The Endothelial Saga: Regenerate to …be old Paul M. Vanhoutte M.D., Ph.D. University of Hong Kong Nice June 12 th , 2011
The Endothelial Saga: Regenerate to …be old
Paul M. Vanhoutte M.D., Ph.D.University of Hong Kong
Nice June 12th , 2011
… « THE RESULTS INDICATE THATFUNCTIONAL COUPLING BETWEENENDOTHELIAL CELLS AND THE ARTERIALMEDIA INVOLVES AT LEAST THREEDIFFERENT PATHWAYS, TWO OF WHICHDEPEND ON THE METABOLISM OF THEFATTY ACID ».
J.G. De Mey, M. Claeys and P.M. VanhoutteJPET, 222 : 166, 1982
A23187
Contraction
Ca2+
ACh
P
mADPPLA2
Membrane lipids
Endoperoxides
PGD2
PGF2α
PGI2
TXA2
PGE2
COX-1
TXAS
TP
AA
PGIS
COX
X+XO
H2O2
1
Endothelial Cell
Smooth muscle Cell
↑
↑
↑ ↑
ROS2 ↑↑
↑ ↑
Summary
isoprostanes
Cole et al. (1999) J Mol Biol 289(1):139-57
• 25kDa pro-inflammatory glycoprotein
• ↑in obese mice and humans• ↓by rosiglitazone• In humans, lipocalin-2 is
positively associated with several parameters of the metabolic syndrome [adiposity, hypertriglyceridemia, hyperglycemia, insulin resistance index], and the levels of the inflammatory marker, C-reactive protein. [Wang et al.(2007) Clin. Chem 53: 34-41]
Lipocalin-2
-10 -9 -8 -7 -6 -5
0
25
50
75
100
wild type
Lcn2-KO
Acetylcholine, log M
Dec
reas
es in
tens
ion
(% U
4661
9 co
ntra
ctio
n)Relaxations to acetylcholine in mouse aortae
[with endothelium; 12-13 weeks]
-10 -9 -8 -7 -6 -5
0
25
50
75
100
wild type
Lcn2-KO
**
* * * *Acetylcholine, log M
Control diet High fat diet
SMOOTH MUSCLE
ENDOTHELIUM
ACETYLCHOLINE or Calcium Ionophore A23187
NO EDCF
RELAXATIONCONTRACTION
L-NAME
Acute
inhibition
Native cells Regenerated cells
30 mm30 mm
Elevated cytoplasmic detection of b-galactosidase in regenerated endothelial cells (P1)
0
50
100
150
200
250
300
350
-7.0
0
-5.3
0
-4.8
0
-4.1
0
-3.6
0
-3.1
0
-2.6
0
-2.1
0
-1.6
0
-1.1
0
-0.6
0
-0.1
0
0.5
0
1.0
0
1.5
0
2.0
0
2.6
0
3.4
0
4.7
0
Num
ber o
f gen
es
Signal Log 2 Ratio
Figure 2A
-5.0
-4.5
-4.0
-3.5
-3.0
-2.5
-2.0
-1.5
-1.0
-0.5 0.0
CAV1*Arginase, type II*ABCA1*IB*TFPI (Lp-associated)*Apo B*GPIIIa*Lrp 1*Acyl-CoA oxidase*Mn SOD*ECE 1*TXNIP*GPX3*LDL receptor *Collagen XIV*COX-1/PGHS-1*Cadherin-11*Lrp 6*E-selectin*GSR*PAI-2*ACAT*TGF-b1R-II*VLCAD*LOX receptor 1*
Oxidative stress
Cholesterol/fatty acid metabolism
Extracellular matrix
Coagulation
Adhesion molecules
Vasomotor tone
Inflammation
Signal Log 2 Ratio
Ɨ
Ɨ
Ɨ
Ɨ
Ɨ
Downregulation of genes in regenerated endothelial cells
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0
*Galactosidase, beta 1*Gp VII PLA2
*PPAR gamma 1a*GADD45B
*Growth arrest specific 61bTGF-*
*GM-CSF*COL1A1
5bintegrin, **MMP 23A&B
*CXCL 12 *F2
*Ang II receptor, type I *GADD45A
*Adenosine deaminase*HPGD*MMP 7
*A-FABP
Cholesterol/fatty acid metabolism
Extracellular matrix
Growth regulation and senescence
Coagulation
Adhesion molecules
Vasomotor tone
Oxidative stress
Signal Log 2 Ratio
Ɨ
Ɨ
Ɨ
Ɨ
ƗƗ
Ɨ
Ɨ
Ɨ
Ɨ
Upregulation of genes in regenerated endothelial cells
0
25
50
75
100P1P4
**
eNO
S(%
of c
ontr
olm
ean)
Real time-PCR A
0
25
50
75
100
*P1P4
eNO
S(%
cha
nge
from
con
trol)
eNOS140 kDa
B Western Blotting
b-actin
C
0
5000
10000
15000
*
P1P4
fmol
/mill
ion
EC
Cyclic GMP
0
50000
100000
150000
BK A23187
**
fmol
/mill
ion
EC
D
mRNA expression, protein presence of eNOS, cyclic GMP levels under basal
conditions and upon stimulation with bradykinin (BK; 10-6M) and A23187 (10-6M)
LKB1
Endothelial Aging
AMPK
SIRT1
N-terminal Kinase domain C-terminal
44 3091 436
Lysine * * * * 48 62 64 122
-14 -12 -10 -8 -6 -4 -2 0 2 4 6 8 10 12 14 16
BI
SOD2
-inducible proteinInterferon,
Thioredoxin interacting protein
Interferon regulatory factor 7
Selectin L
Interleukin 6
Calsarcin 1
Fold-change
Reg
P4
Similar expression of genes in cultures derived from regenerated endothelial
cells (Reg) and senescent endothelial cells (P4) compared to the young cells
(P1)
-2 -1 0 1 2 3 4 5 6 7 8 9 10 11 12
Prostaglandin-endoperoxide synthase 1
p53 protein
Glutathione reductase
Fatty acid binding protein 5
Scavenger receptor class B member 2
Transforming growth factor beta receptor II
Peroxisome proliferator-activated receptor gamma
Matrilysin-related protein (MMP7)
Coagulation factor II
Fatty acid binding protein 4 adipocyte
Fold-change
Reg
P4
Differential expression of genes in cultures derived from regenerated
endothelial cells (Reg) and senescent endothelial cells (P4) compared to the
young cells (P1)
Identification of A-FABP in mouse aortae by quantitative RT-PCR, immunohistochemical and immunofluorescent staining
ApoE-/- 8 weeks old
vWFA-FABP
C57 w
k18
ApoE-/- wk8
ApoE-/- wk1
2
ApoE-/- wk1
80
2
4
6
8
10
*
*
A-F
AB
P e
xpre
ssio
n(n
orm
aliz
ed to
GA
PD
H)
A-FABP + vWF
vWFA-FABP A-FABP + vWF
C57 WT 12 weeks old
ApoE-/- 12 weeks old
ApoE-/- 12 weeks old
Relaxations in aortae (with endothelium) of18 weeks old ApoE-/- mice treated with BMS309403 (15 mg/kg/day; ApoE-BMS) or vehicle for six weeks. Effect of pertussis toxin (PTX; 400ng/ml)
-10 -9 -8 -7 -6 -5
-20
0
20
40
60
80
100
** *** **** **
UK14304 (Log M)
% R
elax
atio
n
-9 -8 -7 -6 -5 -4
-20
0
20
40
60
80
100
120
C57 wk 18vehicleBMS309403
Sodium nitroprusside (Log M)
% R
elax
atio
n
C57 wk 18 ApoE ApoE+BMS0
100
200
300 ***
Area
und
er c
urve
(arb
itrar
y un
it)
vehicl
e
vehicl
e+PTX
BMS3094
03
BMS3094
03+P
TX0
100
200
300 *****
Area
und
er c
urve
(arb
itrar
y un
it)