Development of an in vitro human liver system for interrogating nonalcoholic steatohepatitis - PubMed (original) (raw)
Development of an in vitro human liver system for interrogating nonalcoholic steatohepatitis
Ryan E Feaver et al. JCI Insight. 2016.
Abstract
A barrier to drug development for nonalcoholic steatohepatitis (NASH) is the absence of translational preclinical human-relevant systems. An in vitro liver model was engineered to incorporate hepatic sinusoidal flow, transport, and lipotoxic stress risk factors (glucose, insulin, free fatty acids) with cocultured primary human hepatocytes, hepatic stellate cells (HSCs), and macrophages. Transcriptomic, lipidomic, and functional endpoints were evaluated and compared with clinical data from NASH patient biopsies. The lipotoxic milieu promoted hepatocyte lipid accumulation (4-fold increase, P < 0.01) and a lipidomics signature similar to NASH biopsies. Hepatocyte glucose output increased with decreased insulin sensitivity. These changes were accompanied by increased inflammatory analyte secretion (e.g., IL-6, IL-8, alanine aminotransferase). Fibrogenic activation markers increased with lipotoxic conditions, including secreted TGF-β (>5-fold increase, P < 0.05), extracellular matrix gene expression, and HSC activation. Significant pathway correlation existed between this in vitro model and human biopsies. Consistent with clinical trial data, 0.5 μM obeticholic acid in this model promoted a healthy lipidomic signature, reduced inflammatory and fibrotic secreted factors, but also increased ApoB secretion, suggesting a potential adverse effect on lipoprotein metabolism. Lipotoxic stress activates similar biological signatures observed in NASH patients in this system, which may be relevant for interrogating novel therapeutic approaches to treat NASH.
Figures
Figure 1. Adaptation of the in vitro human liver system to mimic human nonalcoholic fatty liver under lipotoxic stress.
(A) Liver sinusoidal hemodynamics were applied to the human liver system using a cone-and-plate viscometer incorporated into a transwell multiculture model of nonparenchymal cells (NPCs) (top of transwell) and hepatocytes (bottom of the transwell). Rotation of the cone (orange triangle) imparts shear stress onto the transwell. Medium is continually perfused to recapitulate interstitial flow, as indicated by the inflow and outflow ports. (B) Representative photomicrographs (original magnification, ×20) of NPCs and hepatocytes are shown. NPCs include hepatic stellate cells (reelin+, green) and macrophages (CD68+, red). Hepatocytes are stained for E-cadherin (green). Nuclei stained with DAPI (blue). Scale bars: 100 μm and 50 μm for NPCs and hepatocytes, respectively. (C) Hepatocyte protein-protein interaction network was visualized and colored according to signaling community. (D) The relative directionality of many pathways identified in C were explored by calculating the FDR-scaled fold change for each, revealing key signaling pathways perturbed at the RNA level by lipotoxic stress. *Rotation gene set tests (ROAST), FDR < 5%.
Figure 2. De novo lipogenesis and cholesterol synthesis are increased in the human liver lipotoxic system.
(A) Representative photomicrographs (original magnification, ×20) of hepatocytes exposed to the healthy or lipotoxic milieu are shown. Hepatocytes are stained for E-cadherin (green), lipid (Nile red+, red), and nuclei (DAPI+, blue). Insets have been magnified to provide greater clarity of lipid droplets. Scale bars: 50 μm. (B) Representative photomicrograph (original magnification, ×100) of hepatocytes exposed to the lipotoxic milieu reveals adipophilin staining (green) around lipid droplets (Nile red+, red). Nuclei stained with DAPI (blue). Scale bar: 100 μm. (C) Nile red staining intensity from hepatocyte images were quantified and represented as fold change relative to healthy controls. n ≥ 11 experiments, 3 donors. ***P < 0.01, Student’s 2-tailed t test. (D and E) Hepatocyte expression of genes of the fatty acid (D) and cholesterol (E) biosynthesis pathways are represented as log2-fold change of lipotoxic vs. healthy milieu (red = upregulation, blue = downregulation). n = 6 experiments, 3 donors.
Figure 3. Hepatic lipid changes in lipotoxic stress and clinical nonalcoholic fatty liver disease.
All values are log2-fold changes in lipid concentrations relative to controls. Red indicates upregulated and blue indicates downregulated expression. Abbreviations: FC, fold change; NAFL, nonalcoholic fatty liver; NASH, nonalcoholic steatohepatitis; SFA, saturated fatty acid; MUFA, mono-unsaturated fatty acid; PUFA, polyunsaturated fatty acid; n-6/n-3, omega-6 fatty acids/omega-3 fatty acids; FFA, free fatty acid; DAG, diacylglycerol; TAG, triacylglycerol; CE, cholesterol ester; PC, phosphatidylcholine; PE, phosphatidylethanolamine; SM, sphingomyelin.
Figure 4. Glucose utilization and synthesis is altered in the human liver lipotoxic system.
(A) Baseline levels of secreted glucose were measured from hepatocytes exposed to the healthy or lipotoxic milieu for 10 days and represented as fold change relative to healthy controls. n ≥ 12 experiments, 5 donors. (B) Hepatocyte expression of genes of the glycolysis and gluconeogenesis pathways are represented as log2-fold change of lipotoxic vs. healthy milieu (red = upregulation, blue = downregulation). n = 6 experiments, 3 donors. (C) Hepatocytes exposed to the healthy or lipotoxic milieu for 10 days were serum starved with or without 100 nM insulin for 3 hours and secreted glucose was measured and represented as fold change relative to baseline healthy controls. n ≥ 9 experiments, 5 donors. (D) Hepatocytes exposed to the healthy or lipotoxic milieu for 10 days were serum starved and then stimulated with or without 10 nM insulin for 10 minutes to measure Akt phosphorylation. Levels of phosphorylated Akt induced by insulin are represented as fold change relative to baseline phosphorylated Akt in the absence of insulin challenge. n = 5 experiments, 3 donors. *P < 0.05, **P < 0.01, Student’s 2-tailed t test. NS, not significant.
Figure 5. Cellular stress and inflammation are induced in the human liver lipotoxic system.
(A and B) Secreted alanine aminotransferase (ALT) (A) and caspase-cleaved cytokeratin 18 (CK18) (B) were measured in the media effluent from devices at day 10. n ≥ 4 experiments, 4 donors. (C) ATP was measured from hepatocytes exposed to the healthy or lipotoxic milieu for 10 days and represented as fold change relative to healthy controls. n ≥ 13 experiments, 5 donors. *P < 0.05, **P < 0.01, Student’s 2-tailed t test. (D) Secreted analytes were measured in the media effluent at days 5, 7, and 10. n ≥ 4 experiments, 4 donors. Triangles indicate samples that were below the lower limit of quantification. *P < 0.05, **P < 0.01, 2-way ANOVA.
Figure 6. Evidence for extracellular matrix remodeling in the human liver lipotoxic system.
(A, C, and D) Secreted TGF-β (A), osteopontin (OPN) (C), and procollagen 1α1 (D) were measured in the media effluent from devices at the indicated days. n ≥ 4 experiments, 4 donors. (B) Hepatocyte expression of genes of the collagen formation pathway are represented as log2-fold change of lipotoxic vs. healthy milieu (red = upregulation, blue = downregulation). n = 6 experiments, 3 donors. (E) Representative photomicrographs (original magnification, ×20) of nonparenchymal cells (NPCs) are shown. Macrophages (CD68+, red), nuclei (DAPI+, blue), and hepatic stellate cells (smooth muscle α-actin+ (SMAA+), green). Scale bars: 100 μm. SMAA staining intensity from immunofluorescent NPCs images were quantified and represented as fold change relative to healthy controls. n ≥ 4 experiments, 3 donors. Triangles indicate samples that were below the lower limit of quantification. *P < 0.05, **P < 0.01, Student’s 2-tailed t test.
Figure 7. Obeticholic acid restores homeostasis in the human liver lipotoxic system.
Nonparenchymal cells (NPCs) and hepatocytes on device were exposed to the lipotoxic milieu containing 0.5 μM obeticholic acid (OCA) or DMSO vehicle control (Veh) for 10 days. (A) Secreted FGF19 was measured in the media effluent from devices at day 10. n = 3 experiments, 1 donor. (B) Nile red staining intensity from hepatocyte images were quantified and represented as fold change relative to healthy controls. n = 4 experiments, 1 donor. (C) Lipids from hepatocytes from device exposed to the healthy or lipotoxic milieu with 0.5 μM OCA or vehicle control were measured by metabolomics. Scatterplot representation of differentially expressed lipids in these hepatocytes are shown as log2-fold change and colored by response similarity index (RSI). Triacyglycerols (○), cholesterol esters (×), all other lipids (•). n = 4 experiments, 2 donors. (D and E) Secreted analytes were measured in the media effluent from devices at day 10. n ≥ 5 experiments, 3 donors. (F) Secreted apolipoproteins were measured in the media effluent from devices at day 10. n = 4 experiments, 2 donors. Triangles indicate samples that were below the lower limit of quantification. *P < 0.05, **P < 0.01, Student’s 2-tailed t test.
References
- U.S. National Institutes of Health . https://clinicaltrials.gov Accessed October 31, 2016.
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