Development of an in vitro human liver system for interrogating nonalcoholic steatohepatitis

The lipidomic signature of the in vitro lipotoxic model was significantly similar to that of NAFL/NASH patient biopsies.

“a lipidomics signature similar to NASH biopsies.”

• Also stated in: § Hepatic steatosis. · plain

  “Comparing our in vitro data with previously published lipidomics performed on NAFL/NASH patient livers (20) revealed significant similarity (Pearson correlation P values < 0.002 compared with NAFL and NASH biopsies) between lipidomic signatures (Figure 3).”

• cites [20] sourceNot checked yet

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Exposure to the lipotoxic milieu increased hepatocyte lipid accumulation about fourfold compared with healthy conditions.

“The lipotoxic milieu promoted hepatocyte lipid accumulation (4-fold increase, P < 0.01)”

• Also stated in: § Hepatic steatosis. · plain

  “a 4-fold increase in steatosis was observed in lipotoxic conditions relative to healthy conditions (Figure 2C).”

Akt phosphorylation was significantly lower in hepatocytes exposed to the lipotoxic milieu than in those exposed to the healthy milieu.

“Akt exhibited a significant reduction in phosphorylation in hepatocytes exposed to the lipotoxic compared with the healthy milieu (12-fold versus 4-fold, respectively, Figure 4D)”

Obeticholic acid mostly repressed triacylglycerol species that were elevated under lipotoxic conditions.

“TAG species were elevated in lipotoxic conditions (Figure 3) and mostly repressed by OCA”

Obeticholic acid reduced inflammatory and fibrotic secreted factors in the lipotoxic model.

“reduced inflammatory and fibrotic secreted factors”

• Also stated in: § T E C H N I C A L A D V A N C E · plain

  “circulating cell stress and inflammatory factors were reduced at the conclusion of the experiment, including ALT (16% reduced, P = 0.10), MCP1 (66% reduced), IL-6 (48% reduced), and IL-8 (25% reduced) (Figure 7D).”

ApoCII and ApoE were also increased with obeticholic acid treatment.

“Other apolipoproteins, including ApoCII and ApoE were also increased (Figure 7F).”

The in vitro liver system maintained differentiated cell types, as shown by CD68 and reelin expression in macrophages and hepatic stellate cells and E-cadherin in hepatocytes.

“CD68 and reelin, markers for MΦs and HSCs, respectively (12,13), were expressed in our system at the end of the experiment, reflecting the maintenance of the cell types in a differentiated state.”

• Also stated in: § Recapitulation of liver tissue architecture. · plain

  “E-cadherin, a marker of cell-cell junctions, depicts the retention of polarized morphology by differentiated hepatocytes in our system.”

• cites [12] sourceNot checked yet

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• cites [13] sourceNot checked yet

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YKL40 increased sixfold and fourfold at days 7 and 10, respectively, in the lipotoxic milieu compared with healthy conditions.

“YKL40 increased 6-and 4-fold at days 7 and 10, respectively, in the lipotoxic milieu over the healthy milieu conditions (Figure 5D).”

Hepatocytes exposed to the lipotoxic milieu produced about threefold more glucose than those exposed to a healthy milieu.

“Hepatocyte glucose output increased with decreased insulin sensitivity.”

• Also stated in: § Dysregulation of glucose/insulin signaling. · plain

  “these hepatocytes produced 3-fold more glucose than those exposed to a healthy milieu (Figure 4A).”

ApoB was increased twofold with obeticholic acid treatment.

“ApoB, primarily found in LDL, was increased 2-fold (Figure 7F).”

Obeticholic acid reduced circulating cell stress and inflammatory factors including ALT, MCP1, IL-6, and IL-8.

“circulating cell stress and inflammatory factors were reduced at the conclusion of the experiment, including ALT (16% reduced, P = 0.10), MCP1 (66% reduced), IL-6 (48% reduced), and IL-8 (25% reduced) (Figure 7D).”

Obeticholic acid at 0.5 micromolar promoted a healthy lipidomic signature in the lipotoxic model.

“0.5 μM obeticholic acid in this model promoted a healthy lipidomic signature”

• Also stated in: § T E C H N I C A L A D V A N C E · plain

  “OCA treatment in the lipotoxic milieu promotes a change in lipid profile towards that of the healthy controls”

Obeticholic acid potently induced FGF19 secretion by 25-fold.

“OCA was found to potently induce secretion by 25-fold (Figure 7A).”

VEGF levels increased at day 5 in the lipotoxic milieu.

“we found levels of VEGF to be increased at day 5.”

Of 767 detectable lipid species, 449 were significantly regulated under lipotoxic conditions.

“Out of 767 detectable lipid species, 449 were significantly regulated (FDR ≤ 5%).”

Over the tested time course, proinflammatory cytokines IL-8, IL-6, and CXCL10 increased in response to the lipotoxic milieu.

“Over the tested time course, several proinflammatory cytokines increased, including IL-8, IL-6, and CXCL10”

The in vitro lipotoxic model showed strong agreement with NASH patient biopsy datasets for key pathways including fatty acid synthesis, gluconeogenesis, and inflammatory signaling.

“Strong agreement was observed across all 3 datasets for these key pathways associated with NASH, particularly with the fatty acid synthesis, gluconeogenesis, and inflammatory signaling pathways.”

Secreted procollagen 1alpha1 showed no significant changes between healthy and lipotoxic conditions at the tested time points.

“Secreted procollagen 1α1 showed no significant changes at the tested time points between healthy and lipotoxic conditions (Figure 6D).”

There was significant pathway correlation between the in vitro model and human NASH biopsies.

“Significant pathway correlation existed between this in vitro model and human biopsies.”

• Also stated in: § Recapitulation of liver tissue architecture. · boosted

  “Strong agreement was observed across all 3 datasets for these key pathways associated with NASH, particularly with the fatty acid synthesis, gluconeogenesis, and inflammatory signaling pathways.”

Obeticholic acid decreased lipid accumulation by 25% relative to vehicle.

“Lipid accumulation was decreased by 25% with OCA treatment relative to the vehicle (Figure 7B)”

Lipotoxic conditions increased secretion of inflammatory analytes, including IL-6, IL-8, and ALT.

“These changes were accompanied by increased inflammatory analyte secretion (e.g., IL-6, IL-8, alanine aminotransferase).”

• Also stated in: § Dysregulation of glucose/insulin signaling. · plain

  “we observed small, but significant increases in both ALT (1.4-fold, Figure 5A) and CK18 (2.6-fold, Figure 5B) with the lipotoxic milieu compared with the healthy condition on day 10.”

The lipotoxic milieu significantly regulated 639 pathways spanning a broad range of metabolic functions.

“639 pathways were significantly regulated (complete list in Supplemental Materials), spanning a broad range of metabolic functions.”

Obeticholic acid treatment increased total intracellular cholesterol levels.

“total intracellular levels of cholesterol were increased with OCA treatment (Supplemental Figure 9).”

Elevated ATP levels correlated with upregulation of the beta-oxidation gene expression pathway.

“elevated ATP levels correlated with an upregulation of the β-oxidation gene expression pathway (Supplemental Figure 5).”

Obeticholic acid increased ApoB secretion in the lipotoxic model.

“but also increased ApoB secretion”

• Also stated in: § T E C H N I C A L A D V A N C E · plain

  “ApoB, primarily found in LDL, was increased 2-fold (Figure 7F).”

Several cholesterol ester species were regulated in the opposite direction to the healthy response, i.e., increased, with obeticholic acid.

“Several species of CEs were regulated in the opposite direction (RSI < 0, green) of the healthy response (i.e., increased) as seen in the RSI plot (Figure 7C)”

Lipotoxic conditions promoted a significant fourfold increase in ATP levels.

“lipotoxic conditions promoted a significant 4-fold increase (Figure 5C).”

Obeticholic acid significantly reduced procollagen 1alpha1 levels by 59%.

“procollagen 1α1 levels were significantly reduced (59% reduced) with OCA treatment in lipotoxic conditions relative to the vehicle (Figure 7E).”

PCK1 was upregulated by the lipotoxic milieu.

“PCK1 was upregulated by the lipotoxic milieu (Figure 4B)”

Average gene expression for key pathways, except cholesterol biosynthesis, was significantly elevated under lipotoxic conditions.

“The average gene expression for each of these pathways (with the exception of cholesterol biosynthesis) was significantly elevated (FDR < 5%).”

Cellular stress and inflammation are induced in the human liver lipotoxic system.

“Cellular stress and inflammation are induced in the human liver lipotoxic system.”

Glucose utilization and synthesis are altered in the human liver lipotoxic system.

“Glucose utilization and synthesis is altered in the human liver lipotoxic system.”

Obeticholic acid restores homeostasis in the human liver lipotoxic system.

“Obeticholic acid restores homeostasis in the human liver lipotoxic system.”

Lipotoxic stress perturbs key signaling pathways at the RNA level in the human liver system.

“revealing key signaling pathways perturbed at the RNA level by lipotoxic stress.”

Extracellular matrix remodeling occurs in the human liver lipotoxic system.

“Evidence for extracellular matrix remodeling in the human liver lipotoxic system.”

The response to obeticholic acid in the lipotoxic system correlated with outcomes observed in clinical trials.

“Overall, the response to OCA in the lipotoxic system correlated to outcomes observed in clinical trials that have not been recapitulated in other in vitro systems, validating the system for evaluation of new therapeutic targets in the future.”

Hepatocytes can secrete FGF19.

“we have previously found that hepatocytes can also secrete FGF19 (Dash et al., unpublished data).”

• cites Dash et al., unpublished dataNot checked yet

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The lipotoxic milieu concentrations were derived from differences between plasma levels of healthy and NASH patients.

“at concentrations derived from the differences between the plasma levels of healthy and NASH patients (14,15).”

• cites [14] sourceNot checked yet

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• cites [15] sourceNot checked yet

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Obeticholic acid improved histological features of NASH and fibrosis in phase IIb clinical trials.

“OCA is a semisynthetic bile acid analogue that improved histological features of NASH and fibrosis in phase IIb clinical trials (35).”

• cites [35] sourceNot checked yet

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Applying liver sinusoidal hemodynamics and interstitial fluid transport parameters restores a mature, differentiated, in vivo hepatocyte phenotype and function.

“We previously described an approach that applies liver sinusoidal hemodynamics and interstitial fluid transport parameters to restore a mature, differentiated, in vivo hepatocyte phenotype and function”

• cites [7] sourceNot checked yet

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• cites [8] sourceNot checked yet

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The transcriptomic response of hepatocytes to the lipotoxic milieu was reproducible across repeated studies, with a highly significant gene correlation.

“the study was repeated and reproducibility was evaluated using a gene correlation plot, where the correlation was highly significant (Pearson correlation = 0.90, P value < 2.2 × 10 -16”

The in vitro lipotoxic system mimics important signaling aspects of NAFL and early NASH, with the exception of late-stage fibrosis, cirrhosis, and oncogenesis.

“These comparisons provide clinical validation that our in vitro lipotoxic system mimics important signaling aspects of NAFL and early NASH, as outlined in Supplemental Table 1, with the exception of late-stage fibrosis, cirrhosis, and oncogenesis.”

The lipotoxic liver system shows responsiveness to in vivo-relevant concentrations of risk factors and drugs.

“This lipotoxic liver system shows responsiveness to in vivo-relevant concentrations of risk factors and drugs”

The upregulation of PCK1 suggests that the hepatocytes became unresponsive to insulin.

“suggesting that the cells have become unresponsive to insulin.”

Lipotoxic stress in this system activates biological signatures similar to those observed in NASH patients.

“Lipotoxic stress activates similar biological signatures observed in NASH patients in this system”

Obeticholic acid at 0.5 micromolar approximates the steady-state blood serum concentration observed in clinical trials.

“at a concentration of 0.5 μM, which approximates the steady-state blood serum concentration of OCA observed in clinical trials (36).”

• cites [36] sourceNot checked yet

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Primary human hepatocytes, hepatic stellate cells, and macrophages are key players in the progression of NAFL/NASH.

“our multicellular system is composed of primary human hepatocytes, HSCs, and MΦs, which are all key players in the progression of NAFL/NASH (9).”

• cites [9] Paschos, 2009, Non alcoholic fatty liver disease and metabolic syndrome, HippokratiaNot checked yet

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Macrophage phenotype was confirmed by expression of CD68 and CD163.

“MΦ phenotype was confirmed by expression of MΦ-specific markers, CD68 and CD163 (BD Pharmingen, 556059 and Abcam, ab174867, respectively).”

The relevance of these models to humans is questionable because they most often essentialize isolated aspects of human pathophysiology.

“The relevance of these models to humans is questionable because they most often essentialize isolated aspects of the human pathophysiology (6).”

• cites [6] Sanches, 2015, Nonalcoholic steatohepatitis: a search for factual animal models, Biomed Res Int sourceNot checked yet

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Activation of liver-resident macrophages and hepatic stellate cells significantly contributes to NASH pathogenesis by promoting inflammation and fibrosis.

“Activation of liver-resident MΦs (i.e., Kupffer cells) and HSCs significantly contribute to the pathogenesis of NASH by promoting inflammation and fibrosis locally in the liver and systemically via the secretome (10).”

• cites [10] sourceNot checked yet

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The human liver in vitro system was configured to replicate nonalcoholic fatty liver in humans when exposed to lipotoxic stress.

“Adaptation of the in vitro human liver system to mimic human nonalcoholic fatty liver under lipotoxic stress.”