ABSTRACT
BACKGROUND: Chronic ethanol overconsumption promotes alcohol-associated liver disease (ALD), characterized by hepatocyte injury, inflammation, hepatic stellate cell (HSC) activation, and fibrosis. Hyaluronan (HA) concentration is greater in livers and blood from advanced ALD patients than patients with advanced non-ALD. In the liver, HSCs are the major HA producers. The relationship between ethanol, HA, and HSC activation is incompletely understood. Thus, here, we tested the hypothesis that ethanol enhances HSC activation in a HA-dependent manner. METHODS: Liver tissue microarrays (TMAs) containing steatotic livers from donors with or without a history of alcohol consumption were used to measure HA and collagen content. Mice were fed a moderate (2%, v/v) ethanol-containing diet or pair-fed control diet for 2 days, after which they were given a single carbon tetrachloride (CCl4 ) injection. To inhibit HA synthesis, we provided 4-methylumbelliferone (4MU) daily. We used LX2 cells, a human HSC cell line, to determine the impact ethanol had on LPS responses, with or without concurrent 4MU exposure. RESULTS: CCl4 induced liver injury, but it did not differ between ethanol or control diet fed mice with or without 4MU treatment. Ethanol feeding enhanced CCl4 -induced hepatic HA content, which was paralleled by HA synthase (Has)2 transcript abundance; 4MU treatment normalized both. Consistently, HSC activation, assessed by measuring αSMA mRNA and protein, was induced by CCl4 exposure, enhanced by ethanol feeding, and normalized by 4MU. Hepatic transcripts, but not protein, for Ccl2 were enhanced by ethanol feeding and normalized by 4MU exposure. Finally, ethanol-exposed LX2 cells made more LPS-stimulated CCL2 mRNA and protein than cells not exposed to ethanol; 4MU prevented this. CONCLUSION: These data show that ethanol augments HSC activation through HA synthesis and enhances hepatic profibrogenic features. Therefore, targeting HSC HA production could potentially attenuate liver disease in ALD patients.
ABSTRACT
Hyaluronan (HA) is a ubiquitous component of the extracellular matrix. The spatial-temporal localization of HA can be visualized in situ using biotinylated HA binding proteins (HABPs). This assay is sensitive to fixation conditions, and there are currently no best practices for HA detection. Thus, the goal of this study was to optimize fixation conditions for visualizing HA in the ovary, kidney, and liver through analysis of six commonly used fixatives for HA detection: Bouin's Solution, Carnoy's Solution, Ethanol-Formalin-Glacial Acetic Acid (EFG), Histochoice, Modified Davidson's Solution, and 10% Neutral Buffered Formalin. Organs were harvested from CB6F1 mice and fixed with one of the identified fixatives. Fixed organs were sectioned, and the HABP assay was performed on sections in parallel. Hematoxylin and eosin staining was also performed to visualize tissue architecture. HABP signal localization and intensity varied between fixatives. EFG and Carnoy's Solution best preserved the HA signal intensity in the ovary and liver, showing HA localization in various sub-organ structures. In the kidney, only Modified Davidson's Solution was less than optimal. Our findings demonstrate that fixation can alter the ability to detect HA in tissue macro- and microstructures, as well as localization in a tissue-specific manner, in situ.
