anti-vascular endothelial growth factor therapy attenuates
Post on 25-May-2015
347 Views
Preview:
TRANSCRIPT
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
top related