Transcript Διαφάνεια 1

Anti-fibrotic effect of azathioprine on
primary human hepatic stellate cells
P. Manousou1, T. Shepherd1, L. Longato 1, K. Rombouts 1, A.Tzallas 3,N.
Giannakeas2, M. Tsipouras 2, E. V. Tsianos 2, M. Pinzani 1
1
The Royal Free Sheila Sherlock Liver Unit, Royal Free Hospital and
UCL Institute for Liver and Digestive Health, London, UK
2 1st Division of Internal Medicine and Division of Gastroenterology, University of
Ioannina, Greece
3 School of Applied Technology, Department of Computer Engineering,
Technological Educational Institute of Epirus, Greece
Background
p=0.045
p=0.045
Our results favouring the TT group were supported by our findings of
HVPG measurement.
This finding gives internal validation to the more severe fibrosis found
in this group.
Why did we use AZA in one arm ?
The study design was made in 1999 when MMF was not used
in our unit, so azathioprine was our standard adjunctive
immunosuppression.
Why did we get criticism ?
Early use of MMF to prevent ACR episodes
The registration study for MMF in LT was an international multicenter
randomized double-blind comparison of MMF versus AZA, each
combined with cyclosporine and steroids:
The incidence of ACR or graft loss, biopsy-proven and treated rejection
was greater in AZA arm.
Wiesner
Liver Transpl 2001
Early use of MMF to prevent ACR episodes
1-year patient and graft survival rates were not different:
MMF 88.8% and 85.3% versus AZA 87.1% and 85.4%.
The dose of MMF used was 3 g/day
Currently, most units use only 2 g/day MMF, so the comparison with AZA
in the registration study does not mirror today’s clinical practice.
Wiesner
Liver Transpl 2001
Hazard curves
for Ishak stage 4
p=0.005 by Mantel-Cox
p=0.003 by Mantel-Cox
MT
MT
TT
17 patients discontinued
TT – AZA discont
azathioprine (10 – 37m) post LT.
n=17
TT – still on AZA
n=31
time to
to reach
reach S4
S4 (months
(months post
post LT)
LT)
time
Manousou
Gut
2014
Transplant free survival in PSC is influenced by AZA and
steroids, independent of the presence of IBD
333 patients were diagnosed with PSC, 16 with small-duct PSC, 21 with
autoimmune overlap.
IBD: 208 ulcerative colitis, 30 Crohn’s disease, 8 indeterminate colitis
Factors associated with transplant-free survival at Cox analysis were:
AZA treatment
(p=0.012, OR=2.3, 95%CI=1.2–4.4),
steroids
(p=0.001, OR=4.7, 95%CI=2.2–10.1) and
concomitant IBD (p=0.023, OR=2.11, 95%CI=1.11–3.4).
Aim of the study
The possible antifibrotic effect of azathioprine could be due
to a direct effect on hepatic stellate cells, the cellular
effector of liver fibrogenesis.
Therefore, we investigated the potential antifibrotic effect of
azathioprine in cultured human Hepatic Stellate Cells
(hHSC).
Materials and methods
Human hepatic stellate cells (HSCs) isolated from normal liver tissue and
maintained in culture, were pre-exposed to TGFb1 (5ng/ml) and then
treated with different doses of AZA or MMF.
Hepatic Stellate Cells
Kupffer Cells
We have managed to successfully
optimise the isolation and cell culture of
hepatic stellate and Kupffer cells
obtained from 10 normal control
patients as well as from the explants of
patients undergoing LT.
A single protocol was used successfully
for the isolation of stellate cells and
Kupffer cells
Materials and methods
Cell culture viability with and without AZA or MMF was estimated
with the MTT assay.
Cell proliferation was examined by BrdU incorporation.
The expression of the myofibroblast differentiation marker, asmooth muscle actin (SMA) was examined by Western Blot.
Real time PCR was used to assess the mRNA expression of TIMP1, Collagen 1a1, LOX and MMP2 genes.
Results ….. MTT assay
O.D.
1.2
1
0.8
AZA
0.6
MMF
0.4
0.2
0
medium serum free
vehicle
10(-6)
10(-5)
10(-4)
10(-3)
10(-2)
0.1
1
10
Azathioprine concentrations:10-6 to 10 nM
MMF concentrations:
10-6 to 10 ngr/ml
Tetrazolium salts are metabolized by NAD dependent dehydrogenase activity to
form a colour reaction product. The ammount of dye formed corellates to the
number of viable cells.
Results ….. BrDU assay
0.6
0.5
*
*
0.4
*
0.3
AZA
MMF
0.2
0.1
0
medium serum free
vehicle
10(-6)
10(-5)
10(-4)
10(-3)
10(-2)
0.1
1
10
There was a significant decrease in hHSC proliferation when AZA was used at
24hrs and 48 hrs (median 0.58 for SF vs 0.23 with AZA, p=0.034) and at 72hrs
(median 0.47 for SFM vs 0.18 for aza respectively, p=0.031).
The results were not significant for MMF: median 0.54 for serum free medium vs
0.50 for MMF treated cells at 48 hrs and 0.43 (serum free medium) vs 0.406 (MMF
treated) at 72hrs.
Results
HSCs
Serum free
Vehicle
Aza 0.1 nΜ
Aza 1nM
TGF-β
Aza 0.1 nΜ
+ TGF-β
Aza 1nM +
TGF-β
HSCs
Serum free
Vehicle
MMF 0.1
ngr/ml
MMF
1ngr/ml
TGF-β
MMF 0.1
+ TGF-β
MMF 1
+ TGF-β
Collected for RNA and protein at different time points: 6, 12, 24, 48 hours
Results … Western Blot
HSCs
Serum free
Aza 0.1 nΜ
Aza 1nM
Vehicle
Aza 10nM HSCs
MMF 0.1
ngr/ml
SFM
AZA1
Serum free
Vehicle
MMF
1ngr/ml
MMF
10ngr/ml
AZA2 COMB1 COMB2 TGF
Western Blot for a-SMA revealed a 2-fold reduction in TGF pre-treated
myofibroblasts when azathioprine was added.
There was no difference when MMF was used.
Results … RT - PCR
TIMP1 / GAPDH
7
6.2
6.3
*
*
6
5
3.94
4
3.3
2.86
3
2.5
2.41
*
2
1
0
SM
(20%
FBS)
SFM
Vehicle
AZA 1
nM
TGF
TGF+
AZA 1
Veh
MMF
10 ng
MMF
TGF+
MMF
The proteins encoded by this gene family are natural inhibitors of the
matrix metalloproteinases (MMPs), a group of peptidases involved in
degradation of the extracellular matrix.
Results … RT - PCR
COL1A1 / GAPDH
2.0
1.7
1.8
1.6
1.6
1.4
1.4
1.2
*
1.2
1.0
1.0
1.0
1.0
0.8
**
0.6
0.5
0.4
0.4
**
*
0.2
0.7
0.0
SM
SFM
Veh
AZA
AZA 1
nM
TGF
TGF+
AZA 1
Veh
MMF
TGF
10
ng/ml
MMF
TGF+
MMF
This gene encodes the pro-alpha1 chains of type I collagen. Type I is a fibrilforming collagen.
Results … RT - PCR
Lox / GAPDH
0.30
0.27
0.26
0.26
0.25
*
*
0.20
**
0.19
0.17
0.16
0.15
0.10
0.05
0.13
0.13
*
*
**
0.14
0.12
0.00
SM
SFM
Vehicle
AZA 1 nM
TGF
TGF+ AZA 1
Veh MMF
TGF
10 ng/ml MMF TGF+ MMF
LOX (lysyl oxidase) is a protein-coding gene. The protein encoded by this gene
is an extracellular copper enzyme that initiates the crosslinking of collagens
and elastin.
Results … RT - PCR
MMP2 / GAPDH
*
**
**
**
*
Proteins of the matrix metalloproteinase (MMP) family are involved in the
breakdown of extracellular matrix
Conclusion
There was a significant decrease in hHSC proliferation when AZA was
used at 24hrs, 48 hrs and at 72hrs
We were not able to demonstrate the same for MMF.
*
**
Western Blot for a-SMA revealed a 2-fold reduction in TGF pre-treated
** when azathioprine was added.
myofibroblasts
** was used.
There was no difference when MMF
There was a significant down-regulation (q-PCR) of LOX and Collagen
* was applied in SFM as well as in TGF
1A1 genes, when AZA 1nΜ
induced HSCs.
Conclusion
Azathioprine exerted an anti-fibrotic effect in cell
proliferation assays *as well as in**protein and RNA level in
h HSC in culture.
**
**