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Regeneration and zonation by ZEB2 of Liver Endothelium

Periodic Reporting for period 1 - REZONABLE (Regeneration and zonation by ZEB2 of Liver Endothelium)

Reporting period: 2016-01-01 to 2017-12-31

The liver plays an essential role in metabolism. It is responsible for the uptake and detoxification of ingested toxins and drugs as well as the removal of waste products from the body. In addition, the liver plays a key role in glucose and lipid metabolism and produces various bioactive proteins including albumin and clotting factors. Liver disease is a common health problem and can be caused by various hepatotoxins including alcohol and drugs, by fat accumulation and by viral infection. The early stage of liver disease is liver fibrosis. It is a slow and reversible condition but remains asymptomatic until it progresses to cirrhosis. Cirrhosis reduces liver function and patients show various symptoms including jaundice, fatigue, bleedings and abdominal fluid accumulation and they have a high risk to develop liver cancer. There is no medication available to cure or reduce cirrhosis, the only cure for patients with liver cirrhosis is liver transplantation.

To accommodate its functions, the liver has a unique vascular system. It receives a mix of nutrient-rich blood from the portal vein and oxygen-rich blood from the hepatic artery, which flows through the hepatic sinusoids and is eventually collected in the central vein. All blood vessels are lined on the inside with endothelial cells (ECs). The hepatic sinusoids are lined with an endothelium consisting of highly specialised liver sinusoidal ECs (LSECs). To facilitate transport of agents to and from the hepatocytes, LSECs have fenestrae, intracellular gaps and lack a basement membrane. In addition, they express LSEC marker genes including several scavenger receptors to take up macromolecules from the blood. LSECs are known to play a role in liver disease, since they lose their specific characteristics already before the onset of fibrosis. We therefore hypothesised that LSECs represent a promising target to develop medication to cure and diagnose liver disease in an early stage.

The liver is zonated, i.e. the hepatocytes around the portal triad where the blood enters have a different function than those around the central vein. For example, the hepatocytes on the portal side are important in glucose production whereas the hepatocytes around the central vein are more important for triglyceride production and toxin removal. LSECs also show morphological signs of zonation but little is known about how zonation is regulated in LSECs and how LSEC marker genes are distributed across the hepatic sinusoids.

Transcription factors (TFs) are proteins that regulate the expression of other proteins. Previously, the host lab identified 7 TFs that are highly expressed in LSECs and 23 LSEC-enriched marker genes. In this study, we aimed to evaluate the role TFs in LSEC marker expression and the zonation of these proteins. In addition, we evaluated how one of the identified TFs (‘TFA’) affects the liver vasculature in healthy and diseased livers.
Our first aim was to study the role of TFs in the regulation of LSEC marker gene expression. Therefore, we isolated ECs from umbilical cords and from blood of adult donors. In these ECs, we enforced the expression of each of the TFs via viruses. We found that a combination of 3 of the 7 TFs (TFB, C and D) regulated together 50% of the LSEC marker genes. TFA, on the other hand, only regulated the expression of one LSEC marker gene.

Our second aim was to study whether the expression pattern of LSECs is zonated and whether this involved TFs. From mouse livers we isolated and separated LSECs from the periportal and the pericentral side. Interestingly, we found that most of the LSEC marker genes and TFs were expressed mainly at the periportal side with the exception of TFA and 2 other LSEC marker genes. We concluded that LSECs are indeed very heterogeneous within the liver and that periportal and pericentral LSECs show a differential expression pattern, potentially regulated by TFs.

Our final aim was to evaluate the role of TFA in the liver vasculature in healthy mice and mice with liver disease. Therefore, we generated transgenic mouse models with decreased or increased expression levels of TFA in ECs. We studied the livers of adult mice and found that mice lacking TFA in ECs had more blood vessels in the liver. When we gave these mice a toxic compound to induce liver fibrosis, they showed more liver fibrosis whereas mice with more TFA in ECs had less fibrosis. Also, when we stopped giving the toxic compound, mice without endothelial TFA expression recovered slower than mice with normal levels of TFA. More detailed studies looking at the blood vessels showed that the LSECs in mice lacking TFA lost some of the normal LSEC characteristics and that they showed increased LSEC damage. We are currently evaluating which genes are affected by TFA and how this can lead to changes in liver disease.
From the first part of our study, we found that the LSEC gene signature is indeed in part regulated by LSEC-enriched TFs in ECs. Of the 7 TFs highly enriched in LSECs, mainly 3 significantly determined LSEC marker gene expression by together regulating 50% of the 23 LSEC marker genes. We are currently studying whether we can use this TF combination to generate LSECs from pluripotent stem cells. These LSECs could be useful as the EC component in in vitro liver toxicity models. Furthermore, we also showed here that, like hepatocytes, LSECs have a zonated expression pattern across the liver sinusoids. The latter may have a significant impact on how to interpret and evaluate LSEC function.

Although it has been shown that LSECs change their phenotype early in liver fibrosis, little is known about how they may affect liver fibrosis and therefore they have been disregarded as a potential target in liver disease. We here showed that increasing the expression of TFA specifically in liver ECs reduced liver fibrosis, indicating that targeting LSECs is a potential new strategy to cope with this disease. TFA itself may represent a novel therapeutic target, but also its target genes that we are currently identifying have potential for the development of novel therapeutics for liver disease. In addition, since LSECs change early in the disease process, these TFA target genes may also serve as biomarkers to diagnose patients at risk for liver disease. Our current findings are based on a toxin-induced model of liver disease, yet, non-alcoholic fatty liver disease (NAFLD) is becoming the most prevalent form of liver disease due to the obesity epidemic. Although different in origin, like toxin-induced liver disease, NAFLD commonly involves liver fibrosis. The data generated in this project have set the basis for our ongoing studies in which we test the hypothesis that TFA as well as its targets also play a role in NAFLD and are also potential therapeutic targets for this condition.