Lipid-induced hepatocyte-derived extracellular vesicles regulate hepatic stellate cell via microRNAs targeting PPAR-γ.

Background&aimsHepatic stellate cells (HSCs) play a key role in liver fibrosis in various chronic liver disorders including nonalcoholic fatty liver disease (NAFLD). The development of liver fibrosis requires a phenotypic switch from quiescent to activated HSCs. The triggers for HSCs activation in NAFLD remain poorly understood. We investigated the role and molecular mechanism of extracellular vesicles (EVs) released by hepatocytes during lipotoxicity in modulation of HSC phenotype.MethodsEVs were isolated from fat-laden hepatocytes by differential centrifugation and incubated with HSCs. EV internalization and HSCs activation, migration and proliferation were assessed. Loss- and gain-of-functions studies were performed to explore the potential role of PPAR-γ-targeting miRNAs carried by EVs into HSC.ResultsHepatocyte-derived EVs released during lipotoxicity are efficiently internalized by HSCs resulting in their activation, as shown by marked up-regulation of pro-fibrogenic genes (Collagen-I, α-SMA and TIMP-2), proliferation, chemotaxis and wound healing responses. These changes were associated with miRNAs shuttled by EVs and suppression of PPAR-γ expression in HSC. Hepatocyte-derived EVs miRNA content included various miRNAs that are known inhibitors of PPAR-γ expression with miR-128-3p being the most effectively transferred. Furthermore loss- and gain-of-function studies identified miR-128-3p as a central modulator of the effects of EVs on PPAR-γ inhibition and HSC activation.ConclusionOur findings demonstrate a link between fat-laden hepatocyte-derived EVs and liver fibrosis and have potential implications for the development of novel anti-fibrotic targets for NAFLD and other fibrotic diseases

Caspase-1 as a central regulator of high fat diet-induced non-alcoholic steatohepatitis.

Nonalcoholic steatohepatitis (NASH) is associated with caspase activation. However, a role for pro-inflammatory caspases or inflammasomes has not been explored in diet-induced liver injury. Our aims were to examine the role of caspase-1 in high fat-induced NASH. C57BL/6 wild-type and caspase 1-knockout (Casp1(-/-)) mice were placed on a 12-week high fat diet. Wild-type mice on the high fat diet increased hepatic expression of pro-caspase-1 and IL-1β. Both wild-type and Casp1(-/-) mice on the high fat diet gained more weight than mice on a control diet. Hepatic steatosis and TG levels were increased in wild-type mice on high fat diet, but were attenuated in the absence of caspase-1. Plasma cholesterol and free fatty acids were elevated in wild-type, but not Casp1(-/-) mice, on high fat diet. ALT levels were elevated in both wild-type and Casp1(-/-) mice on high fat diet compared to control. Hepatic mRNA expression for genes associated with lipogenesis was lower in Casp1(-/-) mice on high fat diet compared to wild-type mice on high fat diet, while genes associated with fatty acid oxidation were not affected by diet or genotype. Hepatic Tnfα and Mcp-1 mRNA expression was increased in wild-type mice on high fat diet, but not in Casp1(-/-) mice on high fat diet. αSMA positive cells, Sirius red staining, and Col1α1 mRNA were increased in wild-type mice on high fat diet compared to control. Deficiency of caspase-1 prevented those increases. In summary, the absence of caspase-1 ameliorates the injurious effects of high fat diet-induced obesity on the liver. Specifically, mice deficient in caspase-1 are protected from high fat-induced hepatic steatosis, inflammation and early fibrogenesis. These data point to the inflammasome as an important therapeutic target for NASH

Adiposity is increased in caspase-1 knockout mice.

<p>Mice on the control and high fat diets were analyzed for body adiposity. Fat content of subcutaneous (B) and visceral (C) depots were calculated from three images. Total adipose volume was calculated from subcutaneous and visceral depots (D). Adiposity was normalized to body weight and expressed as% adipose (E). Lean mass was calculated based on the density of adipose tissue and normalized to body weight and expressed as% lean mass (F). Values represent means ± SEM. Values with different superscripts are significantly different from one another (<i>p</i><0.05). n = 5 C57BL/6, n = 4 <i>Casp1^-/-</i>.</p

Caspase-1 and IL-1β expression is increased after high fat feeding.

<p>Livers of wild-type mice on the control or high fat diet were analyzed for pro-caspase-1 and IL-1β expression by immunoblot (A). Densitometric analysis was done for each protein normalized to HSC70 (B and C). mRNA for IL-1 expression in the liver of mice on the control or high fat diet was analyzed by RT-PCR (D). IL-1 was assessed by ELISA in whole liver tissue (E). Values with different superscripts are significantly different from one another (<i>p</i><0.05). n = 4 control, n = 6 high fat.</p

High fat-induced expression of inflammatory cytokines and chemokines is attenuated in caspase-1 knockout mice.

<p>Inflammatory cytokine/chemokine production in the liver is associated the early stages of NASH. mRNA expression for Tnfα (A), Mcp-1 (B) and F4/80 (C) in the liver of wild-type mice on control or high fat diets was performed by RT-PCR. Values represent means ± SEM. Values with different superscripts are significantly different from one another (<i>p</i><0.05). n = 4 control, n = 6 high fat.</p

Caspase-1 knockout mice are protected from high fat-induced hepatic steatosis.

<p>Representative images of H&E stained livers in wild-type and <i>Casp1^-/-</i> mice on control or high fat diet (10x) (A). NAFLD activity score of wild-type and <i>Casp1^-/-</i> mice on the high fat diet was assessed by histopathology of H&E stained livers in a blinded-fashion by BPG (B). Hepatic triglyceride (TG) levels were measured biochemically from mice on the control or high fat diets (C). Plasma alanine aminotransferase (ALT) levels were analyzed from mice on the control or high fat diets (D). Plasma cholesterol (E), FFA (F) and TG levels (G) were measured biochemically as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056100#s2" target="_blank">methods</a> section. Values represent means ± SEM. Values with different superscripts are significantly different from one another (<i>p</i><0.05). n = 4 control, n = 6 high fat.</p

Histopathological analysis of mice on the high fat diet.

<p>Pathologist: BPG. Data are represented as Mean ± SEM.</p><p>H&E slides of the livers of mice on the control and high fat diets were analyzed for steatosis, inflammation, and ballooning. H&E sections were scored in a blinded fashion by BPG. Values represent means ± SEM. n = 4 control, n = 6 high fat.</p