The Endothelial Saga: Regenerate to …be old

Paul M. Vanhoutte M.D., Ph.D.University of Hong Kong

Nice June 12th , 2011

Is NO the only answer ?

… « 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

NN

From Taddei et al 1996

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

IRS-1 IRS-1 P

Akt

PI-3K PI-3K P

P

eNOS eNOS

insulin

Insulin receptor

Lipocalin-2

Deficiency+

Akt

P

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)

Regenerate to be old…

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

Cell proliferation Telomerase activity

Senescence-associated beta-galactosidase staining

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)

Regenerate in vivo to be old…

Makowski L; Nature Medicine, 2001

A-FABP and atherosclerosis (1)

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)