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, UK2 1st Division of Internal Medicine and Division of Gastroenterology, University of

Ioannina, Greece3 School of Applied Technology, Department of Computer Engineering,

Technological Educational Institute of Epirus, Greece

Background

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.

p=0.045

p=0.045

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 use AZA in one arm ?

Why did we get criticism ?

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/dayCurrently, 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

Early use of MMF to prevent ACR episodes

Hazard curves for Ishak stage 4

MT

TT

p=0.005 by Mantel-Cox

time to reach S4 (months post LT)

17 patients discontinued azathioprine (10 – 37m) post LT.

time to reach S4 (months post LT)

MT

TT – still on AZAn=31

TT – AZA discontn=17

p=0.003 by Mantel-Cox

Manousou Gut 2014

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).

Transplant free survival in PSC is influenced by AZA and steroids, independent of the presence of IBD

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, 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.

Results ….. MTT assay

Azathioprine concentrations:10-6 to 10 nMMMF concentrations: 10-6 to 10 ngr/ml

O.D.

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

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.

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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

HSCs Serum free Vehicle

MMF 0.1 ngr/ml

MMF 1ngr/ml

MMF 10ngr/ml

Results … Western Blot

HSCs Serum free Vehicle

Aza 0.1 nΜ Aza 1nM Aza 10nM

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.

SFM AZA1 AZA2 COMB1 COMB2 TGF

Results … RT - PCR

TIMP1 / GAPDH

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The proteins encoded by this gene family are natural inhibitors of thematrix metalloproteinases (MMPs), a group of peptidases involved in

degradation of the extracellular matrix.

Results … RT - PCR

COL1A1 / GAPDH

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This gene encodes the pro-alpha1 chains of type I collagen. Type I is a fibril-forming collagen.

Results … RT - PCR

Lox / GAPDH

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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

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Proteins of the matrix metalloproteinase (MMP) family are involved in the breakdown of extracellular matrix

Conclusion

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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. 

Conclusion

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**Azathioprine exerted an anti-fibrotic effect in cell

proliferation assays as well as in protein and RNA level in h HSC in culture.