BaCKgRoUND aND aIMS:In clinical and experimental NASH, the origin of the scar-forming myofibroblast is the HSC. We used foz/foz mice on a Western diet to characterize in detail the phenotypic changes of HSCs in a NASH model. appRoaCH aND ReSUltS:We examined the singlecell expression profiles (scRNA sequencing) of HSCs purified from the normal livers of foz/foz mice on a chow diet, in NASH with fibrosis of foz/foz mice on a Western diet, and in livers during regression of NASH after switching back to a chow diet. Selected genes were analyzed using immunohistochemistry, quantitative real-time PCR, and short hairpin RNA knockdown in primary mouse HSCs. Our analysis of the normal liver identified two distinct clusters of quiescent HSCs that correspond to their acinar position of either pericentral vein or periportal vein. The NASH livers had four distinct HSC clusters, including one representing the classic fibrogenic myofibroblast. The three other HSC clusters consisted of a proliferating cluster, an intermediate activated cluster, and an immune and inflammatory cluster. The livers with NASH regression had one cluster of inactivated HSCs, which was similar to, but distinct from, the quiescent HSCs. CoNClUSIoNS: Analysis of single-cell RNA sequencing in combination with an interrogation of previous studies revealed an unanticipated heterogeneity of HSC phenotypes under normal and injured states. (Hepatology 2021;74:667-685).C hronic liver injury of any etiology produces inflammation with the subsequent activation of mesenchymal cells to become myofibroblasts. In turn, myofibroblasts secrete the extracellular matrix (ECM), which produces the fibrous scar of liver fibrosis. (1,2) In experimental liver fibrosis including chronic carbon tetrachloride (CCl 4 ), chronic ethanol intake, and NASH, HSCs activate to become myofibroblasts. (3)(4)(5) Similarly, in human liver diseases, such as HBV, HCV, and NASH, the origin of myofibroblasts appears to be activated HSCs (aHSCs). (1,6,7) Other cell types such as mesothelial cells (important in capsular fibrosis), (8) portal fibroblasts (important in cholestatic fibrosis), (9) and fibrocytes (recruited to the inflamed liver) (10) make small contributions to hepatotoxic-induced liver fibrosis. Cell fate mapping in the injured and recovered liver has identified three distinct HSC phenotypes; the quiescent HSC (qHSC) in the normal liver, the aHSC in the fibrogenic liver, and the inactivated HSC (iHSC) in the liver with regression of fibrosis.

Background & Aims: Although there are associations among oxidative stress, NADPH oxidase (NOX) activation, and hepatocellular carcinoma (HCC) development, it is not clear how NOX contributes to hepatocarcinogenesis. We studied the functions of different NOX proteins in mice following administration of a liver carcinogen. Methods: Fourteen-day-old Nox1−/− mice, Nox4−/– mice, Nox1−/−; Nox4−/− (double knockout) mice, and wild-type (WT) C57BL/6 mice were given a single intraperitoneal injection of diethylnitrosamine (DEN) and liver tumors were examined at 9 months. We also studied the effects of DEN in mice with disruption of Nox1 specifically in hepatocytes (Nox1ΔHep), hepatic stellate cells (Nox1ΔHep), or macrophage (Nox1ΔMac). Some mice were also given injections of the NOX1 specific inhibitor ML171. To study the acute effects of DEN, 8–12 week old mice were given a single intraperitoneal injection, and liver and serum were collected at 72 hrs. Liver tissues were analyzed by histology, quantitative PCR, and immunoblots. Hepatocytes and macrophages were isolated from WT and knockout mice and analyzed by immunoblots. Results: Nox4−/− mice and WT mice developed liver tumors within 9 months after administration of DEN, whereas Nox1−/− mice developed 80% fewer tumors, which were 50% smaller than those of WT mice. Nox1ΔHep and Nox1ΔHSC mice developed liver tumors of the same number and size as WT mice, whereas Nox1ΔMac developed fewer, smaller tumors, similar to Nox1−/− mice. Following DEN injection, levels of tumor necrosis factor (TNF), interleukin 6 (IL6) and phosphorylated STAT3 were increased in livers from WT, but not Nox1−/− or Nox1ΔMac mice. Conditioned medium from necrotic hepatocytes induced expression of NOX1 in cultured macrophages, followed by expression of TNF, IL6, and other inflammatory cytokines; this medium did not induce expression of IL6 or cytokines in Nox1ΔMac macrophages. WT mice given DEN followed by ML171 developed fewer and smaller liver tumors than mice given DEN followed by vehicle. Conclusions: In mice given injections of a liver carcinogen (DEN), expression of NOX1 by macrophages promotes hepatic tumorigenesis by inducing the production of inflammatory cytokines. We propose that upon liver injury, damage-associated molecular patterns released from dying hepatocytes activate liver macrophages to produce cytokines that promote tumor development. Strategies to block NOX1 or these cytokines might be developed to slow HCC progression.

Kinetic study of ethanol steam reforming over a commercial nickel−magnesia−alumina (Ni/MgO/Al2O3) catalyst was conducted in a fixed-bed reactor 15 mm in diameter. The effects of temperature (673−873 K), molar ratio of steam to ethanol in the feed (in the range of 3:1 to 18:1), feed flow rate (W/F EtOH = 46.2−555.25 g-cat min/mol), catalyst particle size (2.25−0.75 mm), and time-on-stream study was studied. Maximum conversion (>95%) was obtained at 873 K, with a molar ratio of steam to ethanol of 12:1 and a W/F EtOH value of >185 g-cat min/mol at atmospheric pressure. A maximum yield of 3.0 moles of hydrogen per mole of ethanol fed was obtained at a temperature of 873 K, a steam-to-ethanol molar feed ratio of 12:1, and a W/F EtOH value of >110 g-cat min/mol. The acquired data was fitted to a power-law kinetic model and the kinetic parameters were evaluated. The activation energy was determined to be 23 kJ/mol. The average absolute deviation (AAD) for the predicted rates of reaction was determined to be 10.2%. The work also tested the feasibility of using the Eley−Rideal mechanism proposed in the literature and concludes that a more-elaborate scheme of reactions is necessary to describe the complex reactions that occur during the steam reforming process. A considerable amount of coke formation was observed during the process; yet, the catalyst showed a negligible loss of activity, exhibiting the feasibility of using this catalyst for ethanol steam reforming. In an attempt to reduce this coke formation, it is suggested that the process may be performed in the presence of hydrogen gas.

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