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

2. 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.

3. 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 ?

4. 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

5. 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

6. 17 patients discontinued azathioprine (10 – 37m) post LT.
Hazard curves for Ishak stage 4
time to reach S4 (months post LT) MT
TT – still on AZA
n=31
TT – AZA discont
n=17
p=0.003 by Mantel-Cox MT TT
p=0.005 by Mantel-Cox
time to reach S4 (months post LT)
17 patients discontinued azathioprine (10 – 37m) post LT.
Manousou Gut

7. 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).

8. 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).
However, there is no data accessing in vitro, any possible anti-fibrotic effect of the medication.

9. 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

10. 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, a- smooth muscle actin (SMA) was examined by Western Blot. Real time PCR was used to assess the mRNA expression of TIMP-1, Collagen 1a1, LOX and MMP2 genes.

11. Results ….. MTT assay O.D. Azathioprine concentrations:10-6 to 10 nM
The overall metabolic activity is determined in a cell population.
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.
Azathioprine concentrations:10-6 to 10 nM
MMF concentrations: 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.

12. * * * Results ….. BrDU assay
Quantitate cell proliferation based on the measurement of BrdU incorporation during DNA synthesis in proliferating cells.
Determination of the inhibitory or stimulatory effect of various compounds
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 (MMF treated) at 72hrs.

13. 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
Collected for RNA and protein at different time points: 6, 12, 24, 48 hours

14. Results … Western Blot HSCs Serum free Vehicle Aza 0.1 nΜ Aza 1nM
MMF 0.1 ngr/ml
MMF 1ngr/ml
MMF 10ngr/ml
SFM AZA1 AZA2 COMB1 COMB2 TGF
Quantitate cell proliferation based on the measurement of BrdU incorporation during DNA synthesis in proliferating cells.
Determination of the inhibitory or stimulatory effect of various compounds
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.

15. * * * Results … RT - PCR TIMP1 / GAPDH
Quantitate cell proliferation based on the measurement of BrdU incorporation during DNA synthesis in proliferating cells.
Determination of the inhibitory or stimulatory effect of various compounds
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.

16. * ** ** * Results … RT - PCR COL1A1 / GAPDH
Quantitate cell proliferation based on the measurement of BrdU incorporation during DNA synthesis in proliferating cells.
Determination of the inhibitory or stimulatory effect of various compounds ** *
This gene encodes the pro-alpha1 chains of type I collagen. Type I is a fibril-forming collagen.

17. * * ** * ** * Results … RT - PCR Lox / GAPDH
Quantitate cell proliferation based on the measurement of BrdU incorporation during DNA synthesis in proliferating cells.
Determination of the inhibitory or stimulatory effect of various compounds *
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.

18. * ** * * ** * Results … RT - PCR MMP2 / GAPDH
Quantitate cell proliferation based on the measurement of BrdU incorporation during DNA synthesis in proliferating cells.
Determination of the inhibitory or stimulatory effect of various compounds *
Proteins of the matrix metalloproteinase (MMP) family are involved in the breakdown of extracellular matrix

19. 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 myofibroblasts when azathioprine was added.
There was no difference when MMF was used.
There was a significant down-regulation (q-PCR) of LOX and Collagen 1A1 genes, when AZA 1nΜ was applied in SFM as well as in TGF induced HSCs. * ** * * **
Quantitate cell proliferation based on the measurement of BrdU incorporation during DNA synthesis in proliferating cells.
Determination of the inhibitory or stimulatory effect of various compounds *

20. Conclusion
Azathioprine exerted an anti-fibrotic effect in cell proliferation assays as well as in protein and RNA level in h HSC in culture. * ** * * **
Quantitate cell proliferation based on the measurement of BrdU incorporation during DNA synthesis in proliferating cells.
Determination of the inhibitory or stimulatory effect of various compounds