designed to include an intervening intron between the sense and antisense primers, The aim of the microarray experiments was to screen the thereby eliminating the possibility of amplifying any genomic effects of beta-carotene (with or without arachidonic acid) on DNA, and checked for specificity by BLAST searches. All gene expression patterns in HUVECs. For microarray real-time PCR reactions were performed on the DNA Engine 226 A. Dembinska-Kiec et al. / Biochimica et Biophysica Acta 1740 (2005) 222–239

Table 1 Sequence of primers for real-time PCR Gene symbol mRNA sequence Forward primer Reverse primer MEOX2 NM_005924 5V-CTGCGGAGGCGGAGAA-3V 5V-GTAATTTCCTTCCTGGGAGTCTGA-3V MAD1L1 NM_003550 5V-ACAGTCTCTGTAATCGCGAAAGC-3V 5V-TCAGGGTGGATAAAACCATGG-3V EGR-1 NM_001964 5V-AGCACCTGACCGCAGAGTCT-3V 5V-GGTCTCCACCAGCACCTTCTC-3V IL-8 NM_000584 5V-CTGGCCGTGGCTCTCTGG-3V 5V-TTAGCACTCCTTGGCAAAACTG-3V CXCR4 NM_003467 5V-CACCGCATCTGGAGAACCA-3V 5V-TCCTGCGTGTAGTTATCTGAAGTGTATATAC-3V VCAM1 NM_080682 5V-AGGCTGGAAGAAGAAGCGGAAAGG-3V 5V-AGCTGTAAGTTTTATGTCTTTTGGAGTAAC-3V BIRC5 NM_001168 5V-TTAACCCTTGGTGAATTTTTGAAACT-3V 5V-TTCTTATTGTTGGTTTCCTTTGCAA-3V BCMO NM_017429 5V-TGCCAGCCGGAATTTCTTTAT-3V 5V-TCGGTATTGCTTTCCATGTGA-3V GAPDH NM_002046 5V-GCCAGCCGAGCCACATC-3V 5V-GCGCCCAATACGACCAAA-3V

Opticon II (MJ Research). The thermal profile included initial augmented the BC uptake (Fig. 1B). The BC concentration denaturation for 15 min at 95 8C, followed by 40 amplifica- of 3 AM was used for the further experiments, since the tion cycles of denaturation for 30 s at 94 8C, annealing for 30 s higher concentrations of BC caused endothelial cell toxicity at 60 8C, and elongation for 30 s at 72 8C. Following PCR (especially in the presence of AA), as evidenced by the amplification, melting curve analysis was performed with a LDH leakage from HUVECs after a 24-h incubation (data temperature profile slope of 1 8C/s from 35 8Cto958C. A not shown). negative control without cDNA template was run with every assay to ensure overall specificity. The expression rates were 3.2. Effects of beta-carotene and arachidonic acid on calculated as the normalized CT difference between a control HUVEC apoptosis probe and a sample with the adjustment for the amplification efficiency relative to the expression level of the housekeeping Unlike in the cells treated with proapoptotic stauroporine gene GAPDH. Calculation was performed using the program (1 nM), no proapoptotic activity measured by caspase Calculation Matrix for PCR Efficiency REST-XL (gene. [email protected]) [22].

2.13. Statistical analysis

Statistical analysis was made with the Microsoft EXCELL 5 program and by one-way ANOVA. All results are expressed as mean valuesFstandard error (S.E.). Before statistical analysis, the normal distribution and homogeneity of variables were tested. Parameters that did not fulfil these tests were logarithmically-transformed. Statistical compar- isons were made by unpaired t-tests for comparisons of quantitative variables. Pb0.05 was considered significant.

2.14. Bioinformatics

Sequence data were assembled and analyzed using promotor sequences retrieved from the database of Tran- scriptional Start Sites DBTSS (http://dbtss.hgc.jp/index. html) website transcription factor consensus sequences and databases were accessed using the TRANSFAC (http:// transfac.gbf.de/TRANSFAC/) website. Calculation of real- time PCR reaction efficiency was performed using the program REST-XL ([email protected]). Fig. 1. The beta-carotene uptake by HUVECs. Cellular beta-carotene content was analyzed by HPLC. (A) Concentration dependent beta-carotene uptake by HUVECs incubated with vehicle (THF/EtOH) and different 3. Results concentrations of beta-carotene (0.3–3.0 AM BC) for 24 h. (B) Fatty acid dependent beta-carotene uptake of HUVECs incubated with control 3.1. Uptake of beta-carotene by HUVECs (medium with 0.075% THF/EtOH), arachidonic acid 3 AM (AA), beta- carotene 3 AM (BC) or in combination at given concentrations (BC/AA) for 24 h. Data represent mean valuesFS.E. of three independent experiments BC uptake by endothelial cells in culture was concen- done in triplicates. *Significantly different from the corresponding control tration-dependent (Fig. 1A) and the presence of AA (3 AM) cells; *Pb0.05. A. Dembinska-Kiec et al. / Biochimica et Biophysica Acta 1740 (2005) 222–239 227 activation was detected in HUVECs incubated for 24 h with 3.6. The proangiogenic activity of beta-carotene in the in the BC or AA at 3 AM, as well as with the mixture of both vivo mouse model compounds (Fig. 2). The presence of BC at 3 AM in the matrigel administered 3.3. Effect of beta-carotene and arachidonic acid on subcutaneously significantly increased the density of capil- HUVEC proliferation lary network (with and without lumen) and the amount of endothelial cells penetrating to the matrigel plug stimulated Unlike VEGF (0.2 nM) and bFGF (0.5 nM), which by bFGF (Fig. 6). stimulated cell proliferation, both BC as well as AA used in the non-toxic concentrations did not influence 3.7. The microarray analysis HUVEC proliferation measured by the BrdU incorporation (Fig. 3). Using the criteria described in Methods, we identified 838 genes, whose expression changed only in response to 3.4. Effect of beta-carotene and arachidonic acid on the stimulation with BC (446 down-regulated, 393 up- HUVEC migration regulated). The expression of 644 genes was regulated by AA (375 down-regulated, 269 up-regulated) and the BC (3 AM) four-fold increased the migration of HUVECs expression of 740 genes was regulated by beta carotene (Fig. 4). Arachidonic acid alone did not stimulate HUVEC and AA together (498 down-regulated, 242 up-regulated). migration, but the cell migration was further increased (six- The analysis of microarray data, including genes regulated fold) when AA was used in combination with BC (Fig. 4). (up or down) from 1.4-fold up to 10-fold, provided a list of Sphingosine-1-phosphate (S1P) the potent activator of the selected significantly regulated genes coding for endothelial cell migration [18] was used as the positive proteins, which belong to the cellular pathways such as: control to confirm HUVEC chemotactic potential in our pathways contributing to proangiogenic activity (cell cycle, assay. adhesion, matrix remodeling, chemotaxis), apoptosis, recep- tor-mediated signal transduction, as well as transcription 3.5. Effect of beta-carotene and arachidonic acid on factors and regulators of protein synthesis (zinc finger tubulogenesis in the 3D matrigel in vitro model proteins, ribosomal proteins), xenobiotic metabolism, inflammatory response (Table 2). The 3D matrigel assay of tubulogenesis was used to According to the recent data concerning the proangio- verify the angiogenic property of BC and AA in vitro [19]. genic activity of endothelial cells [1,23], special attention Only the trace tubulogenic activity of HUVEC was detected was paid to the BC-regulated genes involved in the cell in cells cultured in matrigel covered with medium without cycle-proliferation. BC weakly up-regulated the key genes factors. No tubulogenic activity of BC and AA incubated coding for proteins participating in the regulation of a cell alone as well as together was observed in the above in vitro cycle such as MCM5, MAD1L1 connected with G1/S check model (Fig. 5A,B). The proangiogenic VEGF (0.2 nM) and point and polo-like kinase Plk1, NUCKS related to G2/M bFGF (0.5 nM) potently increased the number of tubules in check point. BC up-regulated such important inhibitors of matrigel HUVEC suspension (Fig. 5A,B). cell cycle as Wee1, PKMyt1 (Table 2) [24].

Fig. 2. Influence of beta-carotene and arachidonic acid on the HUVEC apoptosis. The rate of apoptosis was estimated by the measurement of global cellular caspase activity following a 24-h incubation of HUVEC with the studied factors (control: medium with 0.075% THF/EtOH), beta-carotene (BC) 3 AM, arachidonic acid (AA) 3 AM or both compounds (BC/AA). Data represent mean valuesFS.E. of three independent experiments done in triplicates, expressed as the percentage of activity detected in the control. *Significantly different from the corresponding control, *Pb0.05. 228 A. Dembinska-Kiec et al. / Biochimica et Biophysica Acta 1740 (2005) 222–239

Fig. 3. Effects of beta-carotene and arachidonic acid on HUVEC proliferation. HUVEC cultured in EBM medium containing 10% FBS and antibiotics were treated with medium with 0.075% THF/EtOH (control), beta-carotene (BC) 3 AM, arachidonic acid (AA) 3 AM or a combination of both (BC/AA) for 24 h were labeled with BrdU for 3 h before the end of incubation. BrdU incorporation was determined by collorimetric immunoassay. The results are given as the percent of the control proliferation. Data represent mean valuesFS.E. of three independent experiments performed in triplicates. Significantly different from the corresponding control, *Pb0.05, **Pb0.005.

BC also weakly up-regulated the expression of apoptosis cules (VCAM1, SELP, CD24) [26], were generally down- inhibitors associated with the FASL pathway such as regulated by both BC and AA (Table 2). On the contrary, the CFLAR (Flip), BIRC5 (IAP) TRAF4 genes and down- expression of genes coding for proteins associated with cell- regulated the expression of apoptosis activators OPTN, adhesion, such as integrins (ITGA6), PAR1 and PAR4 [25]. The parallel up-regulation of the scavenger receptor SCARB1, was up-regulated by BC expression of a number of pro- and anti-apoptotic members (Table 2). of the Bcl-2 family was observed. BC up-regulated the The expressions of genes encoding extracellular matrix proapoptotic Bcl2L11/BAM, anti-apoptotic MCL-1 and degrading and stimulators of chemotaxis, which TEGT-BAX inhibitor (Table 2). may regulate the matrix-degradation [27], receptor shading The group of BC-regulated genes, which code for [28] and cell migration, such as ADAMTS1, ADAMTS18, proteins participating in cell–cell interactions, such as MMP10, MMP12, MMP14, MMP24, were differentially cadherins (CELSR1), catenins (CTNNA1L, CTNNB1), regulated. The down-regulation of different types of and the leukocyte–endothelium adhesion mediating mole- extracellular matrix components , fibrillin 1

Fig. 4. Beta-carotene-induced chemotaxis of HUVEC. Cells were seeded onto upper well membranes of Transwell plate inserts and then placed into wells prefilled with EBM medium with 0.5% FBS containing the chemoattractant beta-carotene (BC) 3 AM or arachionic acid (AA) 3 AM or BC/AA and sphingosine-1-phosphate (S1P) 500 nM. Cells treated with S1P were used as positive controls. After incubation for 24 h, cells that had migrated across micropore inserts (5-Am pore size) to the lower surface of the membranes were stained by anti-human CD31 antibody coupled with phycoerythrin (PE). Chemotaxis index: the ratio of stimulated migration divided by that of basal, unstimulated migration of HUVEC in control medium sample. Values are mean valuesFS.E.; n=3 done in triplicates. Significantly different from the corresponding control, *Pb0.05, **Pb0.005. A. Dembinska-Kiec et al. / Biochimica et Biophysica Acta 1740 (2005) 222–239 229

Fig. 5. Influence of beta-carotene on tube-like structure formation in vitro in the 3D matrigel model. The formation of microtubules by HUVECs suspended in matrigel incubated 24 h with beta-carotene (BC) 3 AM or arachidonic acid (AA) 3 AM or both (BC/AA) as well as VEGF 0.2 nM, bFGF 0.5nM was investigated (positive controls). (A) Tube-like structures were observed under the optical microscope (at magnitude Â10) and photographed. (B) Lengths of the analyzed tubule-like structures were calculated and expressed as the average sum of the total length of tubules visible under the optical microscope in the random five fields of each matrigel. Values are mean valuesFS.E., n=10 done in triplicates. Significantly different from the corresponding (medium with 0.075% THF/EtOH) control, *Pb0.05, **Pb0.005.

(FBN1), laminin beta1 (LAMB1), matrilin 2 (MATN2), regulated genes was performed using the database of matrix Gla protein (MGP) was also observed (Table 2). transcriptional start sites to recognize a common transcrip- The up-regulated expression of genes of proteins tional regulatory network for the reconstitution of up/down- involved in endothelial homing/chemotaxis (IL-8; CXCR- stream signalling pathways possibly influenced by BC in 4) [29,30] by BC was also found (Table 2). HUVECs. It was analysed by the comparison of certain Among the proteins participating in intracellular signal- promoter sequences retrieved from the database of Tran- ling pathways the most evident changes in BC-induced gene scriptional Start Sites DBTSS (http://dbtss.hgc.jp/index. expression were found within the members of G-protein html) website transcription factor consensus sequences and coupled receptors (GPCR) such as GPR12, CXCR4, ITGA6, database TRANSFAC (http://transfac.gbf.de/TRANSFAC/) DTR, the Rho-like small GTPase family or their regulators, website. The promoter analysis revealed that BC-activated and secreted factors such as IL-8, CXCL2. AA alone did not genes are modified predominantly by transcription factors affect expression of above mentioned genes (Table 2). regulated by p38 MAP kinase pathway (e.g. STAT, Max, The list of the other genes regulated significantly by BC cMyc, Elk1, CHOP, MEF2, ATF2, PPAR, CREB, SP-1, and AA, including transcription factors, xenobiotic metab- cJUN, cFOS, C/EBPa, GATA) [31]. Early growth response olism are presented in Table 2. factor (Egr-1), which was found to mediate the expression Following the identification of differentially regulated of EC genes after vascular injury [32], was the most genes, a detailed promoter analysis of the significantly frequently recognized as common transcription factor for 230 A. Dembinska-Kiec et al. / Biochimica et Biophysica Acta 1740 (2005) 222–239

The direction of beta-carotene expression regulation was found similar to the microarray data with respect to the factors, whose activity is related to proliferation (MAD1L1, MEOX2) [24,33], chemotaxis (IL-8, CXCR-4, VCAM1), [30,29,26], and inhibition of tubulogenesis (MEOX2) (Fig. 7). Additionally, the BC-induced difference in the EGR-1 gene expression was detected, consistent with the up- regulation of DR1 and NAB1 (the inhibitors of the transcription factor EGF-1, which is important for endothe- lial cell proangiogenic activity) [32] observed in the microarray experiments (Fig. 7). The basal expression of h,h-carotene 15,15V-monoxyge- nase (BCMO, EC 1.13.11.21) in HUVECs detected by real- time PCR and also demonstrated the lack of BC-induced changes was in agreement with the microarray assay (Fig. 8).

3.9. Western blotting analysis of IL-8 protein level

The activation of the protein expression of chemotactive IL-8 by BC was confirmed by Western blot. Fig. 9A shows a Western blot of total cell lysates probed with an antibody to detect IL-8 protein. Densitometric analysis revealed that incubation of HUVEC with BC, AA or BC with AA increased IL-8 protein level (Fig. 9B).

4. Discussion

The main result of presented study is the original demonstration of the proangiogenic activity of BC in vivo and the involvement of BC-induced chemotactic activity of endothelial cells in this process. Beta-carotene, alpha-carotene, h-cryptoxanthin lutein and lycopene belong to the well-characterized family of carotenoids constituting approximately 90% of the total Fig. 6. Effects of beta-carotene on angiogenesis in mouse matrigel model in plasma carotenoids in characterized humans in concentra- vivo. Angiogenesis was assayed in subcutaneously injected matrigel plugs tions ranging from undetectable to 3–10 Amol/l (dependent containing medium with 0.075% THF/EtOH (as control), bFGF 50 nM or upon diet or supplementation) [34]. Dietary lipids are beta-carotene 3 AM (BC) or both isolated after 6 days. Angiogenesis was important for the absorption of carotenoids both in vivo quantified by counting. (A) The number of vessels without lumen. (B) The and in the cell culture system [35]. Also, in our in vitro number of vessels with lumen. (C) The number of endothelial CD31 (PECAM)-positive cells penetrating the matrigel plug, as average in five experimental model, AA increased the uptake of BC by different fields in each of the three slices taken from different regions HUVECs, which was documented by HPLC (Fig. 1B). This throughout the each matrigel plug. For immunostaining, sections were was not related to the increase of the basal expression of incubated with mouse monoclonal anti-CD31 antibody. Values are mean h,h-carotene 15,15V-monoxygenase (BCMO) (the micro- F values S.E., n=6 per group. *Significantly different from the correspond- array experiments, confirmed later by RT-PCR). BCMO is ing control, *Pb0.05. the key that cleaves beta-carotene centrally into two molecules of retinal, the source of retinoic acid or retinol BC-regulated genes or for described transcription factors (Vitamin A). Thus, the observed effects of BC may coactivator or target. be related to its direct free radical scavenger activity [36] and/or possible activity of its metabolites (mainly retinoic 3.8. Changes in relative gene expression (real-time PCR) acid), generated locally by HUVECs. The results of the microarray experiments indicate that In order to confirm the microarray results related to the degree of change in gene expression regulated by the the biological effects of BC in HUVECs, the expression used concentration of BC in HUVECs is moderate. Also, of selected genes was verified by the quantitative real- the expression of 95% of up- or down-regulated genes was time PCR. changed less than 2-fold. It is important to stress that non- A. Dembinska-Kiec et al. / Biochimica et Biophysica Acta 1740 (2005) 222–239 231

Table 2 The microarray analysis: List of genes related to proliferation, apoptosis, adhesion, cytoskeleton, chemotaxis, intracellular signaling pathways, xenobiotic metabolism, transcription factors regulated by beta-carotene in HUVEC AA vs. BC vs. AA/BC vs. Gene title Gene symbol UniGene ID THF/EtOh THF/EtOH THF/EtOH Proliferation Cell cycle checkpoint regulator NC 1.9 NC MAD1 mitotic arrest deficient-like 1 (yeast) MAD1L1 Hs.7345

S phase cell cycle 1.4 1.4 NC MCM5 minichromosome maintenance deficient 5, cell division cycle 46 MCM5 Hs. 77171 (S. cerevisiae) NC NC 1.6 SET translocation (myeloid leukemia-associated) SET HS. 436687

G1 phase cell cycle NC NC À1.4 DnaJ(Hsp40)homolog, subfamily A, member 2 DNAJA2 Hs.368078

G2/S transaction of cell cycle NC 1.9 NC polo-like kinase (Drosophila) PLK Hs.329989 1.5 1.4 NC membrane-associated tyrosine- and threonine-specific cdc2-inhibitory kinase PKMYT1 Hs.77783 NC 1.4 NC WEE1 homolog (S. pombe) WEE1 Hs.249441 NC NC 2.3 nuclear ubiquitous casein kinase and cyclin-dependent kinase substrate NUCKS Hs.510265

Apoptosis Apoptosis inhibitor NC 3.5 NC myeloid cell leukemia sequence 1 (BCL2-related) MCL1 Hs.86386 NC 1.9 1.7 TNF receptor-associated factor 4 TRAF4 Hs.8375 1.6 1.6 1.5 baculoviral IAP repeat-containing 5 (survivin) BIRC5 Hs.1578 NC 1.4 NC testis enhanced gene transcript (BAX inhibitor 1) regulator TEGT Hs.35052 1.2 1.3 1.4 CASP8 and FADD-like apoptosis CFLAR Hs.355724 NC NC À1.9 ring finger protein RNF7 Hs.512849

Apoptosis activator NC 6.5 NC BCL2-like 11 (apoptosis facilitator) BCL2L11 Hs.84063 NC 1.4 NC signal-induced proliferation-associated gene 1 SIPA1 Hs.7019 À1.5 À1.4 À1.4 optineurin OPTN Hs.390162 NC À1.5 À1.5 coagulation factor II (thrombin)receptor F2R/PAR1 Hs.128087 NC À1.5 À1.2 PRKC, apoptosis, WT1, regulator PAWR/PAR4 Hs.406074 NC NC 1.5 pleckstrin homology-like domain, family A, member 2 PHLDA2 Hs.154036 NC NC 1.4 B-cell CLL/lymphoma 10 BCL10 Hs.193516 NC NC 1.4 caspase recruitment domain family, member 8 CARD8 Hs.446146 NC NC À1.7 pleiomorphic adenoma gene-like 1 PLAGL1 Hs.132911

Chemotaxis Cytokines NC 1.5 1.9 Interleukin 8 IL-8 Hs.624 À1.3 1.5 NC Chemokine (C-X-C motif) ligand 2 CXCL2 Hs.75765

Receptors 2.0 2.1 1.7 Chemokine (C-X-C motig) receptor 4 CXCR4 Hs.421986 NC 1.9 NC Diphteria toxin receptor (heparin-binding epidermal growth factor-like DTR Hs.799 growth factor) 1.6 À2.1 NC Fibroblast growth factor receptor 1 (fms-related tyrosine kinase 2, FGFR1 Hs.748 Pfeiffer syndrome)

Cell–cell adhesion 2.3 2.3 2.0 Tumor necrosis factor (ligand) superfamily, member 4 TNFSF4 Hs.181097 (tax-transcriptionally activated glycoprotein 1, 34 kDa) NC 1.9 NC Endoglin (Osler-Rendu-Weber syndrome 1) ENG Hs.76753 NC 1.5 1.5 Absent in melanoma 1 AIM1 Hs.422550 NC 1.4 NC CD99 antigen CD99 Hs.283477 NC À1.4 NC Cadherin, EGF LAG seven-pass G-type receptor 1 (flamingo homolog, CELSR1 Hs.252387 Drosophila) À2.0 À1.4 À1.1 Catenin (cadherin-associated protein), beta 1, 88kda CTNNB1 Hs.410086 (continued on next page) 232 A. Dembinska-Kiec et al. / Biochimica et Biophysica Acta 1740 (2005) 222–239

Table 2 (continued) AA vs. BC vs. AA/BC vs. Gene title Gene symbol UniGene ID THF/EtOh THF/EtOH THF/EtOH Chemotaxis Cell–cell adhesion NC À1.4 NC Sialophorin (gpl115, leukosialin,CD43) SPN Hs.461934 À2.1 À1.4 À1.7 Selectin P (granule membrane protein 140kDa,CD43) SELP Hs.73800 À2.6 À2.1 NC CD 24 antigen (small cell lung carcinoma cluster 4 antigen) CD24 Hs.375108 À2.6 À2.6 NC CD 24 antigen (small cell lung carcinoma cluster 4 antigen) CD24 Hs.375108 À10.6 À2.6 À5.3 Vascular cell adhesion molecule 1 VCAM1 Hs.109225 NC À5.7 1.6 EphB2 EPHB2 Hs.125124 NC NC À1.4 Activate leukocyte cell adhesion molecule ALCAM Hs.10247 NC NC 1.4 Carcinoembryonic antigen-related cell adhesion molecule 4 CEACAM4 Hs.12 NC NC À1.4 Catenin (cadherin-associated protein), alpha-like 1 CTNNAL1 Hs.58488 NC NC À1.4 Desmoplakin DSP Hs.349499

Cell–matrix adhesion NC 1.9 NC Integrin, alpha ITGA6 Hs.212296 1.5 1.6 NC Scavenger receptor class B, member 1 SCARB1 Hs.130981

Exracellular matrix component 1.7 1.6 1.9 Collagen,type XIII, alpha 1 COL13A1 Hs.211933 1.7 1.6 NC Nidogen 2 (osteonidogen) NID2 Hs.147697 1.5 1.5 1.5 Fibrillin 2 (congenital contractural archnodactyly) FBN2 Hs.79432 À1.4 À1.4 À1.3 Collagen, type V, alpha 2 COL5A2 Hs.283393 À1.3 À1.4 À1.4 Fibrillin 1 (Marfan syndrome) FBN1 Hs.750 NC À1.4 NC Kallman syndrome 1 sequence KAL1 Hs.380850 À1.3 À1.4 À1.2 Laminin, beta 1 LAMB1 Hs.122645 À2.1 À1.9 À2.1 Sulfatase 1 SULF1 Hs.409602 NC À2.0 NC Exostoses (multiple) 1 EXT1 Hs.184161 À2.3 À2.8 À2.5 Collagen, type VIII, alpha COL8A1 Hs.114599 À3.2 À2.8 À3.5 Matrix Gla protein MGP Hs.365706 À4.0 À4.3 À3.7 Matrillin 2 MATN2 Hs.153647 À1.5 NC À1.6 Collagen, type III, alpha 1 (Ehlers-Danlos syndrome type IV, autosomal COL3A1 Hs.443625 dominant)

Cellular NC 9.8 NC Membrane metallo-endopeptidase (neutral endopeptidase, enkephaline, MME Hs.307734 CALLA, CD10) NC 2.5 NC Matrix 14 (membrane-inserted) MMP14 Hs.2399 1.3 1.9 1.6 Serine (or cysteine) proteinase inhibitor, clade B (ovalbumin), member 2 SERPINB2 Hs.75716 NC 1.7 NC Plasminigen activator, tissue PLAT Hs.274404 À1.7 À1.4 À1.7 A disintegrin-like and metalloprotease (reprolysin type) with ADAMTS1 Hs.8230 thrombosposin type 1 motif, 1 À2.1 À2.3 À1.9 Matrix metalloprotainase 12 () MMP12 Hs.1695 NC NC 2.3 A disintegrin and metalloproteinase domain 18 ADAM18 Hs.127930 NC NC 1.4 Matrix metalloprotainase 24 (membrane-inserted) MMP24 Hs.212581 NC NC À1.4 10 () MMP10 Hs.2258

Cytoskeleton NC 1.6 NC Intersectin 1 (SH3 Domain protein) ITSN1 Hs.66392 NC 1.6 NC Lamin B1 LMNB1 Hs.89497 NC 1.6 NC Tubulin, beta polypeptide TUBB Hs.512712 NC 1.5 1.3 Myosin IB MYO1B Hs.121576 NC 1.4 NC Actinin, alpha 4 ACTN4 Hs.443619 NC 1.4 NC Flotillin 2 FLOT2 Hs.18799 NC 1.4 NC Myosin IXB MYO9B Hs.159629 NC 1.4 NC Transgellin 2 TAGLN2 Hs.406504 NC À1.4 NC Transgellin TAGLN Hs.410977 À1.9 À1.7 À2.5 Myosin, heavy polypeptide 10, non-muscle MYH10 Hs.280311 NC NC 1.7 Caveolin 2 CAV2 Hs.139851 NC NC 1.4 Utophin (homologous to dystrophin) UTRN Hs.250607 NC NC À1.4 Kelch-like 3 (Drosophila) KLHL3 Hs.434434 NC NC À1.6 Filamin A,alpha (actin binding protein 280) FLNA Hs.195464 A. Dembinska-Kiec et al. / Biochimica et Biophysica Acta 1740 (2005) 222–239 233

Table 2 (continued) AA vs. BC vs. AA/BC vs. Gene title Gene symbol UniGene ID THF/EtOh THF/EtOH THF/EtOH Receptors G protein coupled receptor 2.0 2.1 1.7 Chemokine (C-X-C motif) receptor 4 CXCR4 Hs.421986 NC 2.0 NC G protein-coupled receptor 12 GPR12 Hs.123034 NC 1.9 NC Diphtheria toxin receptor (heparin-binding epidermal growth factor-like DTR Hs.799 growth factor) NC 1.9 NC Integrin, alpha 6 ITGA6 Hs.212296 NC À1.4 NC Cadherin, EGF LAG seven-pass G-type receptor 1 (flamingo homolog, CELSR1 Hs.252387 Drosophilia) NC À1.5 À1.5 Coagulation factor II (thrombin) receptor F2R/PAR1 Hs.128087 NC À1.5 À1.2 PRKC, apoptosis, WT1, regulator PAWR/PAR4 Hs.406074 NC NC 4.9 Somatostatin receptor 2 SSTR2 Hs.184841 NC NC 2.8 Pyrimidnergic receptor P2Y, G-protein coupled, 4 P2RY4 Hs.248157 NC NC 1.6 Dopamine receptor D5 DRD5 Hs.380681 NC NC 1.5 G protein-coupled receptor 27 GPR27 Hs.356084 À1.4 NC À1.7 Calcitonin receptor-like CALCRL Hs.152175

FGF receptor mediated signalling pathway 1.6 À2.1 NC Fibroblast growth factor receptor 1 (fms-related thyrosine kinase 2,Pfeiffer FGFR1 Hs.748 syndrome)

Eph receptor mediated signalling pathway NC NC 1.6 EphB2 EPHB2 Hs.125124

Interleukin receptor mediated signalling pathway NC 1.5 1.9 Interleukin 8 IL-8 Hs.624 À1.3 1.5 NC Chemokine (C-X-C motif) ligand 2 CXCL2 Hs.75765 À1.5 À1.5 À1.3 Interleukin 1 receptor, type 1 IL1R1 Hs.82112 NC NC À1.4 Interleukin 10 receptor, beta IL1ORB Hs.418291 NC NC À1.5 Interleukin 13 receptor, alpha IL13RA1 Hs.285115

Intracellular signalling Small GTPase mediated signal transdustion NC 1.7 NC CAP, adenylate cyclase-associated protein, 2 (yeast) CAP2 Hs.296341 À1.3 1.5 NC Chemokine (C-X-C motif) ligand 2 CXCL2 Hs.75765 NC 1.5 1.9 Interleukin 8 IL-8 Hs.624 NC 1.4 NC Myosin IXB MYO9B Hs.159629 NC 1.4 NC Res-related C3 substrate 2 (rho family, small GTP binding RAC2 Hs.301175 protein Rac 2) À1.9 À1.3 À1.4 ADP-ribisylation factor-like 4 ARL4 Hs.245540 NC À1.4 NC RAP2C, member of RAS oncogene family RAP2C Hs.225979 NC À1.6 NC Guanine nucleotide exchange factor for Rap 1 GFR Hs.449375 À1.5 À2.0 À1.7 Regulator of G-protein signalling 4 RGS4 Hs.386726 NC NC 1.4 Rho/Rac guanine nucleotide exchange factor (GEF) 2 ARHGEF2 Hs.337774 NC NC À1.4 RAN, member RAS oncogene family RAN Hs.10842 NC NC À1.7 Rap2 interacting protein x RIPX Hs.7972

Protein kinase cascade NC À1.4 À1.9 Mitogen-activated protein kinase kinase kinase 7 MAP3K7 Hs.290346 À1.5 À1.9 À2.1 Transformer-2 alpha TRA2A Hs.445652 NC NC À1.4 Mitogen-activated protein kinase kinase kinase 5 MAP4K5 Hs.246970

NFjB NC 1.7 NC Toll-like receptor 4 TLR4 Hs.174312

JAK/STA NC À1.5 À1.5 Coagulation factor II (thrombin) receptor F2R/PAR1 Hs.128087

JNK NC NC À1.4 Mitogen-activated protein kinase kinase kinase kinase 5 MAP4K5 Hs.246970 (continued on next page) 234 A. Dembinska-Kiec et al. / Biochimica et Biophysica Acta 1740 (2005) 222–239

Table 2 (continued) AA vs. BC vs. AA/BC vs. Gene title Gene symbol UniGene ID THF/EtOh THF/EtOH THF/EtOH Intracellular signalling Phosphatidiloinositol pathway NC 3.5 NC Myotubularin related protein 1 MTMR1 Hs.347187 NC À2.0 NC Dual adaptor of phosphotyrosine and 3-phosphoinositides DAPP1 Hs.62643

Nitric oxide NC 1.4 NC Nitric oxide synthase 3 (endothelial cell) NOS3 Hs.446303 NC 1.6 1.3 NAD(P)H dehydrogenase, quinone 1 NQ01 Hs.406515 NC NC 1.5 GTP cyclohyrolase I feedback regulatory protein GCHFR Hs.245644 NC NC À1.6 Guanylate cyclase 1, soluble, alpha 3 GUCY1A3 Hs.433488

SH2/SH3 NC 1.7 NC SH3-domain binding protein 2 SH3BP2 Hs.167679 NC 1.4 NC 1 SHC1 Hs.433795

Transcription factors NC 3.7 2.5 LIM homebox 3 LHX3 Hs.148427 NC 2.0 1.4 Cbp/p300-interacting transactivator, with Glu/Asp-rich carboxy-terminal CITED2 Hs.82071 domain 2 NC 1.9 NC MAD1 mitotic arrest deficient-like 1 (yeast) MAD1L1 Hs.7345 2.0 1.9 1.3 Mesenchyme homeo box 2 (growth arrest-specific homeo box) MEOX2 Hs.77858 NC 1.7 NC General transcription factor IIIH, polypeptide 4 52 kDa GTF2H4 Hs.102910 NC 1.6 NC Forehead box M1 FOXM1 Hs.511941 NC 1.6 NC Jun B proto-oncogene JUNB Hs.400124 NC 1.6 2.5 Early growth response 1 EGR1 Hs.326035 NC 1.5 NC Heat shock transcription factor 1 HSF1 Hs.132625 NC 1.4 NC CAMP responsive element binding protein 1 CREB1 Hs.22315 NC 1.4 NC Down-regulator o transcription 1, TBP-binding (negative 2) DR1 Hs.348418 1.4 1.4 1.5 Hairy/enhancer-of-split related with YRPW motif 1 HEY1 Hs.234434 NC 1.4 NC Kruppel-like factor 2 (lung) KLF2 Hs.107740 1.3 1.4 NC NGFI-A binding protein 1 (EGR1 binding protein 1) NAB1 Hs.107474 NC 1.4 NC SRY (sex determining region Y)-box 13 SOX13 Hs.201671 NC 1.3 1.5 LIM domain only 4 LMO4 Hs.3844 1.4 1.3 1.4 Pituitary tumor-transforming 1 PTTG1 Hs.350966 NC 1.2 1.4 Hematopoietic cell-specific Lyn substrate HCLS1 Hs.14601 NC À1.3 À1.5 Zinc finger protein 36, C3H type-like 1 ZFP36L1 Hs.85155 NC À1.3 À1.4 Zinc finger protein 24 (KOX 17) ZNF24 Hs.173911 À1.6 À1.4 NC Basic transcription element binding protein 1 BTEB1 Hs.150557 NC À1.4 NC Nuclear receptor subfamily 2, group F, member 2 NR2F2/COU P-TFII Hs.347991 À1.4 À1.4 NC Ubinuclein 1 UBN1 Hs.21479 À1.5 À1.5 NC Delta sleep including peptide, immunoreactor DSIPI Hs.420569 À1.4 À1.5 À1.5 Nuclear receptor subfamily 1, group D, member 2 NR1D2/EAR-1R Hs.37288 À1.2 À1.5 NC Serologically defined colon cancer antigen 33 SDCCAG33 Hs.284217 1.1 À1.5 À1.3 Transcription factor 7 (T-cell specific, HMG-box) TCF7 Hs.169294 À1.4 À1.6 À1.5 Nuclear receptor subfamily 3, group C, member 1 (glucocorticoid receptor) NR3C1 Hs.512414 À1.6 À1.6 À1.3 OLF-1/EBF associated zinc finger gene OAZ Hs.137168 NC NC 1.6 Zinc finger protein 323 ZNF323 Hs.444116 NC NC 1.5 Interferon regulatory factor 7 IRF7 Hs.166120 NC NC 1.4 v-jun sarcoma virus 17 oncogene homolog (avian) JUN Hs.78465 NC NC 1.4 Pre-B-cell leukemia transcription factor 2 PBX2 Hs.93728 NC NC 1.4 Transcription factor 3 (E2A immunoglobin enhancer binding factors E12/E47) TCF3 Hs.371282 NC NC À1.4 v-ets erythroblastosis virus E26 oncogene homolog 2 (avian) ETS2 Hs.292477 NC NC À1.5 Friend leukemia virus integration 1 FLI1 Hs.257049 NC NC À1.5 Metastasis associated 1 MAT1 Hs.101448

Metabolism Xenobiotic metabolism phase I NC 1.6 1.3 NAD(P)H dehydrogenase, quinone 1 NQO1 Hs.406515 NC 1.4 NC Aldehyde dehydrogenase 6 family, member A1 ALDH6A1 Hs.293970 À1.3 NC À1.5 Aldehyde dehydrogenase 1 family, member A1 ALDH1A1 Hs.76392 A. Dembinska-Kiec et al. / Biochimica et Biophysica Acta 1740 (2005) 222–239 235

Table 2 (continued) AA vs. BC vs. AA/BC vs. Gene title Gene symbol UniGene ID THF/EtOh THF/EtOH THF/EtOH Metabolism Xenobiotic metabolism phase II Sulfotransferase À1.7 À2.0 À2.1 Sulfotransferase family, cytosolic, 1B, member 1 SULT1B1 Hs.129742 alkyl or aryl NC NC 1.6 Glutathione S-transferase M5 GSTM5 Hs.75652 Glucuronosyltransferase NC 1.1 1.4 Exostoses (multiple) 2 EXT2 Hs.75334 NC À2.0 NC Exostoses (multiple) 1 EXT1 Hs.184161 Epoxide À1.5 À1.2 À1.4 Leukotriene A4 hydrolase LTA4H Hs.81118 Changes in relative gene expression were calculated versus control (medium with 0.075%THF/EtOH), only genes with the significant change in expression level higher than 1,4 are shown. NC_no change. toxic, physiological concentrations of BC were used in this ation, expression of specific matrix proteins and proteolytic study [7,35]. At the same time, these concentrations of BC, enzymes, and other factors, which are responsible for the mimicking those occurring physiologically, promoted endo- remodeling of matrix and the promotion of the outgrowth of thelial cell chemotaxis in vitro and applied subcutaneously new capillaries [1,3]. Activated EC change their profiles of in matrigel significantly induced the proangiogenic effect of gene expression, switching to proliferative, non-differenti- bFGF in the in vivo mouse model. ated status, investigated already by microarrays [37,38]. Angiogenesis is the crucial event for the remodeling of Several pathways, including retinoid signaling, have been tissues of a growing body in embryonic and adult life, implicated in the development of the cardiovascular system female ovulatory cycle, wound healing, tissue ischemia and in the fetal period. Nutritional deficiency of retinoids, RXR/ inflammatory processes as well as tumor malignancy [1]. RAR, or Raldh2 knock-out mice, is characterized by The induction of angiogenesis involves the activation of multiple developmental malformations, including severe endothelial cells from preexisting capillaries or mobilization cardiovascular defects and lack of ophthalmo-mesenteric of the endothelial progenitor cells [23]. The proangiogenic vessels with disrupted formation of extra-extraembryonic cytokines, such as VEGF, bFGF, PDGF, TGF-h, angiopoie- vessels [39,40]. However, cellular mechanisms, thereby tin 1/2 and several others, activate specific receptors on EC, carotenoid/retinoid participates in the assembly of mamma- inducing endothelial cell detachment, migration, prolifer- lian blood vessels, have not been defined.

Fig. 7. The expression of selected genes verified by the quantitative real-time PCR: influence of beta-carotene and arachidonic acid on CXCR4 (chemokine (C- X-C motif) receptor 4), IL-8 (interleukin 8), VCAM-1 (vascular cell adhesion molecule 1), MAD1L1 (MAD1 mitotic arrest deficient-like 1), MEOX2 (mesenchyme homeo box 2 (growth arrest-specific homeo box)), EGR-1 (early growth response 1) and BIRC5 (baculoviral IAP repeat-containing 5 (survivin) apoptosis inhibitor). Data expressed as the relative gene expression ratio. The mean valuesFS.E., n=3 done in triplicates are shown; *Significantly different from the corresponding control, *Pb0.05. 236 A. Dembinska-Kiec et al. / Biochimica et Biophysica Acta 1740 (2005) 222–239

BC and AA also up-regulated survivin (BIRC5), an anti- apoptotic protein, which binds to pro-caspase-9, preventing its recruitment to Apaf1 [43]. The observed change in the expression of survivin in microarray was later confirmed by the results of real-time PCR. Thus, the proangiogenic activity of BC in HUVECs seems to be related not to proliferation (or inhibition of apoptosis) [41], but to the potent activation of chemotaxis and changes in the expression of genes mediating cell adhesion and matrix assembly [44,45]. The results obtained by the microarray in our model strongly support this Fig. 8. Evidence of beta-carotene 15,15V-monooxygenase 1 (BCMO) gene hypothesis. expression in HUVEC. Gene expression was investigated using quantitative real-time PCR method. PCR products were analyzed also by electrophoresis The microarray data demonstrated changes in the carried out according to standard protocol. expression of several genes coding for group of proteins participating in cell–cell and cell–matrix adhesion, matrix proteins and proteases which regulate cell/matrix interac- Using Raldh2 À/À mice, Lai et al. documented that tion. The list of the BC-responsive genes includes those retinoic acid induced the expression of p21 and p27, the coding for proteins participating in: cell–cell adhesion Cip/Waf family of Cdk inhibitors controlling cell cycle (VCAM1, SELP, CD24), cadherins (CELSR1), and catenins progression in EC. Retinoid treatment in normal EC had no (CTNNA1L, CTNNB1). Expression of those genes was effect on the expression of cyclins: A, B, D3, E, or Cdc2 or mainly down-regulated by both BC and AA. The down- Cdk2 [41]. Using Western blot, those authors found that the regulation of VCAM-1, ICAM and selectin E by carote- formation of the complex between cyclin D1 or D2 and noids, including BC, in human aortic endothelium stimu- Cdk4 was significantly reduced, whereas the formation of lated with Il-1h was suggested to be responsible for the the complex between RA-induced p21 or p27 and Cdk4 as modulatory effect on inflammatory response [46] and may well as cyclin D1 and D2 was greatly enhanced in the reflect the anti-inflammatory, protective effect of BC on presence of retinoic acid. The reduced Cdk/cyclin D endothelium. complex formation resulted in lower levels of phosphory- lated retinoblastoma protein (Rb) in RA-treated EC. Thus, there was no influence on the number of cells (prolifer- ation), but there was a significant decrease in the proportion of endothelial cells in phase S and an increase in the proportion of cells in phase G1 with no evidence of apoptosis in EC [41]. We have not observed any measurable effect of BC on EC proliferation or apoptosis. Results from our microarray experiments indicated that BC and AA weakly up-regulated the key molecules participating in cell cycle regulation, such as MCM-5 as well as polo-like kinase Plk1, or NUCKS controlling the G2/M check point. However, it was paralleled by the up-regulation of the important inhibitors of G2/M cell cycle phase, as Wee1 and PKMyt1. The expression of MAD1L1, participating in G1/S cell cycle check point and inhibition of proliferation [24], was also observed. Although the analysis of the microarray data revealed a strong (6.5-fold) up-regulation of apoptosis facilitator Bcl2L11, a member of the Bcl2 family by BC, we did not observe any change in the rate of apoptosis in HUVECs measured by the caspase activity. The up- regulation of some proapoptotic members of the Bcl-2 Fig. 9. Influence of studied compounds on IL-8 protein expression. Western family, such as Bcl2L11/BAM and anti-apoptotic MCL-1, blot analysis of protein content in the HUVECs treated 24 h with EBM TEGT-BAX inhibitor was noted in the microarray experi- medium with 0.075% THF/EtOH (control), BC 3 AMorAA3AM or both ments in BC treated HUVECs. It has been shown that compounds. Beta-actin was used as a the control of the equally loading of proteins. Values are meanFS.E., n=6 done in triplicates. *Pb0.05. (A) Bcl2L11 releases mitochondrial cytochrome c, but the Representative Western blot of IL-8 protein expression. (B) Quantitative simultaneous increase of the MCL1 expression may have evaluation (densitometric analysis) of analyzed proteins, expressed as attenuated the proapoptotic potential of Bcl2L11 [42]. The percent of control. A. Dembinska-Kiec et al. / Biochimica et Biophysica Acta 1740 (2005) 222–239 237

The expression of the genes coding for proteins interaction and contraction of the cell through the regulation associated with cell-extracellular matrix adhesion, such as of phosphorylation of the myosin light chains (MLCs). integrins (ITGA6) and SCARB1, was up-regulated by BC. MLC phosphorylation occurs in the presence of specific Members of the extracellular matrix degrading enzymes MLC kinases, such as MLCK Ca+2 dependent kinase, Rho involved in the regulation of the matrix degradation kinase (ROCK) and p21-activated kinase (PAK). Rho products promoting cell migration, such MMP14, and the kinases also inhibit dephosphorylation by the inhibition of tissue plasminogen activator gene (PLAT), were similarly myosin phosphatase type I (PP1M) and increase MLC up-regulated, while MMP12 was down-regulated. The phosphorylation [54,55]. The obtained microarray results integrin-mediated stimulation of chemotaxis by the metal- demonstrated that BC, through the regulation of expression loproteinase-mediated matrix degradation products is an of Rho/Rac/Cdc42 pathway members, lead to the activation important step of the capillary network formation [44, of HUVEC migration. 45,47]. The expression of desintegrin ADAMTS1, respon- Despite the evidence of their proangiogenic activity, BC sible for receptor shadding [28], was also inhibited. (and AA) do not seem to influence the differentiation of The BC-treated cells also showed the down-regulation HUVECs as reflected by the microarrays results and in vitro of genes coding for various extracellular matrix compo- 3D model of tubulogenesis. Lai et al. reported that RA nents, such as collagens, fibrillin 1, laminin h1, matrilin 2 deficiency had no effect on the differentiation of immature and matrix Gla protein. The importance of the expression endothelial cell as evidenced by the expression of VEGF, of cell surface proteins, which participate in cell/matrix Flk1-receptor, VE-cadherin or angiopoietin Tie2 receptor (e.g. integrins) and cell/cell (e.g. cadherins, catenins, [41]. We also did not observe any changes in the expression endoglin, ephrins and their receptors) interactions required of genes associated with HUVEC differentiation following for the regulation of proliferation, migration and differ- incubation with BC or AA. entiation during angiogenesis, has been well documented It has been reported that cytokines (bFGF, VEGF) [3]. Additionally BC up-regulates the expression of IL-8, stimulate formation of tube-like structures and endothelial the potent activator of migration of EC [30], what was cell maturation in an in vitro matrigel model, and that confirmed in this study by both real-time PCR and Western vascular repair after injury is associated with the increase of blot. the expression of early growth response transcription factor Remodeling of the extracellular matrix proteins by (EGR-1) [32]. As suggested by the results of our microarray enzymatic degradation and synthesis of proteoglycans experiments, the expression of the bFGF receptor (FGFR1) changes the extracellular matrix composition and regu- was down-regulated by BC. The microarray data revealed lates the migration of endothelial cells. Migration and that BC induced two potent repressors of EGR-1: DR-1 and cellular shape change are also associated with the stress NAB-1 [32]. Additionally, the expression of the tran- fiber formation and reorganization of the cellular cytos- scription factor MEOX2, which has an inhibitory effect on keletal proteins [48,49]. The contribution of nitric oxide the VEGF-stimulated tube-formation of HUVECs [33], was in the chemotactic activity and cytoskeleleton reorganiza- up-regulated by BC and AA in our model. Moreover, the tion of endothelial cells was also reported [50]. The highest expression activation by BC (9.8-fold) was observed induction of the endothelial nitric oxide synthase (eNOS) in gene coding for endopeptidase MME. The membrane- gene by BC in HUVEC was also observed in our bound metallopeptidase, which cleaves and degrades microarray data. angiogenic peptides, such as atrial natriuretic peptide, The G-protein binding receptors and regulators of Rho endothelin, angiotensin I, substance P, and bradykinin, GTPases were the second largest group of genes, whose was also shown to inhibit HUVEC differentiation [45]. expression was significantly changed by BC. The expres- These findings suggests the activation of migration and sion of some of them was confirmed by real-time PCR. The proliferation of non-differentiated EC and may imply the genes up-regulated by BC, such as CXCR4 and IL-8 (whose formation of non-mature and non-functional capillary net- induction was verified by real-time PCR), and others, such work in response to BC. Such a network is characteristic of as GPR12, were recently demonstrated to be activated by tumor vasculature [1]. sphingosine 1-phosphate (S1P)-Edg class receptor [51], In summary, we postulate that the originally observed DTR-heparan-binding epidermal growth factor (EGF)-like proangiogenic activity of BC is related to the activation of receptor, and the integrin alpha 6 (ITGA6) are known chemotaxis of endothelial cells. The effect of BC on EC regulators of HUVEC migration that activate Rho GTPases gene expression as assessed with microarrays is moderate. [52,53]. Proangiogenic factors, such as stromal derived However, we were able to detect gene expression changes factor (SDF-1), IL-8, or S1P, lead to the activation of Rho/ that support the involvement of BC in the regulation of Rac/CDC42 small GTPases through the activation of G- synthesis of extracellular matrix and adhesion molecules protein-coupled receptors and by interaction with adhesion- synthesis, resulting in a potent activation of Rho/Rac/Cdc42 mediated signaling pathway [18,29,30,53]. Rho GTPases GTPase signaling pathway, which in turn may promote regulate cytoskeletal changes responsible for cell motility, HUVEC migration and the formation of not a completely shape and contraction. Rho GTPases promote actin–myosin matured capillary network. 238 A. Dembinska-Kiec et al. / Biochimica et Biophysica Acta 1740 (2005) 222–239

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