Dembińska-Kieć Beta-Carotene and Arachidonic Acid ...home.ueb.cas.cz/COST926/Session 7-6.pdf ·...
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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