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Biochimica et Biophysica Ac

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 Krakow, PolandbDepartment of Pathology, The Jagiellonian University Medical College, Krakow, Poland

cDepartment of Medical Biochemistry, The Jagiellonian University Medical College, Krakow, PolanddDepartment 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 genes. 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-protein 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

Angiogenesis, the formation of new blood vessels from

the preexisting ones, plays a central role in a number of

physiologic and pathologic events including vascular

remodeling of growing ischemic tissue, inflammation,

diabetic retinopathy, as well as in progression of solid

tumor growth and invasiveness [1,2].

The knowledge of the formation of new tubular

structures in angiogenesis is being continuously enriched

0925-4439/$ - see front matter D 2004 Published by Elsevier B.V.

doi:10.1016/j.bbadis.2004.11.017

* Corresponding author. Tel.: +48 12 421 40 06; fax: +48 12 421 40

73.

E-mail address: [email protected] (A. Dembinska-Kiec).

in new observations suggesting the influence of the new

regulators. A number of growth factors (such as VEGF,

bFGF, TGFb, PDGF, IGF and others), cell/matrix (integ-

rins) and cell/cell (VE-cadherins, catenins, endoglins,

ephrins, their receptors or Jagged/Notch pathway) inter-

actions, and environmental factors (such as shear stress,

nutrients, oxygen supply and others) regulate the most

important steps in angiogenesis, which include detachment,

proliferation, migration, homing and differentiation of the

vascular wall cells, mainly endothelial or their progenitors

[3,4].

Carotenoids are lipid soluble hydrocarbon pigments

(C40H56) containing 11 conjugated double bonds. The main

plant carotenoids detected in human blood are: beta-carotene

ta 1740 (2005) 222–239

A. Dembinska-Kiec et al. / Biochimica et Biophysica Acta 1740 (2005) 222–239 223

(BC), alpha-carotene, gamma-carotene and lycopene. They

are isomers varying in the position of double bonds and the

presence of acyclic ring. Carotenoids present in human

tissues function as free radical scavengers, immunomodula-

tors, or some being the substrate for the synthesis of retinol

(Vitamin A) and retinoic acid (RA) are regulators of cell fate

and differentiation [5]. RA, derived from beta-carotene, is a

well known activator of nuclear receptors such as retinoid/

rexinoid receptors (RXR/RAR). RA in cooperation with fatty

acids and fatty acid metabolites regulates the activity of the

PPAR/RXR heterodimer (peroxisome proliferator-activated

receptors, PPARs) [6]. As regulators of transcription they

influence cell differentiation, maturation of tissues and body

organization in both fetal and adult periods of life [5–7].

Epidemiological studies suggested that the intake of

fruit and vegetables rich in carotenoids results in the

reduction of the cancer incidence, decreased risk of heart

diseases, and enhanced immunity due to its free radical

scavenging properties, anti-inflammatory and increased

activity of NK cells [8–11]. Surprisingly, some of the large

clinical studies, undertaken to prove the efficacy of beta-

carotene supplementation in the prevention of coronary

heart disease and cancer, showed that administration of

beta-carotene or vitamin A might increase the risk of lung

cancer, especially in smokers and patients afflicted with

asbestosis (Alpha-Tocopherol Beta-Carotene Cancer Pre-

vention Study (ATBC), [12], Beta-Carotene Retinol Effi-

cacy Trial (CARET) [13], Physician’s Health Study (PHS),

[14]).

Since 15% of beta-carotene ingested reach human target

cells in the non-metabolized form [15] and angiogenesis is

an important part of remodeling of an ischemic tissue as

well as solid tumor malignancy [1], this study was under-

taken to define the direct effects of beta-carotene on

endothelial cells in terms of angiogenic activity and

regulation of gene expression.

2. Methods

The HPLC grade beta-carotene (1 mg) filled with

nitrogen in vials made of dark glass were kindly provided

for the project by the Roche Vitamins AG, Kaiseraugust,

Switzerland.

2.1. The cell culture

Primary endothelial cells (HUVECs) were isolated from

human umbilical veins using collagenase digestion.

HUVECs were cultured in EBM (EGM Bullet Kit,

Clonetics) with supplements: hEGF (10 ng/ml), hydro-

cortisone (1 ng/ml), Bovine Brain Extract (12 Ag/ml),

antibiotics: gentamicin (50 Ag/ml) as well as amphoter-

icin-B (50 ng/ml), and 10% Fetal Bovine Serum (Clonetics).

The resulting cell lines were characterized by morphological

and immunohistochemical criteria such as desmin, alpha-

actin, vWF [16]. Experiments were performed on 70%

confluent cell cultures (up to 5th passage).

For cell culture, 4 mM beta-carotene (BC) stock solution

in tetrahydrofurane (THF) (Sigma) was further diluted in

ethanol (1:1 v/v THF/EtOH ratio) to obtain 2 mM

concentration of BC. The further dilutions were made with

the tissue culture medium (EBM). The final concentration of

THF/EtOH in the cell culture medium was 0.075%.

HUVECs were incubated with the solvent (0.075% THF/

EtOH) containing medium (as the control) and beta-carotene

(BC) (0.3–10 AM), arachidonic acid (AA) (3 AM) (Sigma),

or both BC (3 AM) and AA (3 AM) for 24 h at 37 8C in 5%

CO2 and 95% humidity (Jouan IG 150).

2.2. Beta-carotene uptake by HUVECs (measured by HPLC)

All solvents used for HPLC (THF, ethanol, tBME

(tetra-butylmethylether) (Sigma) and BHT (2,6-Di-tert-

butyl-p-cresol) (Sigma)) were analytical grade. THF and

ethanol were additionally filtered through the Aluminum

Oxide column (Sigma) before use. Following 24 h

incubation of HUVECs with the studied compounds, cells

were detached by trypsinization and washed three times

with Ca+2 and Mg+2 free PBS (centrifugation: 400�g for

10 min, at the room temperature). Cells were counted

using the Burker chamber and spinned down (400�g, 10

min, at room temperature). The resulted pellet was frozen

(at �80 8C) for further analysis. BC concentration in the

cells and BC stability in culture medium (lack of

spontaneous degradation products), were assessed with

the HPLC micromethod developed by Roche Vitamins AG

(Kaiseraugust, Switzerland). Briefly, for extraction, 200 Alof acetone (with 0.025% BHT f.c.) was added to a frozen

cell pellet, mixed for 1 min and dried in a vacuum

centrifuge under argon (30 min at 50 mbar). 200 Al of theBC 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, 8000�g at 4 8C). The supernatant

was used for HPLC.

For the measurement of BC content in the tissue culture

medium, 25 Al of the medium was mixed with 225 Al of theBC extraction solvent described above, mixed for 1 min and

centrifuged for 3 min at 8000�g at 4 8C. The HPLC sample

analysis was isocratically performed on a Vydac 218TP54

column C-18 (Roche) (4.6�250 mm) at a constant column

temperature 20 8C. The sample solvent consisted of ethanol/

THF/tBME 9:1:5, 0.025% BHT f.c. The mobile phase was:

acetonitryl/tert-butylmethylether/ammonium acetate (80

mM)/triethylamine (73:20:7:0.05) at a constant flow of

butylmethylether/1.5 ml/min. Shimadzu SCL-10AVP instru-

ment (Shimadzu, Kyoto, Japan) with the SPD-10AV

detector was set at 450 nm for the carotenoids estimation.

In this HPLC system, 12V-apocarotenal, 8V-apocarotenal, 4V-h-apocarotenal, all-trans-h-carotene and (Z)-h-carotenecould be detected. The amount of BC was expressed as

pmol of BC/106 cells.

A. Dembinska-Kiec et al. / Biochimica et Biophysica Acta 1740 (2005) 222–239224

2.3. Detection of cellular toxicity and apoptosis

The possible toxic effects of used compounds and solvents

on HUVECs were assessed by the cellular lactic dehydrogen-

ase (LDH) presence in the medium after 24-h incubation (Cy-

toTox 96 Non-Radioactive Cytotoxicity Assay, Promega).

For the estimation of apoptosis the ApoFluorR Green

Caspase Activity Assay (ICN Biomedicals Inc.) was used.

This test allows for the measurement of global cellular

caspase activity. Proapoptotic staurosporine (at 1 nM) was

used as the positive control for apoptosis. Following

incubation with the fluorescent dye (1 h) cells were

harvested, washed to remove unbound dye with the Ca+2

and Mg+2 free PBS and 1�106 cells were placed in black

microtiter plates (NUNC A/S, Roskilde). The fluorescence

was detected using a plate reader (Synergy HT, BIO-TEK)

and quantitated at 520 nm emission wavelength following

the excitation with the 490 nm light. Results are expressed

as the percentage of activity detected in the control

(incubated with the solvent only) HUVECs.

2.4. Assay of HUVEC proliferation (The bromo-

deoxyuridyne (BrdU) incorporation assay)

In order to determine the effect of BC and AA on the

HUVEC proliferation, the rate of DNA synthesis was

measured by the incorporation of the thymidine analog,

bromo-deoxyuridine (BrdU) to DNA [17]. HUVECs

(5�104 cells) were incubated with the compounds for 24

h, as described above, and additionally with BrdU for the

last 3 h. Following incubation, cells were fixed and stained

with the anti-BrdU kit (Roche) according to the manufac-

turer’s recommendations. HUVECs proliferation induced by

VEGF (Vascular Endothelial Growth Factor) (0.2 nM)

(Sigma) or bFGF (basic fibroblast growth factor) (0.5 nM)

(Sigma) for 24 h was served as the positive control. Results

are given as the percent of the control BrdU incorporation to

the HUVEC incubated in the medium with solvents only.

2.5. Migration of endothelial cells (The Boyden’s Chamber

assay)

HUVECs harvested from 3–4 passages were suspended

(105 cells/100 Al) in the EBM medium with 0.5% FBS and

seeded into BD Falconk FluoroBlokk Inserts (3 Am pore,

Becton Dickinson). The inserts containing HUVECs were

placed into a 24-well plate with 600 Al of the EBM medium

and 0.5% FBS, and incubated for 24 h at 37 8C. Media with

the addition of BC (3 AM) or AA (3 AM) as well as 1-

phospho-sphingosine (S1P) (500 nM) (Sigma) as the

positive control [18] were used as chemoattractant in the

lower chamber. After 24 h, HUVECs were stained with anti-

CD31 antibody conjugated with phycoerythrin (PE) (10 Ag/ml) (Becton Dickinson) for 30 min at 37 8C. Subsequently,cells in the inserts were washed with the Ca+2 and Mg+2 free

PBS and the fluorescence of the cells, which had migrated

through the pores to the bottom side of the inserts, was

measured with a fluorescence plate reader (Synergy HT,

BIO-TEK). The changes in intensity of fluorescence in a

sample with agents versus control (cells migrated against the

EBM with solvents only) corresponded to the amount of

migrating cells. The chemotactic activity of the endothelial

cells was expressed as the chemotaxis index (CHI) which

represents the ratio of migration stimulated by investigated

compound versus random migration of unstimulated

HUVECs in THF/EtOH control sample.

2.6. The three dimensional [3D] model of tubulogenesis in

matrigel in vitro [19]

HUVECs harvested from 3–4 passages were resuspended

in Matrigel (containing laminin, collagen IV, entactin,

heparan sulfate proteoglycans) (Becton Dickinson) to the

final concentration of 1�106 cells/ml on ice. The cell–

matrigel mixture (50 Al) was placed into the cell culture

dishes and incubated in a humidified CO2 incubator (Jouan)

at 37 8C for 30 min. Subsequently the EBM medium (100

Al) supplemented with solvent (control probe), or BC (3

AM), or AA (3 AM), or BC/AA mixture was applied on top

of the cells immersed in matrigel and was incubated in a

humidified CO2 incubator for 24 h at 37 8C. The number of

tubules formed in the presence of VEGF (0.2 nM) or bFGF

(0.5 nM) served as the positive control. The formation of

tubules by HUVECs suspended in matrigel was assessed

under the light microscope (at magnitude �10) and photo-

graphed. The lengths of the analyzed tubule-like structures

were calculated and expressed as the average sum of total

length of tubules visible under the light microscope.

2.7. Angiogenesis in vivo (the mouse model)

Protocol was accepted by the local University Ethic

Committee. All animal experiments were performed accord-

ing to Polish laws and approved by the Polish Animal

Inspectorate and Institutional Animal Care. Female Balb/c

mice (n=6) received sterile injections of 2�500 Al matrigel

(Becton Dickinson) s.c. (dorsally). The matrigel plug con-

tained solvent or BC (3 AM), bFGF (50 nM), or both BC and

bFGF at the given concentrations. Six days later the animals

were sacrificed by overdosing urethane anesthesia and the

matrigel plug were removed, fixed and immersed in paraffin.

Immunohistochemistry was performed using routine proto-

col. Primary anti-CD31 antibodies (anti-PECAM-1, BD

Pharmingen) at 1:300 dilution were used. The slides were

rehydrated and incubated in 3% peroxide solution for 10 min

to block endogenous peroxidase activity. The Streptavidin–

Biotin (BD Pharmingen) detection system was used. DAB

was used as chromogen. The slides were contra-stained with

Mayer hematoxylin (DAKO, Denmark). The amount of

capillaries was counted under the microscope in five different

fields in each of the three slices taken from different parts of

each plugs. The number of capillaries detected in slices from


the plugs containing the studied compounds was compared to

the plugs with solvents and expressed as the number of

vessels with or without the lumen. Additionally, the number

of separately detected endothelial PECAM-positive single

cells, not connected with capillaries, migrating to the matrigel

was calculated.

2.8. The protein synthesis (Western Blot)

Western-blot was used to detect the synthesis of

interleukin -8 (IL-8) by HUVECs incubated with inves-

tigated compounds.

The cells were rinsed with the Ca+2 and Mg+2 free PBS

and lysed in CelLyticTM-M Mammalian Cell Lysis/Extrac-

tion Reagent (Sigma) containing protease inhibitors (Protease

Inhibitor Cocktail Tablets complete TM, Roche). Protein

content was determined by Bradford’s method [20]. Proteins

were electrophoretically separated on an SDS-polyacryla-

mide gel under reducing conditions and transferred to PVDF

(polyvinylidine difluoride) membranes (BIORAD). Transfer

was performed in 100 V for 2 h. Blots, stained with 2%

Ponceau S (Sigma), in 3% trichloroacetic acid to visualize

proteins, were saturated with 1% blocking solution (Lumi-

LightPLUS Western Blotting Kit Mouse/Rabbit, Amersham

Pharmacia Biotech) and incubated for 1 hwithmouse anti-IL-

8 antibody (Mouse monoclonal anti-IL-8 antibody [B-2]).

Following washing in TBST (Lumi-LightPLUS Western

Blotting Kit Mouse/Rabbit), membranes were incubated for

30min, subsequently with anti-mouse Ig-POD Fab fragments

(Lumi-LightPLUS Western Blotting Kit Mouse/Rabbit,

Roche) or monoclonal anti-goat/sheep IgG Monoclonal

Anti-Goat/Sheep IgG Clone GT-34 peroxidase conjugated

(Sigma). Monoclonal antibodies against actin were used as a

control of protein loading (mouse monoclonal anti-actin

antibodies—Actin [C-2], Santa Cruz Biotechnology, Inc.).

Enhanced chemiluminescence, performed according to

manufacturer’s instructions (Amersham), was used to

demonstrate positive bands that were visualized after

exposure on a transparent medical X-ray film.

2.9. Isolation of total RNA

Following 24-h incubation with the studied compounds,

total RNA was isolated from HUVECs by the guanidine

thiocyanate–caesium chloride method [21] using Trizol

(Invitrogen Life Technologies) and was purified using the

SV total RNA Isolation System Kit (Promega). The quality

of RNA was confirmed by denaturing gel electrophoresis

and an analysis on the Agilent 2100 Bioanalyser (Agilent

Technologies).

2.10. Microarray affymetrix HG-U133A hybridization

The aim of the microarray experiments was to screen the

effects of beta-carotene (with or without arachidonic acid) on

gene expression patterns in HUVECs. For microarray

hybridization, RNAwas reverse transcribed into cDNA with

a primer containing the T7 promoter using Superscript II

(Invitrogen Life Technologies). cDNA was used as a

template in a biotin-labeled transcription reaction (Enzo

BioArray, Affymetrix). The resulting target cRNA was

purified on RNeasy columns (QIAGEN) and fragmented

for hybridization to Affymetrix HG-U133A GeneChips.

Hybridization was done overnight at 45 8C for 16 h in the

GeneChip Hybridization Oven 640 (Affymetrix). The

GeneChips were subsequently processed on the Affymetrix

GeneChip Fluidics Workstation 400 according to the

EukGE-WS2v4 protocol. The GeneChips were scanned with

the Hewlett Packard GeneArray Scanner, and the results

analyzed using Affymetrix Microarray Analysis Suit 4.0.

2.11. Analysis of the microarray data

Changes in relative gene expression were calculated

versus the control (THF/EtOH solvent). Only spots with

significant differences in signal intensity (more than 1.4-fold

and only when the P value was at least 0.05) were included

in the analysis.

In order to identify genes belonging to pathways regulated

by beta-carotene and/or arachidonic acid in HUVECs we

searched promotor sequences of the identified genes to find

similar transcription factors binding sites. The 4-kb upstream

sequences relative to the transcription start site were retrieved

from the database of the Transcriptional Start Sites DBTSS

(http://dbtss.hgc.jp/index.html). Searches of transcription

factor binding sites were done using the TRANSFAC

database at a default threshold setting of 90 (http://

molsun1.cbrc.aist.go.jp/research/db/TFSEARCH.html).

2.12. cDNA synthesis and quantitative real-time PCR

In order to confirm the regulation of the expression of

genes important for demonstration of the proangiogenic

activity of BC in HUVECs, which were identified by the

microarray experiments, the quantitative expression analysis

IL-8, CXCR4, VCAM-1, EGR-1, MAD1L1, BIRC5 and

MEOX2 was performed by real-time PCR using GAPDH as

the reference gene. For the cDNA synthesis 1 Ag of total RNAwas reverse transcribed at 42 8C for 50 min in a total volume

of 40 Al reaction buffer containing 5� First Strand Buffer,

DTT, oligo(dT) (Sigma), deoxy-NTPs (Promega), and 200

units of SUPERSCRIPT II reverse transcriptase (Invitrogen

Life Technologies). The reaction mixture was heated to 70 8Cfor 15 min and immediately chilled on ice. Subsequently,

cDNAwas subjected to real-time PCR in a reaction mixture

containing QuantiTect SYBR Green PCR (Qiagen) mix and

primers. The sequences of the primers used in this study are

presented in Table 1.The primers were designed to include an

intervening intron between the sense and antisense primers,

thereby eliminating the possibility of amplifying any genomic

DNA, and checked for specificity by BLAST searches. All

real-time PCR reactions were performed on the DNA Engine

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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-3VMAD1L1 NM_003550 5V-ACAGTCTCTGTAATCGCGAAAGC-3V 5V-TCAGGGTGGATAAAACCATGG-3VEGR-1 NM_001964 5V-AGCACCTGACCGCAGAGTCT-3V 5V-GGTCTCCACCAGCACCTTCTC-3VIL-8 NM_000584 5V-CTGGCCGTGGCTCTCTGG-3V 5V-TTAGCACTCCTTGGCAAAACTG-3VCXCR4 NM_003467 5V-CACCGCATCTGGAGAACCA-3V 5V-TCCTGCGTGTAGTTATCTGAAGTGTATATAC-3VVCAM1 NM_080682 5V-AGGCTGGAAGAAGAAGCGGAAAGG-3V 5V-AGCTGTAAGTTTTATGTCTTTTGGAGTAAC-3VBIRC5 NM_001168 5V-TTAACCCTTGGTGAATTTTTGAAACT-3V 5V-TTCTTATTGTTGGTTTCCTTTGCAA-3VBCMO NM_017429 5V-TGCCAGCCGGAATTTCTTTAT-3V 5V-TCGGTATTGCTTTCCATGTGA-3VGAPDH NM_002046 5V-GCCAGCCGAGCCACATC-3V 5V-GCGCCCAATACGACCAAA-3V


Opticon II (MJ Research). The thermal profile included initial

denaturation for 15 min at 95 8C, followed by 40 amplifica-

tion cycles of denaturation for 30 s at 94 8C, annealing for 30 sat 60 8C, and elongation for 30 s at 72 8C. Following PCR

amplification, melting curve analysis was performed with a

temperature profile slope of 1 8C/s from 35 8C to 95 8C. Anegative control without cDNA template was run with every

assay to ensure overall specificity. The expression rates were

calculated as the normalized CT difference between a control

probe and a sample with the adjustment for the amplification

efficiency relative to the expression level of the housekeeping

gene GAPDH. Calculation was performed using the program

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-wayANOVA. 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

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

(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

done in triplicates. *Significantly different from the corresponding control

cells; *Pb0.05.

3. Results

3.1. Uptake of beta-carotene by HUVECs

BC uptake by endothelial cells in culture was concen-

tration-dependent (Fig. 1A) and the presence of AA (3 AM)

augmented the BC uptake (Fig. 1B). The BC concentration

of 3 AM was used for the further experiments, since the

higher concentrations of BC caused endothelial cell toxicity

(especially in the presence of AA), as evidenced by the

LDH leakage from HUVECs after a 24-h incubation (data

not shown).

3.2. Effects of beta-carotene and arachidonic acid on

HUVEC apoptosis

Unlike in the cells treated with proapoptotic stauroporine

(1 nM), no proapoptotic activity measured by caspase

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activation was detected in HUVECs incubated for 24 h with

the BC or AA at 3 AM, as well as with the mixture of both

compounds (Fig. 2).

3.3. Effect of beta-carotene and arachidonic acid on

HUVEC proliferation

Unlike VEGF (0.2 nM) and bFGF (0.5 nM), which

stimulated cell proliferation, both BC as well as AA

used in the non-toxic concentrations did not influence

HUVEC proliferation measured by the BrdU incorporation

(Fig. 3).


HUVEC migration

BC (3 AM) four-fold increased the migration of HUVECs

(Fig. 4). Arachidonic acid alone did not stimulate HUVEC

migration, but the cell migration was further increased (six-

fold) when AA was used in combination with BC (Fig. 4).

Sphingosine-1-phosphate (S1P) the potent activator of

endothelial cell migration [18] was used as the positive

control to confirm HUVEC chemotactic potential in our

assay.


tubulogenesis in the 3D matrigel in vitro model

The 3D matrigel assay of tubulogenesis was used to

verify the angiogenic property of BC and AA in vitro [19].

Only the trace tubulogenic activity of HUVEC was detected

in cells cultured in matrigel covered with medium without

factors. No tubulogenic activity of BC and AA incubated

alone as well as together was observed in the above in vitro

model (Fig. 5A,B). The proangiogenic VEGF (0.2 nM) and

bFGF (0.5 nM) potently increased the number of tubules in

matrigel HUVEC suspension (Fig. 5A,B).

Fig. 2. Influence of beta-carotene and arachidonic acid on the HUVEC apoptosis.

caspase activity following a 24-h incubation of HUVEC with the studied facto

arachidonic acid (AA) 3 AM or both compounds (BC/AA). Data represent mean va

the percentage of activity detected in the control. *Significantly different from th

3.6. The proangiogenic activity of beta-carotene in the in

vivo mouse model

The presence of BC at 3 AM in the matrigel administered

subcutaneously significantly increased the density of capil-

lary network (with and without lumen) and the amount of

endothelial cells penetrating to the matrigel plug stimulated

by bFGF (Fig. 6).

3.7. The microarray analysis

Using the criteria described in Methods, we identified

838 genes, whose expression changed only in response to

the stimulation with BC (446 down-regulated, 393 up-

regulated). The expression of 644 genes was regulated by

AA (375 down-regulated, 269 up-regulated) and the

expression of 740 genes was regulated by beta carotene

and AA together (498 down-regulated, 242 up-regulated).

The analysis of microarray data, including genes regulated

(up or down) from 1.4-fold up to 10-fold, provided a list of

the selected significantly regulated genes coding for

proteins, which belong to the cellular pathways such as:

pathways contributing to proangiogenic activity (cell cycle,

adhesion, matrix remodeling, chemotaxis), apoptosis, recep-

tor-mediated signal transduction, as well as transcription

factors and regulators of protein synthesis (zinc finger

proteins, ribosomal proteins), xenobiotic metabolism,

inflammatory response (Table 2).

According to the recent data concerning the proangio-

genic activity of endothelial cells [1,23], special attention

was paid to the BC-regulated genes involved in the cell

cycle-proliferation. BC weakly up-regulated the key genes

coding for proteins participating in the regulation of a cell

cycle such as MCM5, MAD1L1 connected with G1/S check

point and polo-like kinase Plk1, NUCKS related to G2/M

check point. BC up-regulated such important inhibitors of

cell cycle as Wee1, PKMyt1 (Table 2) [24].

The rate of apoptosis was estimated by the measurement of global cellular

rs (control: medium with 0.075% THF/EtOH), beta-carotene (BC) 3 AM,

luesFS.E. of three independent experiments done in triplicates, expressed as

e corresponding control, *Pb0.05.

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

inhibitors associated with the FASL pathway such as

CFLAR (Flip), BIRC5 (IAP) TRAF4 genes and down-

regulated the expression of apoptosis activators OPTN,

PAR1 and PAR4 [25]. The parallel up-regulation of the

expression of a number of pro- and anti-apoptotic members

of the Bcl-2 family was observed. BC up-regulated the

proapoptotic Bcl2L11/BAM, anti-apoptotic MCL-1 and

TEGT-BAX inhibitor (Table 2).

The group of BC-regulated genes, which code for

proteins participating in cell–cell interactions, such as

cadherins (CELSR1), catenins (CTNNA1L, CTNNB1),

and the leukocyte–endothelium adhesion mediating mole-

Fig. 4. Beta-carotene-induced chemotaxis of HUVEC. Cells were seeded onto up

prefilled with EBM medium with 0.5% FBS containing the chemoattractant b

sphingosine-1-phosphate (S1P) 500 nM. Cells treated with S1P were used as p

micropore inserts (5-Am pore size) to the lower surface of the membranes were

Chemotaxis index: the ratio of stimulated migration divided by that of basal, unst

valuesFS.E.; n=3 done in triplicates. Significantly different from the correspondi

cules (VCAM1, SELP, CD24) [26], were generally down-

regulated by both BC and AA (Table 2). On the contrary, the

expression of genes coding for proteins associated with cell-

extracellular matrix adhesion, such as integrins (ITGA6),

scavenger receptor SCARB1, was up-regulated by BC

(Table 2).

The expressions of genes encoding extracellular matrix

degrading enzymes and stimulators of chemotaxis, which

may regulate the matrix-degradation [27], receptor shading

[28] and cell migration, such as ADAMTS1, ADAMTS18,

MMP10, MMP12, MMP14, MMP24, were differentially

regulated. The down-regulation of different types of

extracellular matrix components collagens, fibrillin 1

per well membranes of Transwell plate inserts and then placed into wells

eta-carotene (BC) 3 AM or arachionic acid (AA) 3 AM or BC/AA and

ositive controls. After incubation for 24 h, cells that had migrated across

stained by anti-human CD31 antibody coupled with phycoerythrin (PE).

imulated migration of HUVEC in control medium sample. Values are mean

ng control, *Pb0.05, **Pb0.005.

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 �10) 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),

matrix Gla protein (MGP) was also observed (Table 2).

The up-regulated expression of genes of proteins

involved in endothelial homing/chemotaxis (IL-8; CXCR-

4) [29,30] by BC was also found (Table 2).

Among the proteins participating in intracellular signal-

ling pathways the most evident changes in BC-induced gene

expression were found within the members of G-protein

coupled receptors (GPCR) such as GPR12, CXCR4, ITGA6,

DTR, the Rho-like small GTPase family or their regulators,

and secreted factors such as IL-8, CXCL2. AA alone did not

affect expression of above mentioned genes (Table 2).

The list of the other genes regulated significantly by BC

and AA, including transcription factors, xenobiotic metab-

olism are presented in Table 2.

Following the identification of differentially regulated

genes, a detailed promoter analysis of the significantly

regulated genes was performed using the database of

transcriptional start sites to recognize a common transcrip-

tional regulatory network for the reconstitution of up/down-

stream signalling pathways possibly influenced by BC in

HUVECs. It was analysed by the comparison of certain

promoter sequences retrieved from the database of Tran-

scriptional Start Sites DBTSS (http://dbtss.hgc.jp/index.

html) website transcription factor consensus sequences and

database TRANSFAC (http://transfac.gbf.de/TRANSFAC/)

website. The promoter analysis revealed that BC-activated

genes are modified predominantly by transcription factors

regulated by p38 MAP kinase pathway (e.g. STAT, Max,

cMyc, Elk1, CHOP, MEF2, ATF2, PPAR, CREB, SP-1,

cJUN, cFOS, C/EBPa, GATA) [31]. Early growth response

factor (Egr-1), which was found to mediate the expression

of EC genes after vascular injury [32], was the most

frequently recognized as common transcription factor for


http:\\transfac.gbf.de\TRANSFAC\

Fig. 6. Effects of beta-carotene on angiogenesis in mouse matrigel model in

vivo. Angiogenesis was assayed in subcutaneously injected matrigel plugs

containing medium with 0.075% THF/EtOH (as control), bFGF 50 nM or

beta-carotene 3 AM (BC) or both isolated after 6 days. Angiogenesis was

quantified by counting. (A) The number of vessels without lumen. (B) The

number of vessels with lumen. (C) The number of endothelial CD31

(PECAM)-positive cells penetrating the matrigel plug, as average in five

different fields in each of the three slices taken from different regions

throughout the each matrigel plug. For immunostaining, sections were

incubated with mouse monoclonal anti-CD31 antibody. Values are mean

valuesFS.E., n=6 per group. *Significantly different from the correspond-

ing control, *Pb0.05.


BC-regulated genes or for described transcription factors

coactivator or target.

3.8. Changes in relative gene expression (real-time PCR)

In order to confirm the microarray results related to

the biological effects of BC in HUVECs, the expression

of selected genes was verified by the quantitative real-

time PCR.

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

plasma carotenoids in characterized humans in concentra-

tions ranging from undetectable to 3–10 Amol/l (dependent

upon diet or supplementation) [34]. Dietary lipids are

important for the absorption of carotenoids both in vivo

and in the cell culture system [35]. Also, in our in vitro

experimental model, AA increased the uptake of BC by

HUVECs, which was documented by HPLC (Fig. 1B). This

was not related to the increase of the basal expression of

h,h-carotene 15,15V-monoxygenase (BCMO) (the micro-

array experiments, confirmed later by RT-PCR). BCMO is

the key enzyme that cleaves beta-carotene centrally into two

molecules of retinal, the source of retinoic acid or retinol

(Vitamin A). Thus, the observed effects of BC may

be related to its direct free radical scavenger activity [36]

and/or possible activity of its metabolites (mainly retinoic

acid), generated locally by HUVECs.

The results of the microarray experiments indicate that

the degree of change in gene expression regulated by the

used concentration of BC in HUVECs is moderate. Also,

the expression of 95% of up- or down-regulated genes was

changed less than 2-fold. It is important to stress that non-

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.

THF/EtOh

BC vs.

THF/EtOH

AA/BC vs.

THF/EtOH

Gene title Gene symbol UniGene ID

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

(S. cerevisiae)

MCM5 Hs. 77171

NC NC 1.6 SET translocation (myeloid leukemia-associated) SET HS. 436687

G1 phase cell cycle

NC NC �1.4 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 �1.9 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

�1.5 �1.4 �1.4 optineurin OPTN Hs.390162

NC �1.5 �1.5 coagulation factor II (thrombin)receptor F2R/PAR1 Hs.128087

NC �1.5 �1.2 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 �1.7 pleiomorphic adenoma gene-like 1 PLAGL1 Hs.132911

Chemotaxis

Cytokines

NC 1.5 1.9 Interleukin 8 IL-8 Hs.624

�1.3 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

growth factor)

DTR Hs.799

1.6 �2.1 NC Fibroblast growth factor receptor 1 (fms-related tyrosine kinase 2,

Pfeiffer syndrome)

FGFR1 Hs.748

Cell–cell adhesion

2.3 2.3 2.0 Tumor necrosis factor (ligand) superfamily, member 4

(tax-transcriptionally activated glycoprotein 1, 34 kDa)

TNFSF4 Hs.181097

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 �1.4 NC Cadherin, EGF LAG seven-pass G-type receptor 1 (flamingo homolog,

Drosophila)

CELSR1 Hs.252387

�2.0 �1.4 �1.1 Catenin (cadherin-associated protein), beta 1, 88kda CTNNB1 Hs.410086

(continued on next page)


Table 2 (continued)

AA vs.

THF/EtOh

BC vs.

THF/EtOH

AA/BC vs.

THF/EtOH


Chemotaxis

Cell–cell adhesion

NC �1.4 NC Sialophorin (gpl115, leukosialin,CD43) SPN Hs.461934

�2.1 �1.4 �1.7 Selectin P (granule membrane protein 140kDa,CD43) SELP Hs.73800

�2.6 �2.1 NC CD 24 antigen (small cell lung carcinoma cluster 4 antigen) CD24 Hs.375108

�2.6 �2.6 NC CD 24 antigen (small cell lung carcinoma cluster 4 antigen) CD24 Hs.375108

�10.6 �2.6 �5.3 Vascular cell adhesion molecule 1 VCAM1 Hs.109225

NC �5.7 1.6 EphB2 EPHB2 Hs.125124

NC NC �1.4 Activate leukocyte cell adhesion molecule ALCAM Hs.10247

NC NC 1.4 Carcinoembryonic antigen-related cell adhesion molecule 4 CEACAM4 Hs.12

NC NC �1.4 Catenin (cadherin-associated protein), alpha-like 1 CTNNAL1 Hs.58488

NC NC �1.4 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

�1.4 �1.4 �1.3 Collagen, type V, alpha 2 COL5A2 Hs.283393

�1.3 �1.4 �1.4 Fibrillin 1 (Marfan syndrome) FBN1 Hs.750

NC �1.4 NC Kallman syndrome 1 sequence KAL1 Hs.380850

�1.3 �1.4 �1.2 Laminin, beta 1 LAMB1 Hs.122645

�2.1 �1.9 �2.1 Sulfatase 1 SULF1 Hs.409602

NC �2.0 NC Exostoses (multiple) 1 EXT1 Hs.184161

�2.3 �2.8 �2.5 Collagen, type VIII, alpha COL8A1 Hs.114599

�3.2 �2.8 �3.5 Matrix Gla protein MGP Hs.365706

�4.0 �4.3 �3.7 Matrillin 2 MATN2 Hs.153647

�1.5 NC �1.6 Collagen, type III, alpha 1 (Ehlers-Danlos syndrome type IV, autosomal

dominant)

COL3A1 Hs.443625

Cellular proteases

NC 9.8 NC Membrane metallo-endopeptidase (neutral endopeptidase, enkephaline,

CALLA, CD10)

MME Hs.307734

NC 2.5 NC Matrix metalloproteinase 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

�1.7 �1.4 �1.7 A disintegrin-like and metalloprotease (reprolysin type) with

thrombosposin type 1 motif, 1

ADAMTS1 Hs.8230

�2.1 �2.3 �1.9 Matrix metalloprotainase 12 (macrophage elastase) 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 �1.4 Matrix metalloproteinase 10 (stromelysin 2) 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 �1.4 NC Transgellin TAGLN Hs.410977

�1.9 �1.7 �2.5 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 �1.4 Kelch-like 3 (Drosophila) KLHL3 Hs.434434

NC NC �1.6 Filamin A,alpha (actin binding protein 280) FLNA Hs.195464


Table 2 (continued)

AA vs.

THF/EtOh

BC vs.

THF/EtOH

AA/BC vs.

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

growth factor)

DTR Hs.799

NC 1.9 NC Integrin, alpha 6 ITGA6 Hs.212296

NC �1.4 NC Cadherin, EGF LAG seven-pass G-type receptor 1 (flamingo homolog,

Drosophilia)

CELSR1 Hs.252387

NC �1.5 �1.5 Coagulation factor II (thrombin) receptor F2R/PAR1 Hs.128087

NC �1.5 �1.2 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

�1.4 NC �1.7 Calcitonin receptor-like CALCRL Hs.152175

FGF receptor mediated signalling pathway

1.6 �2.1 NC Fibroblast growth factor receptor 1 (fms-related thyrosine kinase 2,Pfeiffer

syndrome)

FGFR1 Hs.748

Eph receptor mediated signalling pathway

NC NC 1.6 EphB2 EPHB2 Hs.125124

Interleukin receptor mediated signalling pathway



�1.5 �1.5 �1.3 Interleukin 1 receptor, type 1 IL1R1 Hs.82112

NC NC �1.4 Interleukin 10 receptor, beta IL1ORB Hs.418291

NC NC �1.5 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



NC 1.4 NC Myosin IXB MYO9B Hs.159629

NC 1.4 NC Res-related C3 botulinum toxin substrate 2 (rho family, small GTP binding

protein Rac 2)

RAC2 Hs.301175

�1.9 �1.3 �1.4 ADP-ribisylation factor-like 4 ARL4 Hs.245540

NC �1.4 NC RAP2C, member of RAS oncogene family RAP2C Hs.225979

NC �1.6 NC Guanine nucleotide exchange factor for Rap 1 GFR Hs.449375

�1.5 �2.0 �1.7 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 �1.4 RAN, member RAS oncogene family RAN Hs.10842

NC NC �1.7 Rap2 interacting protein x RIPX Hs.7972

Protein kinase cascade

NC �1.4 �1.9 Mitogen-activated protein kinase kinase kinase 7 MAP3K7 Hs.290346

�1.5 �1.9 �2.1 Transformer-2 alpha TRA2A Hs.445652

NC NC �1.4 Mitogen-activated protein kinase kinase kinase 5 MAP4K5 Hs.246970

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

JAK/STA

NC �1.5 �1.5 Coagulation factor II (thrombin) receptor F2R/PAR1 Hs.128087

JNK

NC NC �1.4 Mitogen-activated protein kinase kinase kinase kinase 5 MAP4K5 Hs.246970

(continued on next page)


Table 2 (continued)

AA vs.

THF/EtOh

BC vs.

THF/EtOH

AA/BC vs.

THF/EtOH


Intracellular signalling

Phosphatidiloinositol pathway

NC 3.5 NC Myotubularin related protein 1 MTMR1 Hs.347187

NC �2.0 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 �1.6 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

domain 2

CITED2 Hs.82071

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 cofactor 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 �1.3 �1.5 Zinc finger protein 36, C3H type-like 1 ZFP36L1 Hs.85155

NC �1.3 �1.4 Zinc finger protein 24 (KOX 17) ZNF24 Hs.173911

�1.6 �1.4 NC Basic transcription element binding protein 1 BTEB1 Hs.150557

NC �1.4 NC Nuclear receptor subfamily 2, group F, member 2 NR2F2/COU P-TFII Hs.347991

�1.4 �1.4 NC Ubinuclein 1 UBN1 Hs.21479

�1.5 �1.5 NC Delta sleep including peptide, immunoreactor DSIPI Hs.420569

�1.4 �1.5 �1.5 Nuclear receptor subfamily 1, group D, member 2 NR1D2/EAR-1R Hs.37288

�1.2 �1.5 NC Serologically defined colon cancer antigen 33 SDCCAG33 Hs.284217

1.1 �1.5 �1.3 Transcription factor 7 (T-cell specific, HMG-box) TCF7 Hs.169294

�1.4 �1.6 �1.5 Nuclear receptor subfamily 3, group C, member 1 (glucocorticoid receptor) NR3C1 Hs.512414

�1.6 �1.6 �1.3 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 �1.4 v-ets erythroblastosis virus E26 oncogene homolog 2 (avian) ETS2 Hs.292477

NC NC �1.5 Friend leukemia virus integration 1 FLI1 Hs.257049

NC NC �1.5 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

�1.3 NC �1.5 Aldehyde dehydrogenase 1 family, member A1 ALDH1A1 Hs.76392


Table 2 (continued)

AA vs.

THF/EtOh

BC vs.

THF/EtOH

AA/BC vs.

THF/EtOH


Metabolism

Xenobiotic metabolism phase II

Sulfotransferase

�1.7 �2.0 �2.1 Sulfotransferase family, cytosolic, 1B, member 1 SULT1B1 Hs.129742

Transferase 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 �2.0 NC Exostoses (multiple) 1 EXT1 Hs.184161

Epoxide hydrolase

�1.5 �1.2 �1.4 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

study [7,35]. At the same time, these concentrations of BC,

mimicking those occurring physiologically, promoted endo-

thelial cell chemotaxis in vitro and applied subcutaneously

in matrigel significantly induced the proangiogenic effect of

bFGF in the in vivo mouse model.

Angiogenesis is the crucial event for the remodeling of

tissues of a growing body in embryonic and adult life,

female ovulatory cycle, wound healing, tissue ischemia and

inflammatory processes as well as tumor malignancy [1].

The induction of angiogenesis involves the activation of

endothelial cells from preexisting capillaries or mobilization

of the endothelial progenitor cells [23]. The proangiogenic

cytokines, such as VEGF, bFGF, PDGF, TGF-h, angiopoie-tin 1/2 and several others, activate specific receptors on EC,

inducing endothelial cell detachment, migration, prolifer-

Fig. 7. The expression of selected genes verified by the quantitative real-time PCR:

X-C motif) receptor 4), IL-8 (interleukin 8), VCAM-1 (vascular cell adhesion

(mesenchyme homeo box 2 (growth arrest-specific homeo box)), EGR-1 (early gro

apoptosis inhibitor). Data expressed as the relative gene expression ratio. The me

from the corresponding control, *Pb0.05.

ation, expression of specific matrix proteins and proteolytic

enzymes, and other factors, which are responsible for the

remodeling of matrix and the promotion of the outgrowth of

new capillaries [1,3]. Activated EC change their profiles of

gene expression, switching to proliferative, non-differenti-

ated status, investigated already by microarrays [37,38].

Several pathways, including retinoid signaling, have been

implicated in the development of the cardiovascular system

in the fetal period. Nutritional deficiency of retinoids, RXR/

RAR, or Raldh2 knock-out mice, is characterized by

multiple developmental malformations, including severe

cardiovascular defects and lack of ophthalmo-mesenteric

vessels with disrupted formation of extra-extraembryonic

vessels [39,40]. However, cellular mechanisms, thereby

carotenoid/retinoid participates in the assembly of mamma-

lian blood vessels, have not been defined.

influence of beta-carotene and arachidonic acid on CXCR4 (chemokine (C-

molecule 1), MAD1L1 (MAD1 mitotic arrest deficient-like 1), MEOX2

wth response 1) and BIRC5 (baculoviral IAP repeat-containing 5 (survivin)

an valuesFS.E., n=3 done in triplicates are shown; *Significantly different

Fig. 8. Evidence of beta-carotene 15,15V-monooxygenase 1 (BCMO) gene

expression in HUVEC. Gene expression was investigated using quantitative

real-time PCR method. PCR products were analyzed also by electrophoresis

carried out according to standard protocol.

Fig. 9. Influence of studied compounds on IL-8 protein expression. Western

blot analysis of protein content in the HUVECs treated 24 h with EBM

medium with 0.075% THF/EtOH (control), BC 3 AM or AA 3 AM or both

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)

Representative Western blot of IL-8 protein expression. (B) Quantitative

evaluation (densitometric analysis) of analyzed proteins, expressed as

percent of control.


Using Raldh2 �/� mice, Lai et al. documented that

retinoic acid induced the expression of p21 and p27, the

Cip/Waf family of Cdk inhibitors controlling cell cycle

progression in EC. Retinoid treatment in normal EC had no

effect on the expression of cyclins: A, B, D3, E, or Cdc2 or

Cdk2 [41]. Using Western blot, those authors found that the

formation of the complex between cyclin D1 or D2 and

Cdk4 was significantly reduced, whereas the formation of

the complex between RA-induced p21 or p27 and Cdk4 as

well as cyclin D1 and D2 was greatly enhanced in the

presence of retinoic acid. The reduced Cdk/cyclin D

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

family, such as Bcl2L11/BAM and anti-apoptotic MCL-1,

TEGT-BAX inhibitor was noted in the microarray experi-

ments in BC treated HUVECs. It has been shown that

Bcl2L11 releases mitochondrial cytochrome c, but the

simultaneous increase of the MCL1 expression may have

attenuated the proapoptotic potential of Bcl2L11 [42]. The

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

hypothesis.

The microarray data demonstrated changes in the

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-

tion. The list of the BC-responsive genes includes those

coding for proteins participating in: cell–cell adhesion

(VCAM1, SELP, CD24), cadherins (CELSR1), and catenins

(CTNNA1L, CTNNB1). Expression of those genes was

mainly down-regulated by both BC and AA. The down-

regulation of VCAM-1, ICAM and selectin E by carote-

noids, including BC, in human aortic endothelium stimu-

lated with Il-1h was suggested to be responsible for the

modulatory effect on inflammatory response [46] and may

reflect the anti-inflammatory, protective effect of BC on

endothelium.


The expression of the genes coding for proteins

associated with cell-extracellular matrix adhesion, such as

integrins (ITGA6) and SCARB1, was up-regulated by BC.

Members of the extracellular matrix degrading enzymes

involved in the regulation of the matrix degradation

products promoting cell migration, such MMP14, and the

tissue plasminogen activator gene (PLAT), were similarly

up-regulated, while MMP12 was down-regulated. The

integrin-mediated stimulation of chemotaxis by the metal-

loproteinase-mediated matrix degradation products is an

important step of the capillary network formation [44,

45,47]. The expression of desintegrin ADAMTS1, respon-

sible for receptor shadding [28], was also inhibited.

The BC-treated cells also showed the down-regulation

of genes coding for various extracellular matrix compo-

nents, such as collagens, fibrillin 1, laminin h1, matrilin 2

and matrix Gla protein. The importance of the expression

of cell surface proteins, which participate in cell/matrix

(e.g. integrins) and cell/cell (e.g. cadherins, catenins,

endoglin, ephrins and their receptors) interactions required

for the regulation of proliferation, migration and differ-

entiation during angiogenesis, has been well documented

[3]. Additionally BC up-regulates the expression of IL-8,

the potent activator of migration of EC [30], what was

confirmed in this study by both real-time PCR and Western

blot.

Remodeling of the extracellular matrix proteins by

enzymatic degradation and synthesis of proteoglycans

changes the extracellular matrix composition and regu-

lates the migration of endothelial cells. Migration and

cellular shape change are also associated with the stress

fiber formation and reorganization of the cellular cytos-

keletal proteins [48,49]. The contribution of nitric oxide

in the chemotactic activity and cytoskeleleton reorganiza-

tion of endothelial cells was also reported [50]. The

induction of the endothelial nitric oxide synthase (eNOS)

gene by BC in HUVEC was also observed in our

microarray data.

The G-protein binding receptors and regulators of Rho

GTPases were the second largest group of genes, whose

expression was significantly changed by BC. The expres-

sion of some of them was confirmed by real-time PCR. The

genes up-regulated by BC, such as CXCR4 and IL-8 (whose

induction was verified by real-time PCR), and others, such

as GPR12, were recently demonstrated to be activated by

sphingosine 1-phosphate (S1P)-Edg class receptor [51],

DTR-heparan-binding epidermal growth factor (EGF)-like

receptor, and the integrin alpha 6 (ITGA6) are known

regulators of HUVEC migration that activate Rho GTPases

[52,53]. Proangiogenic factors, such as stromal derived

factor (SDF-1), IL-8, or S1P, lead to the activation of Rho/

Rac/CDC42 small GTPases through the activation of G-

protein-coupled receptors and by interaction with adhesion-

mediated signaling pathway [18,29,30,53]. Rho GTPases

regulate cytoskeletal changes responsible for cell motility,

shape and contraction. Rho GTPases promote actin–myosin

interaction and contraction of the cell through the regulation

of phosphorylation of the myosin light chains (MLCs).

MLC phosphorylation occurs in the presence of specific

MLC kinases, such as MLCK Ca+2 dependent kinase, Rho

kinase (ROCK) and p21-activated kinase (PAK). Rho

kinases also inhibit dephosphorylation by the inhibition of

myosin phosphatase type I (PP1M) and increase MLC

phosphorylation [54,55]. The obtained microarray results

demonstrated that BC, through the regulation of expression

of Rho/Rac/Cdc42 pathway members, lead to the activation

of HUVEC migration.

Despite the evidence of their proangiogenic activity, BC

(and AA) do not seem to influence the differentiation of

HUVECs as reflected by the microarrays results and in vitro

3D model of tubulogenesis. Lai et al. reported that RA

deficiency had no effect on the differentiation of immature

endothelial cell as evidenced by the expression of VEGF,

Flk1-receptor, VE-cadherin or angiopoietin Tie2 receptor

[41]. We also did not observe any changes in the expression

of genes associated with HUVEC differentiation following

incubation with BC or AA.

It has been reported that cytokines (bFGF, VEGF)

stimulate formation of tube-like structures and endothelial

cell maturation in an in vitro matrigel model, and that

vascular repair after injury is associated with the increase of

the expression of early growth response transcription factor

(EGR-1) [32]. As suggested by the results of our microarray

experiments, the expression of the bFGF receptor (FGFR1)

was down-regulated by BC. The microarray data revealed

that BC induced two potent repressors of EGR-1: DR-1 and

NAB-1 [32]. Additionally, the expression of the tran-

scription factor MEOX2, which has an inhibitory effect on

the VEGF-stimulated tube-formation of HUVECs [33], was

up-regulated by BC and AA in our model. Moreover, the

highest expression activation by BC (9.8-fold) was observed

in gene coding for endopeptidase MME. The membrane-

bound metallopeptidase, which cleaves and degrades

angiogenic peptides, such as atrial natriuretic peptide,

endothelin, angiotensin I, substance P, and bradykinin,

was also shown to inhibit HUVEC differentiation [45].

These findings suggests the activation of migration and

proliferation of non-differentiated EC and may imply the

formation of non-mature and non-functional capillary net-

work in response to BC. Such a network is characteristic of

tumor vasculature [1].

In summary, we postulate that the originally observed

proangiogenic activity of BC is related to the activation of

chemotaxis of endothelial cells. The effect of BC on EC

gene expression as assessed with microarrays is moderate.

However, we were able to detect gene expression changes

that support the involvement of BC in the regulation of

synthesis of extracellular matrix and adhesion molecules

synthesis, resulting in a potent activation of Rho/Rac/Cdc42

GTPase signaling pathway, which in turn may promote

HUVEC migration and the formation of not a completely

matured capillary network.


Acknowledgements

This work was supported by the F5 EU DLARFID

project QLK1-CT-2001-00183 The Author would like to

thank Phd R. Goralczyk, Phd K. Wertz and Phd G. Riss

(DSM Nutritional Products, Human Nutrition and Health,

Carotenoid Section, Basel, Switzerland) for the kind supply

of HPLC-grade BC as well as for the training in the HPLC

methodology of measuring the levels of carotenoids.

References

[1] P. Carmeliet, Angiogenesis in health and disease, Nat. Med. 9 (2003)

653–660.

[2] Z.A. Khan, S. Chakrabarti, Growth factors in proliferative diabetic

retinopathy, Exp. Diabesity Res. 4 (2003) 287–301.

[3] G. Bazzoni, E. Dejana, M.G. Lampugnani, Endothelial adhesion

molecules in the development of the vascular tree: the garden of

forking paths, Curr. Opin. Cell Biol. 11 (1999) 573–581.

[4] J. Gerhart, 1998, Warkany lecture: signaling pathways in develop-

ment, Teratology 60 (1999) 226–239.

[5] D. Simoni, M. Tolomeo, Retinoids, apoptosis and cancer, Curr.

Pharm. Des. 7 (2001) 1823–1837.

[6] B.A. Stoll, Linkage between retinoid and fatty acid receptors:

implications for breast cancer prevention, Eur. J. Cancer Prev. 11

(2002) 319–325.

[7] S.A. Ross, P.J. McCaffery, U.C. Drager, L.M. De Luca, Retinoids in

embryonal development, Physiol. Rev. 80 (2000) 1021–1054.

[8] W. Stahl, N. Ale-Agha, M.C. Polidori, Non-antioxidant properties of

carotenoids, Biol. Chem. 383 (2002) 553–558.

[9] J.A. Fontana, A.K. Rishi, Classical and novel retinoids: their targets in

cancer therapy, Leukemia 16 (2002) 463–472.

[10] E. Dragnev, K.H. Rigas, J.R. Dmitrovsky, The retinoids and cancer

prevention mechanisms, Oncologist 5 (2000) 361–368.

[11] J.J.M. Castenmiller, C.E. West, Bioavailability and bioconversion of

carotenoids, Annu. Rev. Nutr. 18 (1998) 19–38.

[12] D. Albanes, O.P. Heinonen, P.R. Taylor, J. Virtamo, B.K. Edwards, M.

Rautalahti, A.M. Hartman, J. Palmgren, L.S. Freedman, J. Haapa-

koski, M.J. Barrett, P. Pietinen, N. Malila, E. Tala, K. Liippo, E.R.

Salomaa, J.A. Tangrea, L. Teppo, F.B. Askin, E. Taskinen, Y. Erozan,

P. Greenwald, J.K. Huttunen, Alpha-tocopherol and beta-carotene

supplements and lung cancer incidence in the alpha-tocopherol, beta-

carotene cancer prevention study: effects of baseline characteristics

and study compliance, J. Natl. Cancer Inst. 88 (1996) 1560–1570.

[13] G.S. Omenn, G. Goodman, M. Thornquist, J. Grizzle, L. Rosenstock,

S. Barnhart, J. Balmes, M.G. Cherniack, M.R. Cullen, A. Glass, The

beta-carotene and retinol efficacy trial (CARET) for chemoprevention

of lung cancer in high risk populations: smokers and asbestos-exposed

workers, Cancer Res. 54 (1994) 2038s–2043s.

[14] N.R. Cook, I.M. Le, J.E. Manson, J.E. Buring, C.H. Hennekens,

Effects of beta-carotene supplementation on cancer incidence by

baseline characteristics in the physicians’ health study (United States),

Cancer Causes Control 11 (2000) 617–626.

[15] D.S. Goodman, R. Blomstrand, B. Werner, H.S. Huang, T. Shiratori,

The intestinal absorption and metabolism of vitamin A and beta-

carotene in man, J. Clin. Invest. 45 (1966) 1615–1623.

[16] E.A. Jaffe, R.L. Nachman, C.G. Becker, C.R. Minick, Culture of

human endothelial cells derived from umbilical veins. Identification

by morphologic and immunologic criteria, J. Clin. Invest. 52 (1973)

2745–2756.

[17] G. Plickert, M. Kroiher, Proliferation kinetics and cell lineages can be

studied in whole mounts and macerates by means of BrdU/anti-BrdU

technique, Development 103 (1988) 791–794.

[18] F. Wang, J.R. Van Brocklyn, J.P. Hobson, S. Movafagh, Z.

Zukowska-Grojec, S. Milstien, S. Spiegel, Sphingosine 1-phosphate

stimulates cell migration through a G(i)-coupled cell surface receptor.

Potential involvement in angiogenesis, J. Biol. Chem. 274 (1999)

35343–35350.

[19] B. Vailhe, D. Vittet, J.J. Feige, In vitro models of vasculogenesis and

angiogenesis, Lab. Invest. 81 (2001) 439–452.

[20] M. Bradford, A rapid and sensitive method for the quantitation of

microgram quantities of protein utilizing the principle of protein-dye

binding, Anal. Biochem. 72 (1976) 248–254.

[21] P. Chomczynski, N. Sacchi, Single-step method of RNA isolation by

acid guanidinium thiocyanate-thiocyanatephenol-chloroform extrac-

tion, Anal. Biochem. 162 (1987) 156–159.

[22] M.W. Pfaffl, A new mathematical model for relative quantification in

real-time RT-PCR, Nucleic Acids Res. 29 (2001) 9–45.

[23] T. Asahara, H. Masuda, T. Takahashi, C. Kalka, C. Pastore, M. Silver,

M. Kearne, M. Magner, J.M. Isner, Bone marrow origin of endothelial

progenitor cells responsible for postnatal vasculogenesis in physio-

logical and pathological neovascularization, Circ. Res. 85 (1999)

221–228.

[24] R.N. Eisenman, Deconstructing myc, Genes Dev. 15 (2001)

2023–2030.

[25] D.R. Schultz, W.J. Harrington Jr., Apoptosis: programmed cell death

at a molecular level, Semin. Arthritis Rheum. 32 (2003) 345–369.

[26] B.R. Alevriadou, CAMs and Rho small GTPases: gatekeepers for

leukocyte transendothelial migration. Focus on bVCAM-1-medi-

ated Rac signaling controls endothelial cell–cell contacts and

leukocyte transmigrationQ, Am. J. Physiol., Cell Physiol. 285

(2003) C250–C252.

[27] M. Anghelina, A. Schmeisser, P. Krishnan, L. Moldovan, R.H.

Strasser, N.I. Moldovan, Migration of monocytes/macrophages in

vitro and in vivo is accompanied by MMP12-dependent tunnel

formation and by neovascularization, Cold Spring Harbor Symp.

Quant. Biol. 67 (2002) 209–215.

[28] B.L. Tang, ADAMTS: a novel family of extracellular matrix

proteases, Int. J. Biochem. Cell Biol. 33 (2001) 33–44.

[29] R. Henschler, A. Piiper, R. Bistrian, D. Mobest, SDF-1alpha-induced

intracellular calcium transient involves Rho GTPase signalling and is

required for migration of hematopoietic progenitor cells, Biochem.

Biophys. Res. Commun. 311 (2003) 1067–1071.

[30] I.U. Schraufstatter, K. Trieu, M. Zhao, D.M. Rose, R.A. Terkeltaub,

M. Burger, IL-8-mediated cell migration in endothelial cells depends

on cathepsin B activity and transactivation of the epidermal growth

factor receptor, J. Immunol. 171 (2003) 6714–6722.

[31] T.S. Lewis, P.S. Shapiro, N.G. Ahn, Signal transduction through MAP

kinase cascades, Adv. Cancer Res. 74 (1998) 49–139.

[32] R.G. Fahmy, C.R. Dass, L.Q. Sun, C.N. Chesterman, L.M.

Khachigian, Transcription factor Egr-1 supports FGF-dependent

angiogenesis during neovascularization and tumor growth, Nat.

Med. 9 (2003) 1026–1032.

[33] D.H. Gorski, A.J. Leal, Inhibition of endothelial cell activation by the

homeobox gene Gax, J. Surg. Res. 111 (2003) 91–99.

[34] P.D. Fraser, P.M. Bramley, The biosynthesis and nutritional uses of

carotenoids, Prog. Lipid Res. 43 (2004) 228–265.

[35] F.J. Schweigert, Metabolism of carotenoids in mammals, in: G.

Britton, S. Liaaen-Jensen, H. Pfandern (Eds.), Carotenoids, Biosyn-

thesis, vol. 3, Birkh7user Verlag, Basel, 1998, pp. 249–284.[36] D.V. Crabtree, A.J. Adler, Is beta-carotene an antioxidant? Med.

Hypotheses 48 (1997) 183–187.

[37] S.C. Bell, A. Avila, R. Salazar, K.J. Bayless, S. Kanagala, S.A.

Maxwell, G.E. Davis, Differential gene expression during capillary

morphogenesis in 3D collagen matrices: regulated expression of genes

involved in basement membrane matrix assembly, cell cycle

progression, cellular differentiation and G-protein signaling, J. Cell.

Sci. 114 (2001) 2755–2773.

[38] J. Kahn, F. Mahraban, G. Ingle, X. Xin, J.E. Bryant, G. Vehar, J.

Schoenfeld, C.J. Grimaldi, F. Peale, A. Draksharapu, D.A. Lewin,


M.E. Gerritsen, Gene expression profiling in in vitro model of

angiogenesis, Am. J. Pathol. 156 (2000) 1887–1900.

[39] P. Kastner, N. Messaddeq, M. Mark, O. Wendiing, J.M. Grondona, S.

Ward, N. Ghyselinck, P. Chambon, Vitamin A deficiency and

mutations of RXR alpha, RXR beta and RAR alpha lead to the early

differentiation of embryonic ventricular cardiomyocytes, Develop-

ment 124 (1997) 4749–4758.

[40] M. Zile, Function of Vit A in vertebrate development, J. Nutr. 131

(2001) 705–708.

[41] L. Lai, B.L. Bohnsack, K. Niederreither, K.K. Hirschi, Retinoic

acid regulates endothelial cell proliferation during vasculogenesis,

Development 130 (2004) 6465–6474.

[42] R.D. Erwert, K.T. Eiting, J.C. Tupper, R.K. Winn, J.M. Harlan, D.D.

Bannerman, Shiga toxin induces decreased expression of the anti-

apoptotic protein Mcl-1 concomitant with the onset of endothelial

apoptosis, Microb. Pathog. 35 (2003) 87–93.

[43] B.B. Aggarwal, U. Bhardwaj, Y. Takada, Regulation of TRAIL-

induced apoptosis by ectopic expression of antiapoptotic factors,

Vitam. Horm. 67 (2004) 453–483.

[44] A. Collen, R. Hanemaaijer, F. Lupu, P.H. Quax, N. van Lent, J.

Grimbergen, E. Peters, P. Koolwijk, V.W. van Hinsbergh, Membrane-

type matrix metalloproteinase-mediated angiogenesis in a fibrin-

collagen matrix, Blood 101 (2003) 1810–1817.

[45] L.A. Cornelius, L.C. Nehring, E. Harding, M. Bolanowski, H.G.

Welgus, D.K. Kobayashi, R.A. Pierce, S.D. Shapiro, Matrix metal-

loproteinases generate angiostatin: effects on neovascularization, J.

Immunol. 161 (1998) 6845–6852.

[46] K.R. Martin, D. Wu, M. Meydani, The effects of carotenoids on the

expression of cell surface adhesion molecules and binding of

monocytes to human aortic endothelial cells, Atherosclerosis (2000)

265–274.

[47] J. Sottile, Regulation of angiogenesis by extracellular matrix,

Biochim. Biophys. Acta 1654 (2004) 13–22.

[48] C. Menager, J. Vassy, C. Doliger, Y. Legrand, A. Karniguian,

Subcellular localization of RhoA and ezrin at membrane ruffles of

human endothelial cells: differential role of collagen and fibronectin,

Exp. Cell Res. 249 (1999) 221–230.

[49] E.S. Wijelath, S. Rahman, J. Murray, Y. Patel, G. Savidge, M.

Sobel, Fibronectin promotes VEGF-VEGFinduced CD34 cell

differentiation into endothelial cells, J. Vasc. Surg. 39 (2004)

655–660.

[50] K. Kawasaki, R.S. Smith Jr., C.M. Hsieh, J. Sun, J. Chao, J.K. Liao,

Activation of the phosphatidylinositol 3-kinase/protein kinase Akt

pathway mediates nitric oxide-induced endothelial cell migration and

angiogenesis, Mol. Cell. Biol. 23 (2003) 5726–5737.

[51] Y. Takuva, Subtype-specific differential regulation of Rho family G

proteins and cell migration by the Edg family sphingosine-1-

phosphate receptors, Biochim. Biophys. Acta 1582 (2002) 112–120.

[52] A. Gohla, R. Harhammer, G. Schultz, The G-protein G13 but not

G12 mediates signaling from lysophosphatidic acid receptor via

epidermal growth factor receptor to Rho, J. Biol. Chem. 273 (1998)

4653–4659.

[53] J.W. Lee, R. Juliano, Mitogenic signal transduction by integrin- and

growth factor receptor-mediated pathways, Mol. Cells 17 (2004)

188–202.

[54] M.V. Hoang, M.C. Whelan, D.R. Senger, Rho activity critically and

selectively regulates endothelial cell organization during angiogenesis,

Proc. Natl. Acad. Sci. U. S. A. 101 (2004) 1874–1879.

[55] G.P. van Nieuw Amerongen, V.W. van Hinsbergh, Cytoskeletal

effects of rho-like small guanine nucleotide-binding proteins in

the vascular system, Arterioscler. Thromb. Vasc. Biol. 21 (2001)

300–311.