Beta-Carotene and Arachidonic Acid-regulated geneexpression in human endothelial cell progenitors

( the microarray results)

Dembińska-Kieć Aldona

Department of Clinical BiochemistryThe Jagiellonian University Medical College,

Krakow/POLAND

Department of Clinical BiochemistryThe Jagiellonian University, Medical College

Cell culture and agiogenesisLab

Biochemistry Lab

Out Patient Clinic of LipidDisorders and Obesity

Molecular biology and genetics Lab

Anna PolusUrszula CiałowiczUrszula Rażny

Urszula Jarosz

Jadwiga Hartwich

Małgorzata Malczewska-MalecIwona Leszczyńska-GołąbekŁukasz PartykaAgata JabrockaMarcin TrzosAneta SobestoZofia Bacz-Gorgoń

Iwona Wybrańska

Magdalena Szopa

Marek BodziochBeata Kieć-Wilk Katarzyna Łapicka-BodziochMałgorzata Strzałka Magdalena Żuczek

Prof. dr hab. med. Aldona Dembińska-Kieć

Anna Zdzienicka Małgorzata KwaśniakAnna Gruca

Joanna Góralska

Office: Katarzyna Sołtys;

Joanna GrzybowskaMagdalena MikołajczykAleksandra KrukiewiczUrszula Czech Katarzyna Koso

Scientific cooperation:Gerd Schmitz Regensburg (Germany),Jaap Keijer Wageningen (The Netherlands)Saverio Cinti (Italy)Karsten Kristansen Odense (Denmark),Regina Goralczyk (Roche /DSM) Switzerland)Andreu Palou ( Spain)Jan Needergaard, Barbara Cannon (Sweden)EU Commission: Prof. Jim Leslie; Dr Jean Marc Chorrout

Prof. Julian Pryjma,** Department of Immunology,Faculty of Biotechnology Jagiellonian University, Kraków, Poland

Background mouse

Flk-1/lacZ mouseTie-2lacZ mouse

Transgenic mouse ofendothelial specific promoterregulated LacZ

BMtransplant

Subletal BMirradiation

4 weeks

Tumor growth Wound healing Myocardial infarction Hindlimb ischemia Ovarian cycle Cornea

neovascularisation

Other somatic cellslacZ (-)

BM derived cells

Non-endotheliallineage cell

Endotheliallineage cell

lacZ (-) lacZ (+)

FLK-1(-)Tie-2(-)

FLK-1(+)Tie-2(+-)

Isner,& Asahara, 1999

Y. Sato/Pharmacology&Therapeutics 87 (2000) 51-60

Some of factors regulating endothelial cell differentiation

VEGF-mediated signal

Angiopoietin-mediated signal

Vasculogenesis AngiogenesisVascular stabilization

(remodeling)

HIFSCL/tal-1HEX

H IF -1IdHOX D3 i HOX B3ETS-1TEL

PEBP2/CBFMEF2CCOUP-TFII

PPAR!

HOXB3LKLFMEF2C

Perycites

differentiation (bFGF,Tie2)

etc.

etc.EDF-1etc.

tran

scrip

tion

fact

ors

Cell membrane

Nucleus

Cytoplasm

PPARα ; PPAR β/δ; PPARγLigands (FFA)

RAR/RXRLigands ( retinoids)

9-cis RA

RXR

RARCA/GA/GA/TCT→(N)1,2←AGGTCA Target gene

sequence

PPRE

PPARPPARαα PPARPPARββ//δδ PPARPPARγγ

·lipid metabolism β-oxidation ( liver) ·anti-inflammatory * cellular

differentiation*muscle lipid utylisation (UCP-1)* Insulin sensitivity

•insulin sensitizers;•cell ( adipocyte) differentiation•anti-inflammatory•anti-angiogenic

PPARs

all-transRA

Studies indicated that retinoid treatment is connectedwith angiogenesis inhibition and decreased vascular

response in vitro nad in vivo.•Lotan et al.. Suppression of melanoma cell motility factor receptorexpression by retinoic acid. Cancer Res.1992;52; 4878-84

•Pienta et al. Treatment of prostate cancer in the rat with the syntheticretinoid fenretinide.Cancer res. 1993;53;224-6

•Majewski et al. Synergistic effect of retinoids nad interferon alpha ontumor-induced angiogenesis: an antiangiogenic effect on hpv-harboringtumor cells.Int J.Cancer;1994;; 57;81-5

•Lingen et al.. Retinoid acid induces cells cultured from oral squamouscell carcinomas to become anti-angiogenic. Am.J.Pathol.1996;149; 247-48

•Ferrari et al. Inhibition of Kaposi’s sarcoma in vivo by fenretinide ClinCancer Res. 2003;9;6020-9

•Ribatti et al. Inhibition of neuroblasyoma-induced angiogenesis byfenretinide.Int J.Cancer 2001;94;314-21

Studies indicated that retinoid treatment is connectedwith activation of angiogenesis in vitro nad in vivo.

•Gaetano et al..Retinoids induce Fibroblast Growth factor-2 production in endothelial cells via Retinoic Receptor α activation and stimulate angiogenesis in vitro nad in vivo. Circ Res 2001;88;e38- e47•Lansink et al.. Effect of steroid hoirmones nad retinoids on the formation of capilary-like tubular structures of human microvascular endothelial cells in fibrin matrices is related to urokinase expression. Blood; 1998; 92; 927-38•Neuville et all.. Retinoic acid regulates arerial smooth muscle cell proliferation nad phenotypic features in vivo nad in vitro through an RAR-α-dependent signalling pathway. Arterioscler. Thromb. Vasc. Biol. 1999;19;1430-36. Suzuki et al.. Physical interaction between retinoic acid receptor and Sp1:mechanism for induction of urokibnase by retinoic acid. Blood; 1999;93;4264-76•Schwarz et al.. Retinoid nad carotenoid angiogenesis. A possible explanation for enhanced oral carcinogenesis. Nutr.Cancer. 1997;27; 192-9

Objectives:

• 1. Search for the regulatory role of theexogenous β-carotene in the differentiation ofendothelial progenitor cells ( EPC) and HUVEC

• 2. Identification of mechanisms (genes) which areup- or down-regulated in the presence of β-carotene in angiogenesis

Schemat of presentation:

• 1. Influence of the β-carotene in the meanproangiogenic activities of endothelial progenitor ( EPC)as well HUVEC cells ( proliferation; chemotaxis; homing)

• 2. Identification of the important for angiogenesis ofmechanisms (genes) which are up- or down-regulated inthe presence of β-carotene (microarray)

• 3. β-carotene in vivo angiogenesis model (mice , ferretadipose tissue)

Material and Methods HUVEC and human umbilical vein AC133 (Ab/magnetic microbits) progenitors (UPC)

were used . After one week UPC culturing (EBM medium plus VEGF (50ng/ml) and SCF(100ng/ml) and antibiotics (penicillin 50 U/ml, streptomycin 50U/ml) (Sigma),endothelial progenitors: EPC (CD34+, VE-catherin+, AC133+, KDR+, flow cytometry)cells were incubated with β-carotene (0,3-3uM), without or in the presence of 3µM ofpalmitic (PA) ; linoleic (LA) and arachidonic (AA) or ciglitazone (10uM) for 24hrs.

Beta-carotene uptake was controlled by HPLC Cytotoxicity of investigated factors was measured after 24 hrs of incubation using

colorimetric method LDH . Gene expression:Total RNA was isolated by guanidine thiocyanate extraction using

Trizol (Sigma). The high grade purity RNA was used for oligonucleotide microarray (Affymetrix HG-

U133a), 14 500 genes, and mouse oligo microarray 10 000 genes.. Microchip analysis(using www.cbrc.jp; www.dbtss.hgc.jp)

The regulation of gene (Notch-1, Notch-2, Notch-4, HoxB3, HoxD3, CD36, Jagged-1,KDR, CD34, AC133, PPARγ, vWF, eNOS) was confirmed by RT-PCR and Real-Time PCRmethod (Opticon MJ-Research) carried out with 500ng of total RNA. Product wasdetermined and quantified by monitoring real-time fluorescence signal from SYBRGreen bound to double-stranded DNA.

Proliferation was established by measuring BrdU incorporation into DNA. Cells wereincubated with factors for 24 hours.

Protein synthesis (receptors, antigens) was confirmed by flow cytometry and WesternBlot analysis

Chemotaxis: was performed in Boyden chamber calculated by FACS or byimmunoassay (PECAM) measurement.

Proangiogenic activity: (the tubule formation in 3D matrigel model in vitro); as well asin mouse sc matrigel pads in vivo

Time- and concentration-dependent uptake ofbeta- carotene by HUVEC (HPLC)

0

50

100

150

200

control THF/EtOH 0,3uM 1uM 3uM beta

caro

tene

pm

ol/

10

6

cells 24h

48h

72h

Fatty acids increased beta-carotene uptake byHUVEC ( 24h, HPLC)

BC FA

PA

0

10

20

30

40

50

60

70

80

control THF/EtOH BC 3uM PA 1uM PA 1uM

/BC 3uM

PA 3uM PA 3uM

/BC 3uM

Bet

a-ca

rote

ne p

mol

/106

cel

ls

AA

01020304050607080

contro

l

TH

F/EtO

H

BC 3

uM

AA

1uM

AA

1uM

/BC 3

uM

AA

3uM

AA

3uM

/BC 3

uM

Bet

a-ca

rote

ne p

mol

/106

cel

ls

LA

0

10

20

30

40

50

60

70

80

control THF/EtOH BC 3uM LA 1uM LA 1uM

/BC 3uM

LA 3uM LA 3uM

/BC 3uM

Bet

a-ca

rote

ne p

mol

/106

cel

ls

cooperation with R Goralczyk, Roche Vitamins/ DSM.

The appearance of Notch-4; VE-Cadherin;PECAM; eNOS gene expression in umbilical

cord EPC (RT-PCR).

UPC

EPC

HUVEC

Mar

ker

Jag

ged

-1

No

tch

-1

No

tch

-2

No

tch

-4

Ho

xD3

Ho

xB3

PP

ARγ

CD

34

AC

13

3

CD

36

vWF

VE

GFR

1

VE

GFR

2

eNO

S

VE

-cad

her

in

PE

CA

M

0

20

40

60

80

100

120

1 day 2 day 5 day 8 day 12 day 14 day

%

of

posi

tive

cells

AC133

CD34

CD14/45

VE-cadherin

KDR

Endothelial progenitors EPC: Maturation of UPC AC133+cells during incubation with 100ng/ml SCF and 50ng/ml VEGFin time(Flow cytometry.Percent of positive cells)

Lack of proapoptotic effect of beta-caroteneand AA in EPC and HUVEC (24h, cytochrome B assay; n=5-10 )

0

20

40

60

80

100

120

140

160

180

200

control THF/EtOH AA 3 uM BC 3uM BC 3uM/AA

3uM

Staurosporine

% o

f con

trol

0

20

40

60

80

100

120

140

160

180

200

control THF/EtOH AA 3 uM BC 3uM BC 3uM/AA

3uM

Staurosporine

% o

f con

trol

EPC

HUVEC

0

50

100

150

200

250

300

350

control VEGF

0,2nM

bFGF

0,5nM

ciglitazone

1uM

ciglitazone

3uM

ciglitazone

10uM

ciglitazone

30uM

%

of

control

Beta-carotene and PPARagonists (FFA, ciglitazone)do not significantlyinfluence proliferation ofEPC/HUVEC

0

50

100

150

200

250

300

350

control VEGF

0,2nM

bFGF

0,5nM

ciglitazone

1uM

ciglitazone

3uM

ciglitazone

10uM

ciglitazone

30uM

%

of

contro

l

EPC HUVEC

0.0

50.0

100.0

150.0

200.0

250.0

300.0

control VEGF

0.2nM

bFGF

0.5nM

BC 3uM PA 3uM AA 3uM LA 3uM

%

of

control

+BC +BC +BC

0

2

4

6

8

10

12

14

control THF/EtOH BC 3uM S1P 500nM

CH

in

de

x

HUVEC

EPC

* *

*** ***

0

2

4

6

8

10

12

14

control THF/EtOH BC 3uM S1P 500nM

CH

in

de

x

HUVEC

EPC

* *

* * * * * *

β-carotene induced chemotaxis of EPC nad HUVEC (Sphingosine-1-phosphate (S1P) as the positive control CHI= ratio of cells in Boyden’s camera chambers)) n=6-10; ** 0.01>p<*0.05

Fatty acids potentiate the Beta-carotene induceschemotaxis of EPC and HUVEC

(Boyden Chamber, 24h)

0

1

2

3

4

5

6

7

8

control

THF/E

tOH

BC 3

uM

cigl

itaz

one

10uM

PA 3

uM

PA 3

uM/B

C 3

uM

LA 3

uM

LA 3

uM/B

C 3

uM

AA 3

uM

AA 3

uM/B

C 3

uM

CH

in

dex

0

1

2

3

4

5

6

7

cont

rol

THF/E

tOH

BC 3

uM

cigl

itaz

one

10uM

PA 3

uM

BC 3

uM/P

A 3

uM

LA 3

uM

BC 3

uM/LA 3

uM

AA 3

uM

BC 3

uM/A

A 3

uM

CH

in

dex

EPC

HUVEC

0

1

2

3

4

5

6

7

THF/EtOH VEGF 0.2nM bFGF 0.5nM BC 3uM

mea

n tu

bule

leng

th [m

m]

HUVEC

EPC

bFGF (0,5nM)

control

HUVEC

bFGF (0,5nM)

control

HUVEC

0

1

2

3

4

5

6

7

THF/EtOH VEGF 0.2nM bFGF 0.5nM BC 3uM

mea

n tu

bule

leng

th [m

m]

HUVEC

EPC

bFGF (0,5nM)

control

HUVEC

bFGF (0,5nM)

control

HUVEC

Lack of the tubulogenic activity of β-carotene in theMatrigel model in vitro. ( n = 5-10)

0

1

2

3

4

5

6

7

VEGF 0.2nM bFGF 0.5nM THF/EtOH ATRA 300nM beta-carotene

3uM

arachidonic

acid 3uM

beta-carotene

3uM

+arachidonic

acid 3uM

mea

n tu

bule

leng

th [m

m]

+VEGF

Lack of tubulogenic activity of investigated factors onthe mean length sum of tubular structures (in vitro

model of angiogenesis)(HUVEC + VEGF)

Analysis of gene expression after 24h incubation of HUVECwith beta-carotene using oligonucleotide microarray method

(Affymetrix, HG-U133A)Upregulation of gene proteins connected with adhesion, cell motility, cell shapes.a.: kinesin-like5,TSP-1, PECAM1, integrin alpha4, alpha6, beta5, JAM2, MMP14, tPA, caldesmon 1, G protein signalling pathway

Downregulation of gene responsible forproapoptotic proteins caspase6, cell-death regulatory protein 19(GRIM 19), CD27-binding (SIVA) protein (inhibition of apoptosis)TGFb signaling pathway, FGFR1, fibronectin, laminin, cadherins,catenin (inhibition of differentiation)

2

2

AA BC BC/AAup-regulation 74 6 7 136

down-regulation 65 7 0 5 6

AA BC AA/BC

proliferation

S-phase

MCM6 1.7 1.7 1.9

MCM2 1.6 1.5 1.7

MCM5 1.4 1.5 1.3

MCM3 1.5 1.4 1.5

MCM3AP NC 1.4 NC

CDC34 1.7 1.3 1.6

MCM7 1.1 1.2 1.4

G1/S

CDK7 1.4 1.6 1.4

BCCIP 1.7 1.5 NC

CHEK1 1.6 1.5 1.6

CDC34 1.7 1.3 1.6

CUL1 1.5 1.3 NC

GSPT1 NC 1.2 1.5

CEB1 -1.5 -1.2 -1.3

CDKN2C -1.7 -1.6 NC

CDKN3 NC -1.6 NC

CKS1B NC -1.6 -1.5

CDC25C -1.5 -1.9 -1.5

CDKN1C -2.1 -2.5 -2.1

CCNA1 -6.5 -5.7 NC

G2/M

CDK7 1.4 1.6 1.4

BCCIP 1.7 1.5 NC

CHEK1 1.6 1.5 1.6

CEB1 -1.5 -1.2 -1.3

CKS1B NC -1.6 -1.5

CDKN3 NC -1.6 NC

CCNB2 NC -1.6 -1.6

CDC25C -1.5 -1.9 -1.5

RGC32 -1.7 -2.1 -1.7

CDKN1C -2.1 -2.5 -2.1

CCNA1 -6.5 -5.7 NC

cell cy cle check point

MAD1L1 24.3 34.3 7.0

NBS1 2.0 1.7 1.9

ATR 1.4 1.5 1.5

CHEK1 1.6 1.5 1.6

RB1 NC 1.4 NC

AA BC AA/BC

chemotaxis

cy tokines

CCL2 68.6 26.0 26.0

AQP3 9.2 13.0 18.4

IL8 8.0 3.7 6.1

LEP 2.5 2.8 2.5

CCL4 2.8 2.6 2.1

CCRL2 1.7 2.3 1.9

ANGPT1 2.3 2.1 2.3

CCL3 1.9 1.5 NC

SCGF -2.0 -2.3 -2.6

VEGF -2.6 -2.5 -2.6

CCL18 -3.0 -3.5 -3.5

CXCL2 4.0 2.3 2.8

ECGF1 2.1 1.5 1.4

IL1A NC -1.7 NC

IL1B 1.6 1.5 NC

receptors

NRP1 3.0 4.3 2.5

CCR7 4.0 3.7 5.3

CCR2 -1.9 -1.5 -1.6

DTR NC 2.6 NC

IL1RN -3.0 -2.8 -2.6

adhesion

cell-cell adhesion

CD69 3.5 3.0 3.5

SIGLEC7 2.3 2.5 1.9

PRG2 2.8 2.3 2.6

DSG2 NC 2.0 NC

LGALS8 1.7 1.9 NC

CD2 NC 1.5 1.5

SN NC 1.5 NC

AIM1 -1.9 -1.5 -1.9

FXYD5 NC -1.5 -2.0

ICAM3 NC -1.5 -1.6

JAG1 -2.0 -1.9 -1.5

DLG5 NC -2.0 -2.6

LGALS1 -1.5 -2.1 -2.1

CSPG2 -2.5 -2.3 -1.7

cell-matrix adhesion

CD36 1.4 1.6 1.3

PPFIA1 1.4 1.5 NC

SN NC 1.5 NC

ITGA4 NC 1.4 NC

AA BC AA/BC

apoptosis

regulation of programed cell death

TOSO 14.9 13.0 9.2

SNCA 5.7 4.3 4.0

HD NC 4.0 2.5

BIRC3 3.5 3.5 4.6

GRIM19 2.8 2.1 1.7

PDCD8 1.9 2.1 1.7

STK17A 1.5 1.9 2.1

MCL1 2.0 1.9 1.5

EI24 1.3 1.6 1.5

API5 NC 1.4 NC

IER3 NC 1.4 1.9

TIA1 NC 1.4 1.4

TRAF3 NC 1.4 NC

CUL1 1.5 1.3 NC

PLAGL1 NC -1.5 -1.6

ASC NC -1.6 NC

MTL5 NC -1.7 NC

TNFRSF6 -1.5 -1.9 -1.6

TNFSF10 -3.2 -2.0 NC

mitochondrial dependent apoptosis

BCL2L1 2.0 2.3 2.3

CYCS 2.5 2.0 2.0

BECN1 1.9 2.0 1.4

BNIP3L 1.7 1.7 NC

BAG1 NC 1.6 1.7

VDAC1 NC 1.6 NC

AZU1 NC -1.9 -2.1

BNIP3 -4.9 -3.5 -3.2

caspase

STAT1 1.5 1.7 1.9

CASP3 1.5 1.7 NC

CASP8AP2 NC 1.6 NC

CASP4 NC -1.5 -1.5

AZU1 NC -1.9 -2.1

AA BC AA/BC

extracellular matrix

LTF 4.0 4.9 4.9

THBS1 5.7 3.0 4.0

SPOCK2 2.5 2.3 2.1

COL6A3 NC 2.0 NC

FGL2 1.9 1.9 1.7

LGALS8 1.7 1.9 NC

SCYE1 2.1 1.9 1.9

FSTL1 1.7 1.7 NC

AGL NC 1.7 NC

MFNG NC 1.6 NC

LYN 1.5 1.5 1.5

AKR1B1 1.6 1.5 1.4

IDE NC 1.5 1.3

ADM NC 1.4 1.2

PSAP 1.2 1.4 NC

TRAPPC4 1.5 1.4 1.4

S100A8 NC -1.4 NC

TPT1 NC -1.4 NC

FBN2 -2.3 -1.5 NC

FYN -1.3 -1.5 NC

SYNGR1 -1.3 -1.6 NC

SPARC -2.0 -1.7 -1.9

LAMC1 -2.3 -1.9 -1.7

C5 -2.3 -1.9 -2.3

AMY2A -2.5 -2.1 -2.0

CSPG2 -2.5 -2.3 -1.7

VAMP1 -2.3 -2.3 -2.5

CEACAM8 -3.0 -2.5 -2.0

SPP1 -3.7 -5.3 -7.0

extracellular proteases

MMP9 3.2 2.8 2.5

ZMPSTE24 1.7 1.4 NC

TIMP1 1.9 1.2 1.5

PLAU -2.3 -1.9 -1.7

MMP2 -2.6 -2.0 -1.9

ADAM28 -1.7 -2.6 -1.4

AA BC AA/BC

cy toskeleton

TBCE 4.9 4.3 NC

KRT1 3.7 3.2 3.5

MARCKS 1.9 2.3 1.3

SPTA1 NC 2.3 3.2

BAF53A 2.3 2.0 1.7

BIN1 2.5 2.0 NC

MYH10 1.9 2.0 2.5

ACTR6 2.3 1.9 1.3

CAPG 2.3 1.9 NC

PFN1 2.5 1.9 2.0

TPM1 1.7 1.9 NC

ACTR2 2.8 1.7 1.2

CAPZA2 1.6 1.7 1.6

CD2AP 1.4 1.7 NC

PTK9 1.7 1.7 1.5

ACTR1A 1.5 1.5 NC

APPBP2 NC 1.5 NC

ARPC3 1.7 1.5 NC

PDLIM1 1.2 1.5 1.5

TUBG1 1.9 1.5 1.4

ACTR3 NC 1.4 1.6

ARHGAP6 NC 1.4 NC

CORO1C NC 1.4 NC

CSPG6 NC 1.4 NC

MAP7 NC 1.4 NC

CAPZB 1.6 1.3 1.3

CKAP1 1.5 1.3 1.3

GAPCENA -1.4 1.3 NC

ARPC2 1.4 1.2 1.2

ACTB 1.6 1.1 NC

DNCL1 1.4 1.1 NC

ACTG1 NC -1.4 -1.3

CENPF NC -1.4 NC

TNS -2.0 -1.4 -1.7

ESPL1 -1.9 -1.5 -1.4

KIF14 NC -1.5 NC

KNS2 -1.6 -1.5 -1.5

MAP4 -1.4 -1.5 NC

AKAP9 NC -1.6 NC

STK6 -1.7 -1.6 -1.5

TPX2 NC -1.6 -1.6

WASF1 -1.5 -1.6 NC

DNAL4 NC -1.7 NC

GSN -1.6 -1.7 -1.6

PRC1 -2.5 -2.0 -1.7

KIF20A -1.7 -2.1 -1.7

CHS1 -1.9 -2.3 -2.0

NEDD9 -3.5 -2.6 -2.0

BC and AA regulated genes in HUVEC(microarray results)

- up-regulation

- down-regulation

Microarray analysis (HUVEC)β-carotene activated expression of genes connected with cell growth, cell cycle,adhesion, cell-cell signaling, chemotaxis , when inhibited genes connected withapoptosis

angiogenesis

Proliferation/differentiation

xenobioticmetabolism

VEGF Receptor 2angiopoietin 2endoglinNOS- 3KDRintegrinsmetalloproteinasescadherinscatenins

WNT signalingMAPK

retinoic acid repressible protein,

HIF-1 responsive RTP801

cytochrome P450

retinoid metabolizing protein

heme oxygenase- 1

chemotactic activityCXCR4 IL-8

cells survival IAP

-4.0

-3.0

-2.0

-1.0

0.0

1.0

2.0

3.0

4.0

5.0

CXCR4 IL8 VCAM1 MAD1L1 MEOX2 EGR1 BIRC5

rela

tiv

e g

en

e e

xp

res

sio

n

THF/EtOH

AA 3uM

BC 3uM

BC 3uM/AA3uM

In HUVEC•beta-carotene and AA inhibit integrity of endothelium(↓VCAM,Connexin 43)• stimulate chemtaxis (↑ IL-8), promote homing (CXCR4)• ↑ transcription factors regulating expression of genes of proteins involved in in cytoskeletal protein activation ( cellular transport, mobility) (qRT-PCR)

Polus,Grzybowska,Dembinska-Kieć, Kieć-Wilk 2004 ( 5 FW ; EU) DLARFID

Analysis of gene expression after 24h incubation of EPCwith beta-carotene using oligonucleotide microarray method

(Affymetrix, HG-U133A)Upregulation of gene proteins connected with adhesion, cell motility, cell shapes.a.: VEGF Receptor AQP3, NRP1,EGR-1, GATA2, CD36, thrombospondin1, angiopoietin 1, metalloproteinasescadherins, integrins, G protein signalling pathway

Downregulation of gene responsible for adhesion ICAM,CEACAM, ALCAM, SELP, CXCR4, VEGFproapoptotic proteins caspase4, BNIP3, BCL2L1,BIRC3 (antiapoptotic), proliferation CCNA1,

2

2

AA BC BC/AAup-regulation 1224 1140 852

down-regulation 990 676 713

AA BC AA/BC

cytokines sygnalling

CCL2 68,6 26,0 26,0

IL7R 9,8 6,5 9,2

CCR7 4,0 3,7 5,3

CD69 3,5 3,0 3,5

CCL4 2,8 2,6 2,1

CD3D 2,3 2,3 2,5

CCRL2 1,7 2,3 1,9

CXCL2 4,0 2,3 2,8

SOCS2 1,9 2,1 2,6

TLR4 NC 2,0 1,4

CD14 3,0 1,9 1,6

CXCL12 1,5 1,9 NC

IGSF6 1,6 1,7 NC

TLR1 1,6 1,7 1,4

IFITM1 2,5 1,6 1,9

CD2 NC 1,5 1,5

SOCS4 NC 1,5 1,5

CCL3 1,9 1,5 NC

LY96 1,4 1,1 NC

CD97 -1,7 -1,6 -1,6

CCL18 -3,0 -3,5 -3,5

AA BC AA/BC

receptors

GPCR

CCL2 68,6 26,0 26,0

H963 21,1 24,3 22,6

CNR1 NC 7,5 7,0

CCR7 4,0 3,7 5,3

IL8 8,0 3,7 6,1

ADORA2A NC 2,6 NC

CCRL2 1,7 2,3 1,9

CXCL2 4,0 2,3 2,8

GPRK6 1,4 2,3 1,9

C5R1 2,1 2,0 1,4

PTGER2 NC 2,0 1,7

CXCL12 1,5 1,9 NC

GPR65 1,9 1,9 1,7

GPRK5 NC 1,9 NC

F2RL1/PAR2 NC 1,7 NC

CCL3 1,9 1,5 NC

PTAFR 1,6 1,5 1,3

CMKLR1 1,4 1,2 NC

CXCR4 -1,5 -1,3 NC

CCR2 -1,9 -1,5 -1,6

GPR27 NC -1,5 NC

CD97 -1,7 -1,6 -1,6

P2RY2 NC -1,6 NC

PTGER4 -2,0 -1,7 -2,0

AZU1 NC -1,9 -2,1

C5 -2,3 -1,9 -2,3

ADRB2 -3,5 -2,0 -2,3

FZD2 -1,6 -2,1 -1,6

insulin receptor pathway

SOCS2 1,9 2,1 2,6

IGF1 NC 1,4 NC

GRB10 -1,9 -1,6 -1,6

INSR -2,5 -1,7 -2,0

VEGF receptor pathway

KIT -1,2 -1,5 -1,3

FLT3 -2,1 -1,7 -2,0

FGF receptor pathway

FGFR1 -1,6 NC NC

ECGF1 2,1 1,5 1,4

Jagged/Notch pathway

PSEN2 NC 1,5 NC

JAG1 -2,0 -1,9 -1,5

WNT pathway

APC 1,6 2,0 1,9

PPP2R5E 1,4 1,3 1,4

LDLR -2,1 -1,6 -1,6

FZD2 -1,6 -2,1 -1,6

TCR receptor

LCK 18,4 9,2 NC

AA BC AA/BC

intracellular signalling

Ras superfamily

RASGRP1 21,1 8,0 8,6

RRAS2 1,5 2,3 1,5

RAB1A 2,1 2,3 1,4

NRAS 1,6 2,1 1,6

RAB20 2,0 2,1 1,9

RAB7L1 1,4 2,0 1,5

ERBB2IP 1,7 1,9 1,2

RGL 1,6 1,9 2,0

RIT1 2,0 1,7 NC

SARA2 1,2 1,6 NC

RAN 2,0 1,6 1,5

ARF3 1,7 1,5 1,9

ARF4 1,9 1,5 1,6

RAB14 1,5 1,5 NC

RAB31 NC 1,5 NC

RAP2C NC 1,5 NC

PSCDBP 1,6 1,4 NC

IGF1 NC 1,4 NC

RASGRP3 NC 1,4 NC

ARF6 NC 1,3 1,5

RAB4A -1,7 -1,5 -1,4

RAF1 -1,5 -1,5 -1,3

RGS19 NC -1,5 NC

RAB32 -1,7 -1,7 -1,5

RGS1 -1,6 -1,7 -1,5

RAB27A -1,7 -1,9 -1,7

RAB13 -2,0 -2,0 -2,0

CENTB1 NC -2,1 -1,5

RAB27A -1,6 -2,1 -1,9

RASGRP2 NC -2,3 NC

Rho pathway

RHOBTB1 NC 2,0 1,4

GPRK5 NC 1,9 NC

G3BP2 1,2 1,5 NC

ARHGAP6 NC 1,4 NC

ARHGDIB NC -1,4 -1,2

ARHG -1,3 -1,9 -1,5

Rac pathway

CHN2 NC 1,4 NC

VAV3 NC 1,4 1,3

NCF4 1,9 1,2 NC

ELMO1 -1,6 -1,6 NC

TYK2 NC -1,6 NC

AA BC AA/BC

smal GTPase

RASGRP1 21,1 8,0 8,6

RAB1A 2,1 2,3 1,4

RRAS2 1,5 2,3 1,5

ARL7 2,5 2,1 2,0

RAB20 2,0 2,1 1,9

RAB7L1 1,4 2,0 1,5

RHOBTB1 NC 2,0 1,4

RGL 1,6 1,9 2,0

LCK 18,4 9,2 NC

MAPK

MAP2K1IP1 1,7 1,7 1,2

MAP3K5 -1,5 NC NC

MAP4K1 NC -1,5 NC

MAP4K3 1,4 1,6 NC

MAP4K5 1,2 1,4 NC

MAPK13 NC 2,3 2,3

MAPK14 -1,9 -1,9 -1,6

MAPKAPK2 -1,4 NC NC

phosphoinositide-3-kinase

PIK3C2A -1,4 NC NC

PIK3CB -1,4 NC NC

PIK3R1 NC -1,9 -1,5

phospholipase C

PLCB1 -2,0 -2,0 -2,1

PLCG2 -1,5 -1,4 -1,4

PLCL2 1,6 1,7 1,5

PLD1 -1,9 NC -1,7

protein kinase, cAMP-dependent

PRKAR1A NC 1,6 NC

PRKAR2B 1,9 1,9 1,5

protein kinase C

PRKCA NC NC 1,6

PRKCI -1,9 NC -1,2

PRKCSH 1,4 NC NC

PLEK 2,5 NC NC

NFkB

CHUK 1,5 2,0 1,9

PRDX4 1,4 1,2 1,4

phosphatase

DUSP2 NC NC 11,3

DUSP6 2,5 2,0 1,9

DUSP10 -1,3 -1,4 -1,4

DUSP22 -2,1 -2,1 -1,9

PTPN6 NC -1,9 -1,9

nitric oxide

GUCY1B3 1,2 1,6 1,4

DDAH1 -1,5 NC NC

SH2/SH3

SH2D2A NC NC 1,9

SH3BP2 NC -1,5 NC

AMSH -1,5 NC NC

SRC

YES1 1,5 NC NC

AA BC AA/BC

regulation of transcription

POU2AF1 39.4 32.0 22.6

TOSO 14.9 13.0 9.2

SMCY 4.6 6.1 5.3

FKBP1B 4.6 5.7 NC

SPIB 3.2 5.3 NC

BACH2 2.6 4.3 3.2

BANK1 2.6 3.5 3.2

LEF1 3.0 3.2 3.5

SP140 2.0 2.8 2.1

DTR NC 2.6 NC

E2F6 NC 2.5 2.1

E2F6 NC 2.5 2.1

IF2 2.3 2.3 2.3

MJD NC 2.3 2.0

SLC38A6 1.5 2.3 1.5

GRIM19 2.8 2.1 1.7

GRIM19 2.8 2.1 1.7

CBF2 2.3 2.1 2.1

PC4 1.9 2.1 1.9

WWP1 1.7 2.0 1.6

RNF14 1.5 2.0 NC

MYCN 2.1 2.0 1.6

PBX1 1.9 2.0 NC

HMGB1 2.5 2.0 1.9

CIAO1 NC 2.0 2.8

MAF 2.0 1.9 1.4

EVI1 1.3 1.9 2.3

BACH1 1.6 1.9 1.4

PAWR/PAR4 1.5 1.7 1.3

ZNF131 1.4 1.7 1.5

PAWR/PAR4 1.5 1.7 1.3

KHSRP 1.4 1.7 1.7

CRSP2 1.7 1.7 NC

NFIB NC 1.7 2.5

FOXO1A NC 1.7 NC

NEUGRIN 1.7 1.7 NC

TFAM NC 1.7 NC

STAT1 1.5 1.7 1.9

RPS26 1.3 1.7 1.6

AHR 1.9 1.7 NC

MCM6 1.7 1.7 1.9

CIRBP NC 1.6 1.6

SMARCA4 1.6 1.6 1.9

CDK7 1.4 1.6 1.4

PCAF NC 1.6 NC

ETS2 1.2 1.6 1.4

COLEC12 1.5 1.6 1.4

SMARCE1 NC 1.6 1.6

HTATSF1 1.5 1.6 1.3

PAI-RBP1 1.9 1.6 1.5

SH2BP1 1.6 1.6 NC

TOX 1.3 1.6 NC

HNRPR 1.7 1.6 1.6

PRKAR1A NC 1.6 NC

TAL1 NC 1.6 NC

BAZ1A 1.9 1.6 1.5

SMARCA5 NC 1.6 1.7

MCM5 1.4 1.5 1.3

VDRIP 1.3 1.5 1.4

JUND 1.5 1.5 1.7

POLR2L 2.5 1.5 1.4

MCM2 1.6 1.5 1.7

MBP 1.7 1.5 1.4

TCF4 1.4 1.5 1.4

CEBPG 1.9 1.5 NC

SCAND1 1.4 1.5 NC

EGR1 1.7 1.5 2.1

MYC 2.5 1.5 1.2

ETV5 1.9 1.5 1.3

KIAA0179 NC 1.5 1.5

NRIP1 NC 1.5 NC

FUBP3 NC 1.5 1.4

SHARP 2.6 1.5 NC

HMGN4 NC 1.5 NC

TRIM33 1.4 1.4 1.4

STAT3 1.1 1.4 1.3

RB1 NC 1.4 NC

C20orf104 NC 1.4 NC

RB1 NC 1.4 NC

ZDHHC3 NC 1.4 NC

STAT3 1.1 1.4 1.3

ZNF161 1.4 1.4 NC

RENT1 NC 1.4 NC

U2AF2 NC 1.4 NC

MCM3 1.5 1.4 1.5

SRP46 NC 1.4 NC

ZNF302 1.3 1.4 NC

AA BC AA/BC

metabo lism

arom atic com pound m etabolism

CPM 2.3 2.3 NC

GCH1 2.1 2.3 2.1

MICAL2 1.7 1.9 2.5

KMO 1.9 1.6 1.3

ECGF1 2.1 1.5 1.4

KYNU 1.7 1.5 1.6

UMPS 1.4 1.5 1.4

QDPR 1.3 1.5 1.3

MTHFD1 1.5 1.4 1.7

MGLL 1.5 1.3 1.3

PAICS 1.5 1.3 1.5

GMPS -2.5 -2.1 -2.1

DDT -2.1 -2.5 -3.5

ALDH

ALDH1A1 1.5 2.0 NC

F ABP

FABP5 3.7 2.3 2.1

RDH11 1.9 2.0 NC

PPAR coactiv ator

PRC 1.5 1.6 NC

AhR

AHR 1.9 1.7 NC

RAR

RARRES1 NC 2.0 NC

RARG-1 1.6 1.7 1.6

RARRES3 1.9 1.7 1.9

ZNF42/MZF1 -1.4 -1.6 -1.4

response to xenobiotic stim ulus

AHR 1.9 1.7 NC

AZU1 NC -1.9 -2.1

BPI NC -1.7 NC

DEFA1 4.0 3.2 3.7

DEFA4 3.5 2.1 2.8

NQO1 2.0 NC NC

PRG2 2.8 2.3 2.6

S100A12 2.5 2.1 2.6

Cy p450

CYP20A1 NC 6.1 NC

CYP27A1 2.0 1.9 1.6

CYP1B1 1.4 1.6 1.3

POR 1.9 1.6 1.3

TBXAS1 1.4 1.4 NC

drug

ABCB1 2.0 1.7 NC

CTPS 2.1 2.3 2.5

SEMA3C 1.3 1.9 1.9

toxin

NQO1 2.0 NC NC

MPST NC -1.6 NC

oxidativ e stress

ATOX1 1.6 1.9 1.6

DUSP1 1.7 1.4 1.5

FOXM1 -1.5 NC NC

GPX1 NC -1.6 -1.4

GPX3 NC NC 1.7

MPO -1.5 -1.4 -1.2

MSRA -1.5 NC NC

MTL5 NC -1.7 NC

OSR1 1.4 NC NC

PDLIM1 1.2 1.5 1.5

SOD1 1.4 1.2 1.2

SOD2 2.6 1.9 2.0

xenobiotic m etabolism phase II

glutatione transf erase

GSTA1 -1.5 NC NC

GSTM3 2.6 2.0 2.8

GSTP1 1.4 NC NC

LOC51064 NC NC -1.5

MGST3 NC -1.5 -1.5

sulf otransf erase

CHST6 1.6 NC NC

GALNAC4S-6ST 2.3 4.3 4.0

HS3ST2 1.5 1.7 1.6

SULT1A1 1.5 NC NC

TPST1 -1.7 -2.1 NC

squalene epoxidase

KMO 1.9 1.6 1.3

gluatatione S-transf erase

GSTA1 -1.5 NC NC

GSTA1 -1.5 NC NC

GSTM3 2.6 2.0 2.8

GSTP1 1.4 NC NC

GSTP1 1.4 NC NC

BC and AA regulated genes in EPC(microarray results)

- up-regulation

- down-regulation

M icroarray analysis  (EPC) (Affymetrix, HG-U133A)

β-carotene activated expression of genes connected with cell growth, cell cycle,

adhesion, cell-cell signaling, chemotaxis , when inhibited. genes connected withapoptosis

angiogenesis

Proliferation/differentiation

xenobioticmetabolism

VEGF Receptor AQP3, NRP1EGR-1, GATA2 CD36thrombospondin1angiopoietin 1metalloproteinasescadherinsintegrins

WNT signaling↑MAPK ↑

AhR, Arnt, PRC ↑

HIF-1 A ↑

cytochrome P450 ↑

retinoid metabolizing protein RDH11 ↑

heme oxygenase 1, GPX ↑

chemotactic activity CXCR4 ↓ IL-8 ↑

cells survival IAP↑

Jagged/Notch system is atransmembrane protein receptorcomplex involved in developmentalcell fate.

Interactions Notch-ligand (Jagged-1)may function as a general arbiter ofcell fate, regulating differentiationpotential, rate of proliferation andapoptosis.

Delta

Serrate

Jagged

C-Delta-1

X-Delta-1

APX-1

LAG-1

Notch

LIN-12

GLP-1

EGF LMDSL

Ligands Notch

extracellularintracellular intracellular

VEGF

(! NO)

proliferating and migrating EC

vitronectin

fibrin

Jagged STOP

PROLIFERATION

DIFFERENTIATION

proliferation

(" apoptosis)

bFGF(tube formation)

Antisense oligomers to Jagged

increase tube formation in bFGF-

induced angiogenesis

Zimrin 1996

Jagged

Notch-4

Participation of Jagged/Notch in angiogenesis induced by bFGF

Notch signaling limits the number of cells in particular tissues and leaves someprogenitor cells in adolescence that can adopt alternative fate

The relative Jagged-1 and Notch-4 gene expression (24h,Real-Time PCR) . Beta-carotene down-regulates both geneexpression in EPC and HUVEC . The oposite effect of AAin EPC and HUVEC

-22

-20

-18

-16

-14

-12

-10

-8

-6

-4

-2

0

2

4

THF/EtOH BC 3uM AA 3uM BC/AA 3uM

rela

tive g

ene e

xpre

ssio

n

-14

-12

-10

-8

-6

-4

-2

0

2

THF/EtOH BC 3uM AA 3uM BC/AA 3uM

rela

tive g

ene e

xpre

ssio

n

Jagged-1 Notch-4

-5

-4

-3

-2

-1

0

1

2

3

4

5

THF/EtOH BC 3uM AA 3uM BC/AA 3uM

rela

tive g

ene e

xpre

sio

n

-3

-2

-1

0

1

2

3

4

5

6

7

THF/EtOH BC 3uM AA 3uM BC/AA 3uM

rela

tive g

ene e

xpre

sio

n

EPC

HUVEC

*

* *

*

*

* * **

„destabilisation by AA”

differentiation

EPCs : BC and AA-induced change in relative expressionof differentiation, but not angiogenesis (endothelium) related genes

(qReal-Time PCR)

-22.0-20.0-18.0-16.0-14.0-12.0-10.0-8.0-6.0-4.0-2.00.02.04.06.08.0

rela

tive g

ene e

xpre

ssio

nJagged-1

Notch-4

KDR

eNOS

vWF

CD36

TSP

Jagged-1 1.0 -2.7 -19.3 -6.0

Notch-4 1.0 -2.1 -4.2 -12.0

KDR 1.0 2.4 -3.8 -1.3

eNOS 1.0 2.3 -2.1 -1.1

vWF 1.0 1.2 -2.6 -1.1

CD36 1.0 4.8 -1.7 1.0

TSP 1.0 2.7 -1.4 2.2

THF/EtOH BC 3uM AA 3uM BC 3uM/AA 3uM

Jagged-1, Notch-4 -inhibition (promotes differentiation)

eNOS, vWF, KDR – inhibition (decreased differentiation to endothelial cells)

CD36, TSP-1 – up-regulatio (inhibition of angiogenesis )

BC and AA-induced changes in relative expressionof chemotaxis-related genes in EPCs (qReal-Time PCR)

MAD1L1 - the cell-cycle inhibitor

EGR1 -the cell differentiation activator

BIRC3 -the inhibitor of apoptosis

IL8, CCL2 -the activators of migration

CXCL12(SDF), CXCR4 -the homing of cell activators

-2

-1

0

1

2

3

4

5

6

7

rela

tive

ge

ne

exp

ress

ion

MAD1L1

EGR1

BIRC3

IL8

CXCL12/SDF1

CXCR4

CCL2

MAD1L1 1 6.43 1.77 1.25

EGR1 1 2.94 1.83 2.35

BIRC3 1 2.45 0.49 0.54

IL8 1 5.08 -1.25 1.37

CXCL12/SDF1 1 2.45 3.88 3.31

CXCR4 1 -1.06 1.59 1.20

CCL2 1 6.54 2.11 1.06

THF/EtOH BC 3uM AA 3uM BC 3uM/AA 3uM

BC 3uM AA 3uM BC 3uM/AA 3uM

MAD1L1 24.3 34.3 7.0

EGR1 1.7 1.5 2.1

BIRC3 3.5 3.5 4.6

IL-8 8 3.7 6.1

CXCL12/SDF1 1.5 1.9 NC

CXCR4 -1.5 -1.3 NC

CCL2 68.6 26.0 26.0

Microarray results

The role of the SDF-1–CXCR4 axis in migration/circulation of normal- and metastasis ofcancer-stem cells.

Ratajczak 2005

-2.0

-1.0

0.0

1.0

2.0

3.0

4.0

5.0

6.0

THF/EtOH BC 3uM AA 3uM BC 3uM/ AA 3uM

rela

tiv

e

gene

exp

ressio

n

0.0

1.0

2.0

3.0

4.0

5.0

THF/EtOH BC 3uM AA 3uM BC 3uM/ AA 3uM

rela

tiv

e

gene

exp

ressio

n

IL-8 SDF-1

Up-regulation of prochemotactic IL-8 and SDF-1gene expression in EPC by beta-carotene and AA(24h) (Confirmation of Microarray by qReal-Time PCR)

IL-8

Beta-actine

THF/

EtO

H

BC

3uM

AA

3uM

BC

/AA

3uM

0

50

100

150

200

THF/EtOH BC 3uM AA 3uM BC/AA 3uM

IL-8

pro

tein

% o

f co

ntr

ol

Up-regulation of IL-8 protein expression by BC andAA in HUVEC (24h) ( Confirmation of gene/protein expression by Western blott)

0

50

100

150

200

THF/EtOH BC 3uM

IL-8

pro

tein

% o

f c

on

tro

l

*

0

50

100

150

200

THF/EtOH BC 3uM

IL-8

pro

tein

% o

f c

on

tro

l

*

THF/

EtO

H

BC

3uM

IL-8

THF/

EtO

H

BC

3uM

IL-8

beta-actin

THF/

EtO

H

BC

3uM

IL-8

THF/

EtO

H

BC

3uM

IL-8

beta-actin

Up-regulation of IL-8 protein expression by BC and AA in EPC (24h) ( Confirmation of gene/protein expression by Western blott)

The animal models

* C57BL/6 mice ( 5 weeks)

* Ferrets: (3 months and 6 months)

Diet code:1- Kliba diet (2- Kliba + placebo (Placebo Beadlets = 0 ppm ß-Carotene)3- Kliba + beta-carotene (Kliba+ ß-Carotene Beadlet 10% = 1200 ppm ß- Carotene)

Diet was kindly supplied by the ROCHE/DSM Vitamins Basel ( dr Regina Goralczyk)

SUBCUTANEOUS WAT

0

20

40

60

80

BC 0.8 BC 3.2

+20.3%+22.7%

CA

PILL

AR

Y D

ENSI

TY

Control

Beta-carotene treatment increase the capillary densityIn ferret subcutaneous adipose tissue (A. Palou, S. Cinti; The DLARFID EU project)

0

5

10

15

20

25

THF/EtOH bFGF 50nM BC 3uM BC 3uM/ bFGF

50nM

Num

ber

of vessels

w ithout lumen

w ith lumen

0

1

2

3

4

5

6

7

8

9

THF/EtOH bFGF 50nM BC 3uM BC 3uM/ bFGF

50nM

Num

ber

of endote

lial cells

PE

CA

M+

In vivo model of angiogenesis (I) (PECAM-1 immunostaining( Non-treated with BC mice subcutaneously injected with matrigelcontaining BC or/and bFGF for one week)

Beta-carotene potentiates effect of b-FGF activation of angiogenesis in mice

**

*

*

Number of cells in matrigele Number of vessels in preparates

Summary:In non- toxic concentrations:

BC and AA did not influence proliferation of HUVECand EPC

BC and AA increase migration of EPC and HUVECBC and AA did not influence tubule formation (3D in

vitro model)AA „ destabilize” HUVEC but promotes (like BC)

differentiation of EPCBC In vivo augments angiogenesis in rodents nad

ferrets

• The Beta-Carotene/AA inducedendothelial cell (progenitor)chemotaxis/ pro-angiogenic activity isbenefit or may bring danger (?)

The main growth factors and angiogenesisCellular functionCellular function

Endothelial cell migrationEndothelial cell migration

Endothelial cell proliferationEndothelial cell proliferation

Smooth muscle cell migrationSmooth muscle cell migration

Smooth muscle cellSmooth muscle cellproliferationproliferation

ExtracellularExtracellular matrix production matrix production

Induction of proteasesInduction of proteases

Induction of Induction of integrinsintegrins

Generation of NO by Generation of NO by eNOSeNOSFolkmanFolkman,,19951995RissonRisson 1990 1990JukovJukov, 1996, 1996

PDGFPDGF

++

++++

++++

++

+?+?

--

bFGFbFGF

++

++

++

++

++

+/-+/-

ααvvββ55

(+)(+)

TGF-TGF-ββ

??

+/-+/-

+/-+/-

++

--

(+)(+)

(by (by TIMPsTIMPs inactivated) inactivated)

VEGFVEGF

++++

++++

00

00

++

++

ααvvββ33

++++

osteopontinosteopontin, , fibronectinfibronectin

t-PAt-PAuPAuPA

Vasculogenesis

STEM CELLS PROGENITORSBone marrow

Capillares(EC, VSMC, Matrix)

The main activators:

GROWTHFACTORS

Angiogenesis

(capillary net)ISHEMIA (HIF-

1)

GROWTHFACTORS

Arteriogenesis(reorganization ofpreexistingcapilaries)

FLOW (SHEARSTRESS)

MCP-1, GM-CSF

GROWTHFACTORS

not organizedflow

organizedflow regulation

Phenotypic analysis of putative EC precursor cells(ECP)

Surface marker

CD34 (disappears in hematopoesis)AC 133

VEGF-R1 (FLT-1)VEGF-R2 (KDR/FLT-1)VEGF-R3 (FLT-4)TIE 1TIE 2

VE-cadherinPECAMICAM-1eNOSAc-LDL uptake (CD36?)Lectin binding (BS-1)von Willebrand factor (vWF)ThrombomodulinIntegrin αvPIH12CD14 (monocyte)CD68VCAM-1Tissue factor (TF)Weibel-Palade bodies

Hematopoetic stem cells(hemangioblast)

++

++/- (subset 27%)

???

-+-

+/- (subset)+/- (subset)

-----

+ (subset)+---

ECP(angioblast-like)

++

?+! (necessary for EC differentiation)

+?+

++/-?+/-?+++++++---?-?+

EC(differentiated)

-/+-

+++++

++

+↑after stimulation+ ↑NO after VEGF

++++++--

-/+ (after activation IL-1, LPS-1)+/-+

in microvasculature

Benefit

Blood supply to growing organs

Functional remodeling

Revascularisation of ischemic

tissue

Healing of wounds (ulcus)

Negative effects Patological remodeling (restenosis,

cardiomiopathy) Tumor malignancy (vascularisation

of solid tumor, metastasis)

Angiogenesis in inflammation (atherosclerosis, diabetes)

Vasculogenesis Angiogenesis Arteriogenesis

ClassEicosanoids

Oxidized LDL

Native LDL

Oxidized linoleic acid products

LOX products

Others

Ligand15d-PGJ2

PGJ2

prostacyclin (PGI2)

PGA1/2

PGB2

PGEs

PGFs

8-HEPE

8-(R)-HETE

8-(S)-HETE

12-HETE

15-HETE

LTB4

9-(R/S) HODE

13-(R/S) HODE

13-(S)HpODE

9-oxoODE

13oxoODE

NSAIDs

PPARα++

++

++

++

++

-

-

++

(++)

+++

(++)

(++)

+

PPARδ++

++

++

++

(+)

-

-

-

-

PPARγ++

++

(+)

+

+

-

-

(+)

(+)

(+)

++

-

+

+

+

+

++

+

The pharmacological activators of PPARs

CD36 multi-ligand receptor for modifiedlong-chain fatty acids, modified LDL,anionic phospholipids, apoptotic cells,thrombospondin (TSP-1), collagens (I andIV) and fibrinogen.

CD36 is involved in the intracellular fataccumulation and cell adhesion to matrixproteins as well as antiangiogenic activityof TSP-1

-3

-2

-1

0

1

2

3

4

5

c o n t r o le t h a n o l

0 , 1 %

e t h a n o l

0 , 1 %

+ T H F

0 , 0 1 %

0,3uM 1uM 3uM 3nM 10nM 30nM 3nM 10nM 30nM

rela

tive

ex

pre

ssio

n r

ati

o

CD36 is up-regulated by beta-carotene in HUVEC (Real-Time PCR,24h)

beta carotene all-trans RA 13-cis RA

-3

-2

-1

0

1

2

3

4

5

control THF/EtOH BC 3uM AA 3uM BC 3uM/

AA 3uM

rela

tiv

e

gene

expre

ssio

n

-3

-2

-1

0

1

2

3

4

5

6

control THF/EtOH BC 3uM AA 3uM BC 3uM/

AA 3uM

rela

tive

gene

expre

ssio

n

EPC HUVEC

CD36 multi-ligand receptor formodified long-chain fatty acids, modifiedLDL, anionic phospholipids, apoptoticcells, collagens (I and IV) and fibrinogen.

CD36 is involved in the intracellular fataccumulation and cell adhesion to matrixproteins as well as antiangiogenic activityof thrombospondin (TSP-1),

Beta-carotene up-regulates when AA down-regulatesthe CD 36 gene expression (Real-Time PCR)

angiogenesis

scavenger receptor

Lipidaccumulati

on

Matrigel plug in in vivo angiogenesis model in mouse(paraffin fixed slides)

hematoxylin + eosin (10x) anti-CD31(PECAM-1) (40x)

Plasma level of beta-carotene in three groups of mice (HPLC test)

Diet code:1- Kliba diet2- Kliba + placebo ( Placebo Beadlets = 0 ppm ß-Carotene)3- Kliba + beta-carotene (Kliba 12 000 mg/kg ß-Carotene Beadlet 10% = 1200 ppmß- Carotene)

Interaction between transcription factors regulated by beta-carotene in EPC(Ingenuity Pathways Analysis, http://www.biocompare.com/itemdetails.asp)

RXR/RAR/RXR/RAR/PPARPPARγγBeta-Beta-carotenecarotene/AA/AA

analysis of promotorsequences(Microarray)

GATA-1 GATA-2 CREB Id2 PPARγ NFκB/cRel

EGR-1DR1 (inhibition of transcription)

NAB1 STATs

Lack of differentiation

Expression of DAMSblocks TGFβ pathway

Early stages of angiogenesisVEGF response

eNOSHOX1integrinstPACXCR4 (homing)IL-8metaloproteinasesKDRAng2

bFGF

Differentiation of EC(angiogenesis)

Pathways with G13 protein receptorsstimulate

Actin reorganisation

chemotaxis

Cytokines BC; AA

EGR-1 activity promotes:

Inhibition of apoptosis

IAP caspase6

but

EGR-1:TF - early growth responceNAB1: EGR-1 binding proteinSELP

VCAM

MECHANISMS?

CBP/P300

CREB

cAMP

myoD

STATfactors

p53AP-1 NFκB

NUCLEAR RECEPTORsPPAR / RAR / RXR GR / TR

SREBP

CholesterolMuscleDifferentiation

Interferonscytokines

Growtharrest

PhorbolestersInflammationGrowth stimulationOxidative stress

CytokinesPhorbolesters

(Lathman 2002)

Retinoic acid

COMPETITION FOR universal coactivator CBP/p300

Inhibiton of induction of genes expression mediated by AP-1 or NFκB

SteroidsGlucocoticoid hormonesThyroid hormones

XXX transcription regulatory molecules

Transcriptional activation by retinoid receptors

RE RE

DBDDBD

Transcriptional repression

Histone deacetylationother mechanisms?

TRLBD

RXRLBD

NCoR/SMRTSin3

HDAC

TRE TRE

DBDDBD

Chromatinremodeling

Histone deacetylationother mechanisms?T3/TR

LBDRXRLBD

RE RE

DBDDBD

Transcriptional activation

vitD/DR, PPARs

LBD

RXRLBD

SRC-1ACTR

TIF2

p160

CBP/p300p/CAF

pCAF

T3 ligand p160,p300/CBP, pCAF

NCoR/SMRT,Sin3, HDAC

p160, p300/CBP,p/CAF

RAR/RXR binds tochromatin on the surfaceof positioned nucleosome.

Corepressors oftranscription: (↑dissociation)NCoR- nuclear corepressorSMRT- silencing mediator forRXR and TRassociated proteins:Sin3 andHDAC- histone deacetylase;pCAF- p300/CBP-associatedfactorCBP- cAMP-rsponnseelement-binding protein(CREB) binding protein;p300-RAR cofactorSRC-1-steroidreceptorcoactivatorTIF2- transcriptionalintermediate factor 2ACTR- SCR/TIF2 cofactortermed activator of retinoidreceptorsCBP+ACTR+p/CAF –trimeric activation complex

DBD- DNA-binding domainLBD- ligand binding domainTRAP-thyroid hormone-associated proteinT3- thyroid hormoneTR thyroid hormone receptorTRE- thyroid hormone-response elementbasal transcriptional machinery

Histone acetylase (HAT)Histone deacetylase (HDAC)

Acetylo CoA + (CH2)4 (lysine)

CH

NH3

HNHISTONE CO

HAT (CBP/p300)

HDACNH

(CH2)4

CHHN

CO

C CH3

O

EXPRESSIONINHIBITION OFEXPRESSION

HAT: CBP(p300) Human general coactivator: (Human Activator of Transcription)

E2FpRb HDAC

cyclinDcdk4/6

P1b

pRb

E2F

P P

E2F

pRb

P P

P P

CELL CYCLEPROLIFERATION

Progression

DIFFERENTIATION

HDAC

PPARδPPARγ1/2

Lucia Altucci and Hinrich Gronemeyer 2001

FOS JUN

AP-1

RA

c/EBPs

Liganded retinoidreceptors (RARs,RXRs) interfere withthe activity of othertranscription factors

transcription factor-Pphosphatases

RA

transcription factorproteinkinases

RARA has direct andretinoid receptors-mediated effects onthe activity ofseveral proteinkinases. ERK

MAPKPKC

Decreased activity of pRb ( hyperphosphorylation , or Rb-/- (ko) ) ( or inhibition of HDAC) promotes dissociation of

PPARγ - pRb- HDAC compex

and stimulates adipocyte differentiation,white adipose tissue growing, (final differentiation).

(Fajas, Auverx 2002, Bonet, Palou , 2004 Christiansen, 2004)

Role of PPARγ in adipogenesis in vitro

(a)

(b)

The BC up-regulation of genes related to EPC cell cycle and Rb protein, but down-regulate

HDAC . (MAD1L1 – the cell cycle universal nihibitor)

MAD1L1

Examples of CXCR4+ tumors that may derive from the normal CXCR4+ tissue/organ specific stem cells

Conclusions : Beta-carotene- induction of chemotaxis/angiogenesis (?) of endothelial cells may be

related to the changes in expression of genes: participating in cell-cell and cell-matrix adhesion, promoting migration promoting homing of cells matrix proteins and metaloproteinases related to G protein –coupled receptor (GPCR) mediated

signalling (trough RhoA and p21) involved in activation of Ras signaling pathway (cytoskeletal protein reorganization) EGR-1 (early growth response-1) transcription factor seems

to be a key regulator of gene expression connected withBeta-Carotene/AA induced endothelial cell (progenitor)chemotaxis/ pro-angiogenic activity

Angiogenesis model in vivo (mice fed with beta-carotene enriched diet)

Female Balb/c mice were fed 5 weeks with two different diet:

The first: with beta-carotene(Kliba 2415 Vitamin A 1400IU/kg + 5% Cornoil , 0.125% sodium cholate 12 000 mg/kg, beta-carotene Beadlet 10% = 1200 ppm beta-carotene)

The second: without addition of beta-carotene(Kliba + placebo (Kliba 2415 Vitamin A 1400IU/kg + 5% Cornoil,0.125% sodium cholate 12 000 mg/kg,Placebo Beadlets = 0 ppm beta-Carotene).

Genes causatively involved in chemotaxis of HUVEC are upreg ulated by BC and in lesser extend by AA

11-down

781 0up

BC/AAAABC

MLC MLC-P actin/myosin

MLCK

PP1M

p21

BC –induced genes activate migratory potential of EPC

ClassEicosanoids

Oxidized LDL

Native LDL

Oxidized linoleic acid products

LOX products

Others

Ligand15d-PGJ2

PGJ2

prostacyclin (PGI2)

PGA1/2

PGB2

PGEs

PGFs

8-HEPE

8-(R)-HETE

8-(S)-HETE

12-HETE

15-HETE

LTB4

9-(R/S) HODE

13-(R/S) HODE

13-(S)HpODE

9-oxoODE

13oxoODE

NSAIDs

PPARα++

++

++

++

++

-

-

++

(++)

+++

(++)

(++)

+

PPARδ++

++

++

++

(+)

-

-

-

-

PPARγ++

++

(+)

+

+

-

-

(+)

(+)

(+)

++

-

+

+

+

+

++

+

The pharmacological activators of PPARs

CAROTENOIDS (1) : the 40-carbon molecules (C40H56On)(n: 0(carotenes)-6), morethan 600); dimers of symetrically joined polyisoprenes, whose array of conjugated doublebounds makes them particullary effective of quenching free radicals . Carotenoids arecleaved centrally or excenrically to retinoids.There are 6 known isomers of retinol: all-trans;11-cis, 13-cis; 9,13-dicis; 9-cis; and 11,13-di-cis. (Xanthophylls : oxygenated carotenoids)

• Vit A ( 20 –carbon retinol) is taken by humans in food of animal origin in a formof retinyl esters or in fruits or vegetables as precursor-carotenoids.

• 50-60 from all carotenoids containing one unsubstituted β-ionone ring and anattached at least 11 carbon antoms polyene side chain are potential precursorsof vitA , β-carotene is the potent source ( 1/6 absorbed is converted inintestinum; 15% uptaked unchanged) ( non-provitamin A: lutein, lycopene,zeaxanthin, canthaxanthin)(Bauenfield 1972)

• Functional retinoids: all-trans RA; 9-cis-RA; 11-cis retinaldehyde; 3,4-didehydro-RA and perhaps: 4-oxo –RA; 14-hydroxy-4,14-retro-retinol; 4-oxo-retinol

• Retinyl esters hydrolize to retinol in duodeum. ( natural plant isomers are all-trans, when, cis-izomers are more polar and may also result from foodprocesing (heating :13-cis, 9-cis)

Absorbtion of β-carotene in intestine is increased by fat and decreased by thepresence of the other carotenoids ( lutein, lycopene, catnthaxanthin) as well asthe excess of β-carotene itself.

Pathways regulated by beta-carotene andarachidonic acid in HUVEC and EPC

↑↑↑↑CDC42/RAC

↑↑↑↑migration

↓↓↓↓apoptosis

↓↓↓↓Cell cycle

↑↓↑↑STAT

↑↓↑↑NFκB

↑↑↑↑G-protein

↑↑↑↑Rho

↑↓↑↑PI3K/AKT

↓↓↑↑TGF-beta

↓↓↑↑WNT

BCAABCAApathways

EPC HUVEC

CAROTENOIDS (2):• ENTEROCYTES: Retinol (R-OH) binds to Retinol Binding Protein II (CRBPII)

which directs its esterification by lecythin:retinol acyltransferase (LRAT) toretinyl esters (RE)

β-carotene may undergo central ( 15,15 dioxygenase) or excentric cleavage(human , monkey, ferret) with formation of retinoids (retinaldehyde (RAL),subsequently reduced by retinal reductase to retinol (ROL) or oxidized to toretinoic acid (RA) and esterified by acyl:coA retinolacyltransferase (ARAT) toretinoid esters (RE). This proceses are regulated by binding to CRBPII. 15% ofadsorbed β-carotene passed to circulation unchnged.

RE and β-carotene are incorporated to chylomicrones and taked up mainly byhepatocytes.

• LIVER. Retinoid esters (RE) are stored in the perisinusoidal stellate cells) oroxidized to RA ( regulation of liver gene expression) , or liberated to circulationtovards tovard target cells by the synthesized in liver retinoid-binding protein(RBP), and transthyretin ( decreased kidney filtration of retinol-RBP). β-carotene is liberated with VLDL converted in circulation to LDL and uptakedperipherally by target cells.

• TARGET CELLS. Retinol (VitA), retinaldehyde (RAL) binds in cells to retinolbinding protein (CRBP) or cellular retinoic acid binding protein (CRABP). Thisprotein regulate the fate ( catabolism – xenobiotic like cytochromes activationin mitochondria, glucoronidation) or the nuclear transport to regulate thetranscription

CRABP higher affinity than RAR for RA!!! CRBP1 and CRABP are regulatorsof the cellular concentration and metabolism as well as direction of activeretinoids.

Beta-carotene and cell culture• Lycopene as well as β-carotene (1-3µM) protected human colon arcinoma cell

lines (HT29) agains xanthin/xanthin oxidase - strand brake (comette assay) . Atthe higher concentration protection is lost (Lowe 1999)

• β-carotene in combination with vitC and vitE prevents the γ-irradiation inducedlymphocyte DNA breacking (Konopacka 1998)

• β-carotene and canthaxanthin decrease proliferation ( 3Hthymidineincorporation) of human mammalian epithelial cells (Rock 1995), human aorticVSMC (lycopene>β-carotene>zeaxanthin, but not lutein or canthaxanthin)(Carpentier 1999)

• α-carotene decreased proliferation of human neuroblastoma cell line (Murokashi 1989)

• Canthaxanthin (1µM) inhibited proliferation and induced apoptosis of humancolon adenocarcinoma and melanoma cells (Palozza 1998)

• Canthaxanthin as well as α- or β-carotene or lycopene ( < 1µM) inhibits the the3-methylcholanthrene-induced tumorigenic transformation (proliferation)inmouse C3H10T1/2 cells . It is not due to antioxidant activity (VitE not active, orvit A – canthaxanthin is not provitamine) (Bertram 1991)

• α- or β-carotene supress of N-mycRNA arresting neuroblastoma cells in G0(Murakoshi 1989), and induces genes of connexin 43 (Zhang,Bertram 1992)as well as haem oxygenase (HO-1) (Obermueller-Jewitz 1999)

Angiopoietins (Ang) Ang-I Ang-IIAngiogenins Angiogenin-1 Angiogenin-2EphrinsE-selectinFibroblast growth factors (FGF) Acidic FGF Basic FGF FGF3–9

HeparinHepatocyte growth factorInsulin-like growth factorPlatelet derived growth factorProstaglandins (PG) PGE1

PGE2

PGFThyroxineThrombospondinTransforming growth factor- b (TGF-b)Tumor necrosis factor-a (TNF-a)Vascular endothelial growth factors (VEGF)

VEGFA 121, 145, 165, 189, 206

VEGFBVEGFCVEGFDVEGFEPlacental growth factor

Pro-angiogenic factors

beta-carotene (BC)

BC

centeric (symmetric) cleavage

eccenteric (asymmetric) cleavage

RA

apocarotenales

beta-oxidation

RXR RAR

RXR/RXR RXR/RAR

PPAR

PPAR/RXR

gene expression

free radicalsrandom degradation of BC

FA(AA)

AhR

ciglitazone

Mechanisms for fatty acid control of gene transcription

Duplus 2000

Possible explanations for perceived plasticity

Angiogenesis (in vivo model)

0

5

10

15

20

25

30

35

control bFGF beta-carotene bFGF + beta-

carotene

nu

mb

er

of

ve

sse

ls

with lumen

without lumen

0

1

2

3

4

5

6

7

8

9

10

control bFGF beta-carotene bFGF + beta-

carotene

num

ber

of endoth

elial cells

*

*

0

5

10

15

20

25

30

35

control bFGF beta-carotene bFGF + beta-

carotene

nu

mb

er

of

ve

sse

ls

with lumen

without lumen

0

1

2

3

4

5

6

7

8

9

10

control bFGF beta-carotene bFGF + beta-

carotene

num

ber

of endoth

elial cells

*

*

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

control PMP 30ug/ml

nu

mb

er

of

ve

sse

ls

with lumen

without lumen

endothelial cells

BC BC

PMPBC- beta-carotenePMP-platelet microparticles

Myc and Mad

down-regulation p15, p21, Gadd 45

up-regulation: Cdk4, cyclin D1, cyclin D2,cyclin A, cyclin E, cdc25A, p19arf, Id2

PhorboestersCytokinesOxidative stressViral InfectionGrowth factorsNeurotransmittersPolypeptides

Ras/Raf/MAPK kinase activation

Extracellular Signal Regulated Kinases

(ERKs)

Jun N -terminal Kinases

proliferation

c-fos and activate ATF2 diferentiation

P

P38-MAPKStress-Activated Protein Kinase (SAPK)

Activate c-jun- ATF2

PP

Apoptosisor mitosis(JNK)

The first discovery:

CREB (CRE binding protein)

CRE ( CAM response element)

CREB

CBP

PKACREB

P

CREB

P

TRANSCRIPTION

CBP:CREB binding protein coactivator

CBP

Embrional carcinoma F9 cells (murine teratocarcinoma)

(Jin 2002)

JDP2 HDAL

c-jun

DifferentiationResponseElement

DRE: ATF2BP/p300

Jun dimerization protein JDP2: represor of AP-1

ATF2CBP/p300

RAR/RXRJDP2 HDAC

c-jun

Differentiation of F9cells

Retinoic acid:

JDP2 suppress p53 expression (Pin 2001)(suppress apoptosis)

p53 expression is regulated by c-Jun (Strickland 1978) c-Jun p53 apoptosis

Embrional carcinoma F9 cells (murine teratocarcinoma)

(Jin 2002)

JDP2 HDAL

c-jun

DifferentiationResponseElement

DRE: ATF2BP/p300

Jun dimerization protein JDP2: represor of AP-1

ATF2CBP/p300

RAR/RXRJDP2 HDAC

c-jun

Differentiation of F9cells

Retinoic acid:

JDP2 suppress p53 expression (Pin 2001)(suppress apoptosis)

p53 expression is regulated by c-Jun (Strickland 1978) c-Jun p53 apoptosis

Cell cycle is inhib ited ( ? ) by BC/AA reg ulated g enes

699down

1 61 92 1up

BC/AAAABC

Hig h sim ilarity of reg ulated g enes

Induction of throm bospond in by BC 453down

477up

BC/AAAABC

Antiapoptot ic ( ? ) act ivity of BC/AA reg ulated g enes

243down

71 07up

BC/AAAABC

Hig h sim ilarity of reg ulated g enes

Regulation of transcription:

RAR (α 1,2; β 1,2 ; γ 1,2) RXR (α; β; γ )

Transactivation

(δ; ε)

Transrepression(API)

Cellular activity of retinoids

all-trans RA;9-cis RA4-oxo ROH

induction:oxitocin, growth factors, CRBP,CRABP, enzymes(phosphoenolopyruvate, alkoholdehydrogenase, collagenases,laminin B)

inhibition:IGF-1; TGF-a; G1a protein(extracellular matrix)

postranscriptional activity↑ stability of mesenger RNA(connexin 43) processing thetranscript

PPRE

PPAR

9-cis RAfatty acids

+/-

mRNA

RXR

target gene

RARE

RAR

all-trans RAor 9-cis RA

+/-

mRNA

RXR

target gene

RARE

RXR

9-cis RA

+/-

mRNA

RXR

target gene

RA isomers bind toand modulate theactivity oftranscription factorsof the nuclear receptorsuperfamily

How do RAisomers affectgeneexpression?

DLARFID QLK1-CT-2001-00183Dietary Lipids as Risk Factors in Development. Mechanistic

Issues.Participant No 1 (Co-ordinator) Prof. Aldona Dembińska-Kieć MD Phd

Krakow, PolandParticipant No 2 (Contractor) Dr Jaap Keijer, Wageningen, The NetherlandsParticipant No 3 (Assistant contractor to no 1) Assoc. Prof. Piotr Laidler PhD

Krakow, PolandParticipant No 4 (Assistant contractor to No 1) Prof. Aleksander Skotnicki MD, Krakow, PolandParticipant No 5 (Contractor) Prof. Gerd Schmitz MD PhD, Regensburg,

GermanyParticipant No 6 (Contractor) Prof. Karsten Kristansen PhD (Lis Mansen/

Norway), Odense, DenmarkParticipant No7 (Contractor) Prof. Jan Nedergaard Ph, Stockholm, SwedenParticipant No 8 (Contractor) Prof. Andreu Palou, Palma di Mallorca, SpainParticipant No 9 (Contractor) Prof. Saverio Cinti MD PhD, Ancona, ItalyParticipant No 10 (Administrative Assistant CMUJ ), Krakow, PolandParticipant No 11 Roche Vitamins DSM Regina Goralczyk, Basel SwitzerlandEU Comission Representatives:Prof. Jim Leslie (PTA); Dr Jean-Marc Chourot , Merel Groet

Homing of progenitor cels: Signal transduction pathways activated by the SDF-1–CXCR4 axis.

CXCR4