ANTI-VASCULAR ENDOTHELIAL GROWTH FACTOR THERAPY

ATTENUATES HEPATIC ISCHEMIA AND REPERFUSION INJURY

ANTI-VASCULAR ENDOTHELIAL GROWTH FACTOR THERAPY

ATTENUATES HEPATIC ISCHEMIA AND REPERFUSION INJURY

S. TsuchihashiS. Tsuchihashi11, B. Ke, B. Ke11, F. Kaldas, F. Kaldas11, , R.W. BusuttilR.W. Busuttil11, D.M. Briscoe, D.M. Briscoe22, ,

J.W. Kupiec-WeglinskiJ.W. Kupiec-Weglinski11

1 1 Dumont-UCLA Transplant Center, Division of Liver and Pancreas Dumont-UCLA Transplant Center, Division of Liver and Pancreas Transplantation, Department of SurgeryTransplantation, Department of Surgery

2 2 Division of Nephrology, Children’s Hospital, Harvard Medical SchoolDivision of Nephrology, Children’s Hospital, Harvard Medical School

BACKGROUNDBACKGROUND

2. Up-regulation of VEGF is associated with hepatic ischemia/reperfusion (I/R) injury.

3. Anti-VEGF treatment attenuates hepatic I/R injury. (Boros P, et al. Transplantation 2001;72: 805)

(Ke B, et al. Transplantation 2005;79:1078)

1. Vascular endothelial growth factor (VEGF) is a pro-inflammatory cytokine implicated in increasing endothelial cell permeability, inducing the expression of endothelial adhesion molecules, and acting as a monocyte chemoattractant in allograft rejection.

(Reinders MEJ, et al. J.Clin.Invest 2003;112:1655)

AIMAIM

To explore the cytoprotective

mechanisms of anti-VEGF therapy

in hepatic I/R injury

Animals:

male C57BL/6 mice. 25~30 g

Model:

Partial (left & middle hepatic lobes) warm I/R injury

Ischemic Time : 90 min

Reperfusion Time : 0, 2, 4, and 6 h (n=6/time point)

METHODS METHODS

Ischemia (90 min)

0 h

Reperfusion

Evaluation: Evaluation:

2 h 4 h 6 h

VEGF mRNA (RT-PCR)

VEGF protein (Western blot)

METHODSMETHODS

Experimental groups:

-24 h

Ischemia (90 min)

0

Reperfusion (6 h)

1) Sham group (n=5)2) Control group (n=8)3) Anti-VEGF group (n=9)

Sacrifice

Anti-VEGF or control serum

(0.8 ml i.p.)

sGPTHistologyMPO activity (neutrophil infiltration)PCR (TNF-, IFN-, E-selectin, IP-10, MCP-1)Immunohistochemistry (CD45, CD3, Mac)TUNEL stainingWestern blot (Bcl-2, Bcl-xl, Bax, HO-1)

Evaluation (6 h after reperfusion):Evaluation (6 h after reperfusion):

42kDa

42kDa

-actin

VEGF

-actin

VEGF

Naïve 0h 2h 4h 6h Naïve 0h 2h 4h 6h

After reperfusion After reperfusion

0

0.2

0.4

0.6

0.8

1

Naive 0h 2h 4h 6h

VE

GF

mR

NA

/-a

cti

n

After reperfusion

** *

#

VE

GF

pro

tein

/-a

cti

n

After reperfusion

0

0.2

0.4

0.6

0.8

1

1.2

Naive 0h 2h 4h 6h

*

*

#

*

Western blotWestern blotRT-PCRRT-PCR

VEGF expression in hepatic I/RVEGF expression in hepatic I/R

*P < 0.05 vs Naive

#P < 0.05 vs 0h or 4h

*P < 0.01 vs Naive#P < 0.01 vs 0h, 2h, 4h

0

500

1000

1500

2000

2500

3000

3500

4000

4500

Sham Control Anti-VEGF

s G

PT

(IU

/L)

*

sGPTsGPT

*p<0.01

Sham Control Anti-VEGF

X 1

00X

400

HistologyHistology

Suzuki’s score = 7.97±1.08 1.00±0.87P<0.01

0123456789

10

Sham Control anti-VEGF

Uni

ts /g

m *

MPO activity(Neutrophil infiltration)MPO activity

(Neutrophil infiltration)

*p<0.01

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

TN

F-

/-a

ctin

Sham Control Anti-VEGF

TNF-TNF-

*

IFN

-/

-act

in0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Sham Control Anti-VEGF

IFN-IFN-

*

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Sham Control Anti-VEGF

E-s

ele

ctin

/-a

ctin

E-selectinE-selectin

*

MCP-1MCP-1

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

Sham Control anti-VEGF

MC

P-1

/-a

ctin

**

IP-10IP-10

0

0.10.2

0.30.40.50.60.7

0.80.91

Sham Control anti-VEGF

IP-1

0/

-act

in **

RT-PCRRT-PCR

*p<0.01

**p<0.05

Pro-inflammatory cytokinesPro-inflammatory cytokines

ChemokinesChemokines

Adhesion moleculeAdhesion molecule

CD

45

CD

3

Sham Control Anti-VEGF

Ma

cImmunohistochemical stainingImmunohistochemical stainingImmunohistochemical stainingImmunohistochemical staining

(Pan

leu

kocy

te)

(T c

ell)

(Mac

rop

hag

e)

0

5

10

15

20

25

30

Sham Control anti-VEGF

TU

NE

L p

os

itiv

e c

ell

s/f

ield

*

TUNELTUNEL

Sham Control Anti-VEGF

*p<0.01

42kDa

32kDa

28kDa

32kDa

23kDa

-actin

HO-1

Bcl-2

Bcl-xl

Bax

Sham Control Anti-VEGF

Western blotWestern blot

1. prevented hepatic warm I/R injury.

2. inhibited leukocyte (neutrophil, T-cell, and monocyte/macrophage) infiltration through the inhibition of cellular adhesion molecule, decreased the expression of pro-inflammatory cytokines/chemokines.

3. prevented apoptosis, up-regulated expression of anti-apoptotic (Bcl-2/Bcl-xl)/antioxidant (HO-1) protective molecules, and depressed pro-apoptotic (Bax) proteins.

SUMMARYSUMMARYAnti-VEGF treatment:

By selectively modulating leukocyte tr

afficking patterns, VEGF plays a param

ount role in the pathophysiology of he

patic I/R injury.

CONCLUSIONCONCLUSION