Adenosine A(2A) receptors play a role in the pathogenesis of hepatic cirrhosis

Abstract

1. Adenosine is a potent endogenous regulator of inflammation and tissue repair. Adenosine, which is released from injured and hypoxic tissue or in response to toxins and medications, may induce pulmonary fibrosis in mice, presumably via interaction with a specific adenosine receptor. We therefore determined whether adenosine and its receptors contribute to the pathogenesis of hepatic fibrosis. 2. As in other tissues and cell types, adenosine is released in vitro in response to the fibrogenic stimuli ethanol (40 mg dl(-1)) and methotrexate (100 nM). 3. Adenosine A(2A) receptors are expressed on rat and human hepatic stellate cell lines and adenosine A(2A) receptor occupancy promotes collagen production by these cells. Liver sections from mice treated with the hepatotoxins carbon tetrachloride (CCl(4)) (0.05 ml in oil, 50 : 50 v : v, subcutaneously) and thioacetamide (100 mg kg(-1) in PBS, intraperitoneally) released more adenosine than those from untreated mice when cultured ex vivo. 4. Adenosine A(2A) receptor-deficient, but not wild-type or A(3) receptor-deficient, mice are protected from development of hepatic fibrosis following CCl(4) or thioacetamide exposure. 5. Similarly, caffeine (50 mg kg(-1) day(-1), po), a nonselective adenosine receptor antagonist, and ZM241385 (25 mg kg(-1) bid), a more selective antagonist of the adenosine A(2A) receptor, diminished hepatic fibrosis in wild-type mice exposed to either CCl(4) or thioacetamide. 6. These results demonstrate that hepatic adenosine A(2A) receptors play an active role in the pathogenesis of hepatic fibrosis, and suggest a novel therapeutic target in the treatment and prevention of hepatic cirrhosis.

Figure 1

Hepatotoxins stimulate adenosine release from the hepatoma cell line HepG2. (a) Ethanol treatment of HepG2 cells for 3 h stimulated adenosine release into the supernate as measured by HPLC. At an ethanol concentration of 40 mg dl⁻¹, mean adenosine concentration rose from a control value of 71.5±32.6 to 231±47.6 nM, and this increase was sustained at an ethanol concentration of 80 mg dl⁻¹ (242±35.7 nM) (one-way ANOVA, n=4, P=0.003). (b) Methotrexate treatment of HepG2 cells for 72 h stimulated extracellular adenosine release as measured by HPLC. Methotrexate, at a concentration of 10 nM, increased HepG2 cell adenosine release by three-fold (from 103±46.1 to 301±33.8 nM) and the increase in adenosine release was maintained at a methotrexate concentration of 100 nM (298±14.2 nM, one-way ANOVA, n=4, P=0.023).

Figure 2

Adenosine A_2A receptor occupancy stimulates collagen production by hepatic stellate cells. (a) Phosphorimager detection of high-molecular weight ¹⁴C band identified as collagen in supernates of LX-2 cells (human hepatic stellate cell line). (b) Adenosine A_2A receptor agonist, CGS-21680, promotes collagen production by rat hepatic stellate cells. Stellate cell lines were treated sequentially with ascorbic acid (50 μg ml⁻¹), β-aminoproprionitrile (50 μg ml⁻¹), ¹⁴C-proline (2 μCi ml⁻¹), CGS-21680 (16 or 24 h for rat or human hepatic cell lines, respectively) in the presence or absence of the adenosine A_2A receptor antagonist, CSC (10 μM). Maximal inhibition of the effect of CGS-21680 on collagen production was 86% (from 2045±470 to 271±83% control value, two-way ANOVA, n=9, P<0.001). The suppressive effect of CSC on CGS-21680-induced collagen production was not seen with the adenosine A₁ receptor antagonist, DPCPX (10 μM), or A_2B receptor antagonist, enprofylline (10 μM). Percentage control values for collagen production with CGS-21680 alone vs CGS-21680 with DPCPX vs CGS-21680 with enprofylline were 2045±470 vs 2033±364 vs 1820±351%, respectively (NS, one-way ANOVA, n=9, 6, 5, respectively, for CGS-21680 alone vs CGS-21680 with DPCPX vs CGS-21680 with enprofylline, P=0.93). (c) Collagen was identified as a high-molecular-weight protein (>220 kDa) that was cleavable by collagenase, and measured by phosphoimager quantification of ¹⁴C after adjustment to relative density of protein in Coomassie Blue-stained gels. (d) CGS-21680 treatment of LX-2 cells for 24 h modulated activities of MMP-9 and MMP-2 by gelatin zymography. (e) CGS-21680 treatment of LX-2 cells for 24 h modulated expression of MMP-14 by Western blotting.

Figure 3

There is increased expression of adenosine A_2A receptors in fibrotic liver. (a) Expression of adenosine A_2A receptor was increased in homogenates from fibrotic murine livers compared with normal murine livers by RT–PCR. Expression in murine brain tissue was shown as a positive control. (b) Expression of adenosine A_2A receptor normalized to GAPDH was increased in liver homogenates from thioacetamide-treated animals (n=2 animals each for control and fibrotic mice).

Figure 4

Agents that promote hepatic fibrosis increase hepatic adenosine release. Ex vivo treatment of murine liver slices with thioacetamide or CCl₄ significantly increased the release of adenosine into supernate (from 119±21 to 480±113 or 371±89 nM adenosine, control vs thioacetamide vs CCl₄, n=11, 6 and 5, respectively, ^**P<0.001, ANOVA).

Figure 5

Adenosine A_2A receptor-deficient mice are protected from CCl₄-induced hepatic fibrosis. (a) Adenosine A_2A receptor- or A₃ receptor-deficient mice were treated with the hepatic toxin CCl₄ (0.05 ml in oil, 50 : 50 v : v, subcutaneously, twice weekly for 6 weeks). Hepatic sections were stained with picrosirius red and H&E. (b) Quantification of picrosirius red staining was performed digitally using SigmaScan Pro v.5.0.0, and data are presented as the percentage of total liver area stained by picrosirius red (one-way ANOVA, P<0.001). Percentage hepatic area stained with picrosirius red in CCl₄-treated animals were 5.2±1.7 and 4.6±0.7% for wild-type control mice and A₃ receptor-deficient mice, respectively, whereas hepatic slices from A_2A receptor-deficient mice showed markedly less fibrosis with 1.2±0.3% picrosirius red staining (n=28, 12, 4 for A_2A receptor-deficient mice, wild-type controls and A₃ receptor-deficient mice, respectively; one-way ANOVA, ^**P<0.001).

Figure 6

Adenosine A_2A receptor-deficient mice are protected from thioacetamide-induced hepatic fibrosis. (a) Adenosine A_2A receptor- or A₃ receptor-deficient mice were treated with thioacetamide (100 mg kg⁻¹, intraperitoneally, three times weekly for 9 weeks). Hepatic sections were stained with picrosirius red. (b) Quantification of picrosirius red staining was performed digitally using SigmaScan Pro v.5.0.0, and data are presented as the percentage of total liver area stained by picrosirius red. Percentage hepatic area stained with picrosirius red in thioacetamide-treated animals were 9.0±2.7 and 10.7±2.8% for wild-type control mice and A₃ receptor-deficient mice, respectively, whereas hepatic slices from A_2A receptor-deficient mice showed markedly less fibrosis with 0.9±0.4% picrosirius red staining (n=28, 12, 4 for A_2A receptor-deficient mice, wild-type controls and A₃ receptor-deficient mice, respectively; one-way ANOVA, ^**P<0.001).

Figure 7

Adenosine A_2A receptor antagonism protects mice from chemical-induced hepatic fibrosis. C57BL/6 mice were treated with the hepatic toxins CCl₄ (0.05 ml in oil, 50 : 50 v : v, subcutaneously, twice weekly for 6 weeks) or thioacetamide (100 mg kg⁻¹, intraperitoneally, three times weekly for 7 weeks) in the presence or absence of adenosine receptor antagonists DPCPX (A₁ receptor, 50 mg kg⁻¹ day⁻¹ orally), enprofylline (A_2B receptor, 50 mg kg⁻¹ day⁻¹ orally), caffeine (nonselective, 50 mg kg⁻¹ day⁻¹ orally) or ZM-241385 (A_2A receptor, 25 mg kg⁻¹ twice daily intraperitoneally in vehicle consisting of 15% Cremophor EL, 15% DMSO, 70% water. All mice also received intraperitoneal injections of vehicle in addition to any other treatments received). Hepatic sections were stained with picrosirius red. Quantification of picrosirius red staining was performed digitally. (a) Percentage area of picrosirius red-stained hepatic tissue in CCl₄-treated mice were 4.6±0.5, 5.1±0.7, 4.4±0.9, 2.6±0.4 and 1.2±0.2% for control, DPCPX, enprofylline, caffeine and ZM-241385-treated mice, respectively (n=12, 8, 8, 16, 8 for control, DPCPX, enprofylline, caffeine and ZM-241385-treated mice, respectively, one-way ANOVA, P<0.001). (b) Percentage area of picrosirius red-stained hepatic tissue in thioacetamide-treated mice were 5.7±1.2, 6.5±1.2, 6.2±0.7, 3.8±1.1 and 1.0±0.2% for control, DPCPX, enprofylline, caffeine and ZM-241385-treated mice, respectively (n=11, 5, 9, 12, 13 for control, DPCPX, enprofylline, caffeine and ZM-241385-treated mice, respectively, one-way ANOVA, ^**P<0.001).