VEGFA mRNA-LNP promotes biliary epithelial cell-to-hepatocyte conversion in acute and chronic liver diseases and reverses steatosis and fibrosis

Background

In humans, the liver is capable of regenerating following injury primarily through the proliferation of mature hepatocytes. In the context of end-stage liver disease (ESLD) in steatosis, cirrhosis or liver failure, the regenerative ability of hepatocytes is impaired, and injury becomes irreversible. As a result, many researchers have postulated alternative routes of regeneration that could be harnessed to rescue liver function in the context of disease.

Apart from hepatocyte self-renewal, an alternative route of hepatocyte production is hypothesised to originate from biliary epithelial cell (BECs) (Rodrigo-Torres et al. 2014). Often referred to as liver progenitor cells (LPCs), BEC precursors are well-documented to differentiate into de novo hepatocytes during liver disease. In these scenarios, this mainly occurs in highly proliferative and reactive regions of the bile duct, in a process termed ductular reaction (DR). This process has been characterised by the presence hepatocyte-like cells that have been described as bi-phenotypic cells, or “ductular hepatocytes”, that express both BEC and mature hepatocyte markers (Haque et al., 1996; Yoon et al., 2011; Deng et al., 2018). Furthermore, lineage-tracing experiments performed in mice and zebrafish have demonstrated this de novo hepatocyte formation from BECs in the absence of self-renewing hepatocytes (Lu et al, 2015; Raven et al., 2017; Russell., 2018). Despite this, there are currently no therapeutic options that can promote this response in ESLD.

In this preprint, Rizvi and colleagues explored the therapeutic benefits of utilising nucleoside-modified mRNA encapsulated lipid-nanoparticles (mRNA-LNPs) to treat acute and chronic liver injury models. Here, mRNA-LNPs delivering vascular endothelial growth factor A (VEGFA) were used to study its effect on BEC-to-hepatocyte conversion and hallmarks of disease, such as steatosis and fibrosis, in the liver.

 

Key Findings

VEGFR signalling regulates BEC-to-hepatocyte conversion during liver regeneration in zebrafish and mice

In this study, the authors utilised zebrafish and mice models to study the mechanism of BEC-to-hepatocyte conversion during liver regeneration. Firstly, the authors genetically ablated hepatocytes with the nitroreductase-metronidazole model and found that inhibition of VEGFR signalling, both pharmaceutically and genetically, led to a significant reduction in the number of hepatocytes found in BEC-positive areas during a critical regeneration window. In support of this, overexpressing human VEGFA specifically in BECs during liver injury was also sufficient to promote hepatocyte differentiation in these regions. Together these findings suggest a mechanism for VEGFR signalling in BEC-to-hepatocyte conversion during liver regeneration in zebrafish.

Next, using a diet-induced liver injury mouse model of non-alcoholic steatohepatitis (NASH), the authors tested the impact of VEGFA activation during liver regeneration. In an elegant experiment, chronically liver-injured mice were subjected to AAV-mediated delivery of p21 (to induce hepatocyte senescence), followed by two injections of mRNA-LNPs containing VEGFA. In mice that received VEGFA mRNA-LNP injections, cross-sections and flow cytometry analysis revealed a greater proportion of newly developed hepatocytes in these livers, compared to livers of mice injected with control LNPs. In addition to this, new hepatocytes in VEGFA mRNA-LNP injected mice were found to be adjacent to KRT7-positive BECs, indicative of their BEC origin. Interestingly, VEGFA mRNA-LNP treatment significantly reduced fibrosis and steatosis in the liver, and in addition, new hepatocytes were found to be as sufficient at storing glycogen when compared to neighbouring pre-existing hepatocytes. VEGFA mRNA-LNP-treated livers were also functionally more efficient at clearing lipids from hepatocytes compared to control LNP livers. Together these findings demonstrate a potential clinical benefit of VEGFA mRNA-LNPs in alleviating symptoms inflicted by NASH. 

 

VEGFA promotes BEC-to-hepatocyte conversion in APAP-induced acute liver injury in mice

To model human acute liver injury, acute acetaminophen (Paracetomol) toxicity (APAP) was induced in mice. Strikingly, after two consecutive injections of VEGFA mRNA-LNPs, the livers of these mice showed a 20-fold increase in the proportion of newly developed hepatocytes, compared to the livers of mice injected with control LNPs. As well as being situated close to bile ducts, these new BEC-derived hepatocytes were also found to be as sufficient at storing glycogen as the pre-existing hepatocytes. Together with the chronic liver injury model, these results demonstrate the potential clinical benefit of VEGFA mRNA-LNPs as a tool to promote liver regeneration.

 

VEGFA-KDR signalling may have a conserved role in BEC-derived hepatocyte formation

The authors next investigated KDR, the receptor for VEGFA, in liver regeneration in the context of both chronic diet-induced injury and acute APAP-induced injury. The identification of a BEC subset that express the VEGFA receptor, KDR, led to the hypothesis that that these cells may be precursors of BEC-derived hepatocytes. Using a lineage tracing tool, the authors reported that transient KDR expression in BECs correlates with improved BEC-driven hepatocyte repopulation following VEGFA mRNA-LNP treatment in both injury models. Furthermore, in the acute APAP-injury model, identification of cells co-expressing hepatocyte markers and a reporter for KDR expression supported this hypothesis. In mice, these results demonstrate that VEGFA overexpression in conjunction with BEC-KDR expression during liver injury allow for improved hepatocyte recovery.

Finally, the authors asked whether VEGFA-KDR signalling has a conserved role in BEC-hepatocyte conversion using samples from end-stage liver disease (NASH or alcoholic cirrhosis) and terminal liver failure (Child-Pugh B and C cirrhosis). From these samples, the authors identified bi-phenotypic BECs at the fibrotic septa that expressed not only BEC markers but also hepatocyte markers and vice versa. Importantly, KDR expression was indicated in BECs adjacent to newly generated hepatocytes. This data is an important proof of concept for the hypothesis that KDR-expressing BECs may give rise to intermediate hepatocytes, which represents a novel therapeutic avenue for preventing and treating liver disease.

 

What I like about this preprint

The elephant in the room is the lipid-nanoparticle (LNP) delivery system. Over recent years it has been gaining attention as a method of mRNA delivery for COVID vaccines and now it is more and more being used in experimental protocols. This preprint is the second study I’ve seen using LNPs to deliver a growth factor (Szöke D, Kovács G, Kemecsei É, et al Nat Commun 2021) and this work is exploring a tool which has an extraordinary range of applications – from animal models to therapeutic delivery.

This preprint combines both zebrafish and mice models with human tissue samples to address a simple question about the VEGFA-KDR pathway in BEC-hepatocyte conversion. Both pharmacological and genetic experiments in zebrafish as well as lineage tracing in clinically-relevant mice models of acute and chronic liver injury allowed the authors to infer the delineating presence of KDR-expressing BECs to KDR-expressing hepatocytes.

 

Questions for the Author(s)

1. Have the authors looked at the local vasculature response to VEGFA-LNP treatment either in healthy or injured mice livers and whether this could be contributing to hepatocyte repopulation?
2. Do you believe that the results could be replicated in injury models not specific to hepatocytes in mice (I.e. liver resection or cryoinjury) and would it be beneficial to enhance BEC-hepatocyte conversion in the presence of healthy hepatocytes with the capacity to regenerate?

 

References:

Rodrigo-Torres, D., Affo, S., Coll, M., Morales-Ibanez, O., Millan, C., Blaya, D., Alvarez-Guaita, A., Rentero, C., Lozano, J.J., Maestro, M.A., et al. (2014). The biliary epithelium gives rise to liver progenitor cells. Hepatology 60, 1367-1377.

Haque, S., Haruna, Y., Saito, K., Nalesnik, M.A., Atillasoy, E., Thung, S.N., and Gerber, M.A. (1996). Identification of bipotential progenitor cells in human liver regeneration. Lab Invest 75, 699-705.

Yoon, S.M., Gerasimidou, D., Kuwahara, R., Hytiroglou, P., Yoo, J.E., Park, Y.N., and Theise, N.D. (2011). Epithelial cell adhesion molecule (EpCAM) marks hepatocytes newly derived from stem/progenitor cells in humans. Hepatology 53, 964-973.

Deng, X., Zhang, X., Li, W., Feng, R.X., Li, L., Yi, G.R., Zhang, X.N., Yin, C., Yu, H.Y., Zhang, J.P., et al. (2018). Chronic Liver Injury Induces Conversion of Biliary Epithelial Cells into Hepatocytes. Cell Stem Cell 23, 114-122.

Lu, W.Y., Bird, T.G., Boulter, L., Tsuchiya, A., Cole, A.M., Hay, T., Guest, R.V., Wojtacha, D., Man, T.Y., Mackinnon, A., et al. (2015). Hepatic progenitor cells of biliary origin with liver repopulation capacity. Nat Cell Biol 17, 971-983.

Raven, A., Lu, W.Y., Man, T.Y., Ferreira-Gonzalez, S., O’Duibhir, E., Dwyer, B.J., Thomson, J.P., Meehan, R.R., Bogorad, R., Koteliansky, V., et al. (2017). Cholangiocytes act as facultative liver stem cells during impaired hepatocyte regeneration. Nature 547, 350-354.

Russell, J.O., Lu, W.Y., Okabe, H., Abrams, M., Oertel, M., Poddar, M., Singh, S., Forbes, S.J., and Monga, S.P. (2018). Hepatocyte-specific beta-catenin deletion during severe liver injury provokes cholangiocytes to differentiate into hepatocytes. Hepatology. 10.1002/hep.30270.

Szőke, D., Kovács, G., Kemecsei, É., Bálint, L., Szoták-Ajtay, K., Aradi, P., Styevkóné Dinnyés, A., Mui, B.L., Tam, Y.K., Madden, T.D., Karikó, K., Kataru, R.P., Hope, M.J., Weissman, D., Mehrara, B.J., Pardi, N., and Jakus, Z. (2021). Nucleoside-modified VEGFC mRNA induces organ-specific lymphatic growth and reverses experimental lymphedema. Nat Commun 12, 3460

Tags: liver regeneration, mice, zebrafish

doi: https://doi.org/10.1242/prelights.35254

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