Loss of liver function in chronic liver disease: An identity crisis

The liver performs vital roles in systemic homeostasis, including bile acid and cholesterol metabolism, detoxification of endo- and xenobiotics, glucose synthesis and storage, lipid turnover, hormone metabolism and plasma protein secretion. Most of these functions are carried out by hepatocytes, parenchymal cells constituting 80% of the liver mass and 60% of its cellular composition. Besides hepatocytes, there are at least six other types of liver cells: biliary epithelial cells, sinusoidal endothelial cells, stellate cells, dendritic cells, macrophages and additional immune cell types. These cells support hepatocellular function, and together with hepatocytes are arranged in three-dimensional anatomical units in the form of hexagonal columns called liver lobules.^, Liver lobules are perfused by two sources of blood, portal venous blood which is oxygen-poor but rich in nutrients, toxins and gut microbiota-derived molecules, and oxygenated blood coming from the hepatic artery.^, These afferent blood vessels enter the lobules from the corners of the hexagonal structures, termed the portal nodes, and form capillary-size vessels known as sinusoids which ultimately drain into a central vein. Interestingly, metabolic labour is divided among hepatocytes according to their position along this porto-central axis. ATP demanding tasks such as albumin and glycogen synthesis, gluconeogenesis and the urea cycle take place in oxygen-rich periportal hepatocytes. Near the central vein, pericentral hepatocytes are responsible for bile acid synthesis and contribute to ammonia metabolism, whereas hepatocytes in the remaining mid zone are involved in a variety of cytochrome P450-mediated metabolic reactions.^, This functional pattern is known as the metabolic zonation of the liver parenchyma. To perform these complex and spatially coordinated functions hepatocytes must express a large complement of enabling genes. This gene expression signature is progressively established from early hepatic development through cell-to-cell signalling and a network of key transcription factors (TFs) and their activation or repression complexes. After birth, an increasingly complex set of cross-regulated TFs, plus signalling systems such as WNT and Notch, and splicing regulators, define the liver zonal architecture and consolidate mature hepatocellular function.^{,,  ,}  An intricate system of signals, TFs and epigenetic mechanisms secure the preservation of the liver-specific gene expression pattern (i.e. hepatocellular identity) in adulthood.^, The robust nature of this network is essential to maintain liver function and systemic homeostasis.

Chronic liver disease, cirrhosis and its complications, including the development of hepatocellular carcinoma (HCC), are estimated to cause over one million deaths per year worldwide. Hepatitis virus infection, alcohol-related liver disease and non-alcoholic steatohepatitis (NASH) are the most common causes. Regardless of the aetiology, the ultimate outcome of cirrhosis and end-stage liver disease (ESLD) is the development of hepatic insufficiency and decompensation.^{,  ,}  In individuals with cirrhosis without evidence of decompensation, the removal of the causative agent (i.e. hepatitis virus clearance, abstinence from alcohol, weight loss) may ameliorate and partially recover liver function. However, currently there are no specific therapeutic strategies to halt or reverse the pathologic progression of chronic liver disease. Understanding the precise mechanisms that lead to the loss of liver function and ESLD is essential for the development of effective therapies. Progressive liver injury involves hepatocellular death and the substitution of the parenchymal component by abundant extracellular matrix, which disrupts the organ’s architecture and impairs blood perfusion.^, Hepatic insufficiency is also frequently associated with reduced liver volume. However, evidence accumulated over the past two decades indicates that breakdown of hepatic function cannot be simply ascribed to reduced parenchymal mass. Functional deficiencies of chronically injured and regenerating hepatocytes may underlie liver failure in cirrhosis, and these can be related to an impairment in the expression of genes that typify the mature hepatocyte phenotype.^, Herein, after summarising the current understanding of how hepatocellular differentiation occurs during development, we will discuss mechanisms that lead to the loss of hepatocellular identity and that contribute to organ dysfunction in chronic liver injury. Innovative therapeutic approaches based on these notions will be presented.

Deterioration and loss of hepatic functions is a hallmark of severe acute injury and chronic damage to the organ.^, In the clinic, the scores most commonly used to predict patient outcomes, the Child-Turcotte-Pugh and model of end-stage liver disease (MELD) scores, include the serum levels and the activity of proteins produced by hepatocytes such as albumin and coagulation factors, as well as bilirubin concentration, which are indicative of liver biosynthetic and secretory functions. Severity of liver disease, independently from the causative agent, can be estimated with these biochemical tools which, by definition, closely reflect overall liver functional capacity. Hepatocyte death, which can be massive in the context of acute liver injury, undoubtedly contributes to the failure of liver functions.^, However, as mentioned, it is now believed that liver dysfunction cannot be solely ascribed to hepatocytes’ demise and the substitution of parenchyma by fibrous tissue. In a context of impaired perfusion, hypoxia, inflammation, oxidative and endoplasmic reticulum (ER) stress, and under abundant mitogenic signals, the surviving hepatocytes undergo profound coping adaptations, changing their metabolic, bioenergetic and quiescent balance.^,,, Indeed, more than two decades ago, we described a marked reduction in the expression of genes involved in methionine, homocysteine and one carbon metabolism, which correlated with disease severity, in the livers of individuals with cirrhosis. The liver plays a unique role in systemic one carbon metabolism, and these findings helped to explain the hypermethioninemia and hyperhomocysteinemia commonly observed in individuals with cirrhosis. Moreover, downregulation of methionine-adenosyltransferase 1A, a liver-enriched gene responsible for the synthesis of S-adenosylmethionine, in human and experimental cirrhosis was accompanied by the hypermethylation of its gene promoter.^, Since these original observations, the gradual decrease in the expression of hepato-specific genes in association with liver disease progression has been repeatedly confirmed in experimental and clinical studies.^{,,  ,  ,  ,  ,  ,  ,}  These findings suggest that active reprogramming of the hepatocellular transcriptome during chronic liver injury leads to the loss of hepatocyte identity. Moreover, as mentioned, reduced expression of hepato-specific genes is accompanied by the reactivation of foetal isoforms, further contributing to the abandonment of fundamental metabolic functions by the injured liver.^,,, The loss of liver-enriched and zonated gene expression, and the upregulation of foetal-specific genes, have recently been confirmed by single-cell RNA-sequencing analyses of hepatocytes isolated from experimental models of liver injury and regeneration, as well as in clinical samples from individuals with cirrhosis or alcoholic hepatitis.^{,,  ,  ,}  Importantly, the reactivation of foetal hepatic genes and foetal isoforms is not only implicated in liver dysfunction but may also herald the development of cancer. For instance, a recent study in individuals with chronic liver disease, including more than 8,200 people, reported that elevated serum levels of alpha-fetoprotein, encoded by a liver oncofoetal gene, can be observed more than 10 years before HCC detection.

As summarised in the previous section, the preservation of hepatocellular phenotype relies on a complex network of interconnected TFs interacting with the CRMs of genes that define the liver identity. Although this complexity provides stability, pathophysiological changes in expression and/or activity of key TFs are likely to impact on the hepatic transcriptional equilibrium. Early reports showed marked downregulation of FOXA2 and C/EBPβ expression in experimental cirrhosis. Reduced FOXA2 expression was subsequently found in the livers of individuals with fibrosis and cirrhosis, as well as cholestatic syndromes, with FOXA2 downregulation contributing to disease progression.^, Conversely, more recent studies did not find lower FOXA2 gene expression in cirrhotic human livers of various non-cholestatic aetiologies.^, However, FOXA2 was shown to be essential for the preservation of ALB gene expression in decompensated cirrhosis, as well as for bilirubin excretion and albumin production in conditions of acute liver failure.^, The hepatic expression of FOXA1 and FOXA3 is also lower in experimental and human chronic liver injury,^, as is C/EBPα in individuals with acute and chronic liver damage, including severe alcoholic hepatitis.^,

Among the core TFs investigated for their involvement in liver failure, the hepatic expression of HNF1α is reduced in experimental cirrhosis and hepatocarcinogenesis, as well as in human cirrhotic and tumoral liver tissues.^{,,  ,  ,}  The impairment of HNF4α has attracted special attention. In 2003, we reported that HNF4α expression was markedly downregulated in human livers with advanced cirrhosis, inversely correlating with the MELD score. Interestingly, in that early study we also described how TGFβ downregulated HNF4α levels in hepatocytes through the induction of the transcription factor WT1 (Wilm’s tumour 1), a foetal gene reactivated during hepatocellular dedifferentiation in human cirrhosis.^, Since then, a plethora of studies have confirmed and extended these original findings, describing the impairment of HNF4α expression and regulatory activity in human chronic liver disease of viral, alcoholic or metabolic origin,^{,,,,,  ,}  as well as in acute liver failure.^,,, Complementary studies in human tissues and experimental models also identified changes in the ratio of HNF4α P1- and P2-derived isoforms and in the subcellular localisation of HNF4α. Upregulation of HNF4α P2 variants may have important functional consequences. These isoforms – normally expressed in the foetal liver – are induced in HCC, and display significant differences with P1-derived variants in their specificity and the potency with which they drive gene expression.^{,  ,}  Cellular and in vivo models have helped to unravel the mechanisms involved in HNF4α dysregulation in liver injury, and to understand its central role in hepatoprotection. Early works reported that mitogen-activated protein kinase activation downregulates HNF4α expression in cultured human liver cells (HepG2). Later on, we reported that epidermal growth factor receptor activation decreases HNF4α-P1 protein levels, potentiating the effect of TGFβ, and that TGFβ stimulated the expression of P2-derived HNF4α isoforms in human liver cell lines via c-Src-dependent signalling. Furthermore, we demonstrated that these effects on HNF4α isoforms significantly influenced hepatocellular gene expression and function, and that P2–HNF4α variants were markedly upregulated in individuals with alcoholic hepatitis. These findings, together with the recently identified key role for HNF4α in the transcriptional control of hepatic sulfur amino acid metabolism, contribute to our understanding of the aforementioned impairment of methionine and homocysteine metabolism in chronic liver injury, including alcohol-related liver disease.^, Interestingly, mislocalisation of HNF4α to the cytoplasm was recently observed in hepatocytes from individuals with advanced cirrhosis. Alterations in HNF4α acetylation and phosphorylation accompanied this increased cytosolic retention of HNF4α, which correlated with hepatocyte dysfunction. In this context, oxidative stress associated with fatty liver disease was recently reported to induce cytoplasmic retention of HNF4α upon protein kinase C-mediated phosphorylation. Moreover, increased extracellular matrix stiffness can, via Rho/ROCK pathway signalling or YAP pathway activation, lead to impaired HNF4α expression.

Therefore, environmental cues triggered during liver injury can indeed drive hepatocellular dedifferentiation. These signals include cytokines, chemokines, growth factors and extracellular matrix proteins, and are mostly produced by inflammatory cells and other non-parenchymal cells, such as activated stellate cells and sinusoidal endothelial cells.^{,,  ,}  Biliary epithelial cells, which are quiescent under physiological conditions, also undergo profound modifications during liver injury, adopting a proliferative, secretory and ultimately senescent phenotype. Activated cholangiocytes are a rich source of chemokines and cytokines, like tumour necrosis factor-α, interleukin 6 and TGFβ, which can have autocrine effects but may also act in a paracrine manner on other liver cells, including hepatocytes.^, Nevertheless, endogenous mechanisms are also involved. This is exemplified by the fundamental role of FOXA2 in the preservation of HNF4α expression, as recently described in human hepatocytes. We also demonstrated how HNF4α protein stability can be critically regulated by a novel antioxidant role of SLU7 in hepatocytes. Consequently, the downregulation of SLU7 in mouse livers results in reduced expression of P1 promoter HNF4α isoforms, and enhanced P2 promoter activity, a response exacerbated by chronic liver injury.^,

In spite of being mostly gathered in pathophysiological contexts, these data indicate that hepatic HNF4α expression and activity are subjected to very precise regulation. This multifarious control may underpin the natural response of the liver to acute injury and inflammation, during which a transient loss of hepatocellular differentiation appears to be necessary to enable the activation of genes involved in stress control and cell cycle progression.^{,,  ,}  Interestingly, hepatocellular plasticity during regeneration also includes the activation of genes and splice variants characteristic of foetal, immature and transformed hepatocytes;^,; interference with the activation of this oncofoetal gene expression programme hampers liver regeneration. As previously alluded to, reduced HNF4α expression occurs upon acute liver injury and ER stress, and a transient repression of HNF4α levels is also observed after PH in mice,^,, concomitantly with inflammatory and ER stress signalling. Interestingly, the expression of SLU7 is also transiently downregulated after PH in mice. Recovery of HNF4α levels at later time-points after liver resection is required for the restoration of liver function and hepatocellular quiescence. Therefore, a persistently impaired function of key liver TFs such as HNF4α will result in the shutdown of hepatocellular functions and the perpetuation of a proliferative and dedifferentiated status, a fertile ground for HCC development.^,