Pre-clinical and clinical investigations of metabolic zonation in liver diseases: The potential of microphysiology systems

Abstract

The establishment of metabolic zonation within a hepatic lobule ascribes specific functions to hepatocytes based on unique, location-dependent gene expression patterns. Recently, there have been significant developments in the field of metabolic liver zonation. A little over a decade ago, the role of β-catenin signaling was identified as a key regulator of gene expression and function in pericentral hepatocytes. Since then, additional molecules have been identified that regulate the pattern of Wnt/β-catenin signaling within a lobule and determine gene expression and function in other hepatic zones. Currently, the molecular basis of metabolic zonation in the liver appears to be a 'push and pull' between signaling pathways. Such compartmentalization not only provides an efficient assembly line for hepatocyte functions but also can account for restricting the initial hepatic damage and pathology from some hepatotoxic drugs to specific zones, possibly enabling effective regeneration and restitution responses from unaffected cells. Careful analysis and experimentation have also revealed that many pathological conditions in the liver lobule are spatially heterogeneous. We will review current research efforts that have focused on examination of the role and regulation of such mechanisms of hepatocyte adaptation and repair. We will discuss how the pathological organ-specific microenvironment affects cell signaling and metabolic liver zonation, especially in steatosis, viral hepatitis, and hepatocellular carcinoma. We will discuss how the use of new human microphysiological platforms will lead to a better understanding of liver disease progression, diagnosis, and therapies. In conclusion, we aim to provide insights into the role and regulation of metabolic zonation and function using traditional and innovative approaches. Impact statement Liver zonation of oxygen tension along the liver sinusoids has been identified as a critical liver microenvironment that impacts specific liver functions such as intermediary metabolism of amino acids, lipids, and carbohydrates, detoxification of xenobiotics and as sites for initiation of liver diseases. To date, most information on the role of zonation in liver disease including, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), cirrhosis, and hepatocellular carcinoma (HCC) have been obtained from animal models. It is now possible to complement animal studies with human liver, microphysiology systems (MPS) containing induced pluripotent stem cells engineered to create disease models where it is also possible to control the in vitro liver oxygen microenvironment to define the role of zonation on the mechanism(s) of disease progression. The field now has the tools to investigate human liver disease progression, diagnosis, and therapeutic development.

Keywords: Liver zonation; hepatocytes; induced pluripotent stem cells; liver diseases; microfluidics; microphysiology systems.

Figure 1

Normal functional zonation and molecular activity in the liver sinusoidal unit. In the normal liver, oxygen tension drops as blood flows from the portal triad to the central vein. Some cellular functions like oxidative phosphorylation, albumin, and urea secretion follow the gradient, while other functions like Cyp2E1 activity and α1AT phosphorylated secretion run opposite to the oxygen gradient. β-catenin activity is under the control of Wnt activation and regulates the establishment of hepatic metabolic zonation. Phosphatidylethanolamne N-methyltransferase (PEMT) is located primary in zone 3 to regulate a step in phosphatidylcholine metabolism for phospholipid synthesis

Figure 2

Immunohistochemistry for glutamine synthetase as a marker of β-catenin activation. (a) indirect immunohistochemistry shows glutamine synthetase (GS) to be localizing to a rim of hepatocytes surrounding central vein in wild-type (WT) mouse livers. However, in liver-specific β-catenin knockout mice, glutamine synthetase staining was absent. (b) Indirect immunohistochemistry shows glutamine synthetase to be localizing to a rim of hepatocytes surrounding central vein in wild-type mouse livers. However, in liver-specific Wnt co-receptors LRP5 and LRP6 double knockout mice, glutamine synthetase staining was absent. (c) Tumor modules were diffusely glutamine synthetase-positive in HCC that occur in both hMet-S33Y-β-catenin and hMet-S45Y-β-catenin livers at seven weeks after injection of both plasmids into tail vein

Figure 3

Zonation in the NASH liver. Phosphatidylcholine metabolism through PMET is diminished and pan-zonal. Although NASH patients have elevated serum bile acids and decreased lysophosphatidylcholine, the relationship between NASH and zone-specific functions remains largely unknown. Mantena et al. found dysregulated oxygen gradient and mitochondrial effects in a high-fat diet NAFLD disease model in the mouse from which he postulates NASH and NAFLD alters the sinusoidal oxygen gradient so that zone 1 hepatocytes consume more oxygen but puts zone 2 and zone 3 hepatocytes into severe hypoxia

Figure 4

An example platform overview of model liver system to study human organ physiology, disease models, ADME, and drug safety. The present liver MPS LAMPS is under optimal perfusion flow rate to create a specific oxygen tension equivalent to either zone 1 to zone 3. The platform consists of: (a) the Liver Acinus Microphysiology System (LAMPS) constructed in a microfluidic device with four human cell types, a fraction of which are ‘‘sentinel’’ cells expressing fluorescent protein biosensors. Optionally, the four cell LAMPS can be constructed entirely from patient iPS-derived cells. Data are collected from the model via (b) high content imaging readouts of transmitted light and/or fluorescence, an example of which shows hepatocyte sentinel cells expressing an apoptosis biosensor in green. Data are also collected (c) from biochemical and mass spectrometry readouts and will include media pH and media oxygen content in the future. (d) The Microphysiology Systems Database (MPS-Db) accesses chemical, genetic, and bioactivity data for test compounds from external databases as part of the analysis and to build predictive models of human efficacy and toxicity and computational interactions of the genes, proteins, and pathways driving disease progression from simple steatosis to NAFLD, NASH, cirrhosis and HCC