Ecto-5'-nucleotidase (CD73) -mediated extracellular adenosine production plays a critical role in hepatic fibrosis Zhongsheng Peng * , Patricia Fernandez * , Tuere Wilder * , Herman Yee † , Luis Chiriboga † , Edwin S. L. Chan * and Bruce N. Cronstein * ,1 * Department of Medicine, Divisions of Clinical Pharmacology and Rheumatology; and † Department of Pathology, New York University School of Medicine, New York, New York, USA 1 Correspondence: Department of Medicine, Division of Clinical Pharmacology, New York University School of Medicine, 550 First Avenue, New York, NY 10016, USA. E-mail: cronsb01@med.nyu.edu Adenosine is a potent endogenous regulator of tissue repair that is released from injured cells and tissues. Hepatic fibrosis results from chronic hepatic injury, and we have previously reported that endogenously generated adenosine, acting at A 2A receptors, plays a role in toxin-induced hepatic fibrosis. Adenosine may form intracellularly and then be transported to the extracellular space or it may form extracellularly from adenine nucleotides released from injured cells. Because ecto-5'-nucleotidase (CD73) catalyzes the terminal step in extracellular adenosine formation from AMP, we determined whether CD73 plays a role in the development of hepatic fibrosis. Mice were treated overnight with PBS, CCl 4 , ethanol, or thioacetamide (TAA); their livers were harvested, and slices were incubated in medium for 20 h before adenosine concentration in the supernatant was measured by HPLC. Hepatic fibrosis was induced by CCl 4 or TAA treatment in CD73 knockout (CD73KO and C57BL/6 background) and C57BL/6 control mice wild-type (WT) mice and quantified by digital analysis of picrosirius red stained slides and hydroxyproline content. mRNA expression was quantified by real-time polymerase chain reaction, and protein was quantified by Western blot or enzyme-linked immunosorbent assay. Livers from WT mice treated with CCl 4 , ethanol, and TAA released 2- to 3-fold higher levels of adenosine than livers from comparably treated CD73KO mice. CD73KO mice were protected from fibrosis with significantly less collagen content in the livers of CD73KO than WT mice after treatment with either CCl 4 or TAA. There were far fewer α-smooth muscle actin positive hepatic stellate cells in CCl 4 -treated KO mice than that in WT mice. After CCl 4 treatment, the mRNA level of A 1 , A 2A , A 2B , and A 3 adenosine receptors, tumor necrosis factor-α, interleukin (IL) -1β, IL-13rα1, matrix metalloproteinase (MMP)-2, MMP-14, tissue inhibitor of metalloproteinase (TIMP) -1, and TIMP-2, and IL-13 level increased markedly in both CD73KO and WT mice, but Col1α1, Col3α1, and transforming growth factor-β1 mRNA increased much more in WT mice than that in KO mice. Moreover, IL-13rα2, MMP-13 mRNA, and MMP-13 protein were higher in KO mice than that in WT mice. These results indicate that adenosine, formed extracellularly from adenine nucleotides, plays a major role in the pathogenesis of hepatic fibrosis and that inhibition of adenosine production or blockade of adenosine receptors may help prevent hepatic fibrosis.—Peng, Z., Fernandez, P., Wilder, T., Yee, H., Chiriboga, L., Chan, E. S. L., Cronstein, B. N. Ecto-5'-nucleotidase (CD73) -mediated extracellular adenosine production plays a critical role in hepatic fibrosis. Key Words: stellate cell • carbon tetrachloride • thioacetamide

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