Polymorphisms Associated With Metabolic Dysfunction-Associated Steatotic Liver Disease Influence the Progression of End-Stage Liver Disease.

BACKGROUND AND AIMS: Chronic liver injury that results in cirrhosis and end-stage liver disease (ESLD) causes more than 1 million deaths annually worldwide. Although the impact of genetic factors on the severity of metabolic dysfunction-associated steatotic liver disease (MASLD) and alcohol-related liver disease (ALD) has been previously studied, their contribution to the development of ESLD remains largely unexplored. METHODS: We genotyped 6 MASLD-associated polymorphisms in healthy (n = 123), metabolic dysfunction-associated steatohepatitis (MASH) (n = 145), MASLD-associated ESLD (n = 72), and ALD-associated ESLD (n = 57) cohorts and performed multinomial logistic regression to determine the combined contribution of genetic, demographic, and clinical factors to the progression of ESLD. RESULTS: Distinct sets of factors are associated with the progression to ESLD. The PNPLA3 rs738409:G and TM6SF2 rs58542926:T alleles, body mass index (BMI), age, and female sex were positively associated with progression from a healthy state to MASH. The PNPLA3 rs738409:G allele, age, male sex, and having type 2 diabetes mellitus were positively associated, while BMI was negatively associated with progression from MASH to MASLD-associated ESLD. The PNPLA3 rs738409:G and GCKR rs780094:T alleles, age, and male sex were positively associated, while BMI was negatively associated with progression from a healthy state to ALD-associated ESLD. The findings indicate that the PNPLA3 rs738409:G allele increases susceptibility to ESLD regardless of etiology, the TM6SF2 rs58542926:T allele increases susceptibility to MASH, and the GCKR rs780094:T allele increases susceptibility to ALD-associated ESLD. CONCLUSION: The PNPLA3, TM6SF2, and GCKR minor alleles influence the progression of MASLD-associated or ALD-associated ESLD. Genotyping for these variants in MASLD and ALD patients can enhance risk assessment, prompting early interventions to prevent ESLD.

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

The liver is known for its remarkable regenerative ability through proliferation of hepatocytes. Yet, during chronic injury or severe hepatocyte death, proliferation of hepatocytes is exhausted. To overcome this hurdle, we propose vascular-endothelial-growth-factor A (VEGFA) as a therapeutic means to accelerate biliary epithelial-cell (BEC)-to-hepatocyte conversion. Investigation in zebrafish establishes that blocking VEGF receptors abrogates BEC-driven liver repair, while VEGFA overexpression promotes it. Delivery of VEGFA via nonintegrative and safe nucleoside-modified mRNA encapsulated into lipid nanoparticles (mRNA-LNPs) in acutely or chronically injured mouse livers induces robust BEC-to-hepatocyte conversion and elimination of steatosis and fibrosis. In human and murine diseased livers, we further identified VEGFA-receptor KDR-expressing BECs associated with KDR-expressing cell-derived hepatocytes. This work defines KDR-expressing cells, most likely being BECs, as facultative progenitors. This study reveals unexpected therapeutic benefits of VEGFA delivered via nucleoside-modified mRNA-LNP, whose safety is widely validated with COVID-19 vaccines, for harnessing BEC-driven repair to potentially treat liver diseases.

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

The liver is known for its remarkable regenerative ability through proliferation of hepatocytes. Yet, during chronic injury or severe hepatocyte death, proliferation of hepatocytes is exhausted. To overcome this hurdle, we propose vascular-endothelial-growth-factor A (VEGFA) as a therapeutic means to accelerate biliary epithelial cell (BEC)-to-hepatocyte conversion. Investigation in zebrafish establishes that blocking VEGF receptors abrogates BEC-driven liver repair, while VEGFA overexpression promotes it. Delivery of VEGFA via non-integrative and safe nucleoside-modified mRNA encapsulated into lipid-nanoparticles (mRNA-LNP) in acutely or chronically injured mouse livers induces robust BEC-to-hepatocyte conversion and reversion of steatosis and fibrosis. In human and murine diseased livers, we further identified VEGFA-receptor KDR-expressing BECs associated with KDR-expressing cell-derived hepatocytes. This defines KDR-expressing cells, most likely being BECs, as facultative progenitors. This study reveals novel therapeutic benefits of VEGFA delivered via nucleoside-modified mRNA-LNP, whose safety is widely validated with COVID-19 vaccines, for harnessing BEC-driven repair to potentially treat liver diseases. Highlights: Complementary mouse and zebrafish models of liver injury demonstrate the therapeutic impact of VEGFA-KDR axis activation to harness BEC-driven liver regeneration.VEGFA mRNA LNPs restore two key features of the chronic liver disease in humans such as steatosis and fibrosis.Identification in human cirrhotic ESLD livers of KDR-expressing BECs adjacent to clusters of KDR+ hepatocytes suggesting their BEC origin.KDR-expressing BECs may represent facultative adult progenitor cells, a unique BEC population that has yet been uncovered.

Biofabrication of synthetic human liver tissue with advanced programmable functions.

Advances in cellular engineering, as well as gene, and cell therapy, may be used to produce human tissues with programmable genetically enhanced functions designed to model and/or treat specific diseases. Fabrication of synthetic human liver tissue with these programmable functions has not been described. By generating human iPSCs with target gene expression controlled by a guide RNA-directed CRISPR-Cas9 synergistic-activation-mediator, we produced synthetic human liver tissues with programmable functions. Such iPSCs were guide-RNA-treated to enhance expression of the clinically relevant CYP3A4 and UGT1A1 genes, and after hepatocyte-directed differentiation, cells demonstrated enhanced functions compared to those found in primary human hepatocytes. We then generated human liver tissue with these synthetic human iPSC-derived hepatocytes (iHeps) and other non-parenchymal cells demonstrating advanced programmable functions. Fabrication of synthetic human liver tissue with modifiable functional genetic programs may be a useful tool for drug discovery, investigating biology, and potentially creating bioengineered organs with specialized functions.

From a Single Cell to a Whole Human Liver: Disease Modeling and Transplantation.

Although the underlying cause may vary across countries and demographic groups, liver disease is a major cause of morbidity and mortality globally. Orthotopic liver transplantation is the only definitive treatment for liver failure but is limited by the lack of donor livers. The development of drugs that prevent the progression of liver disease and the generation of alternative liver constructs for transplantation could help alleviate the burden of liver disease. Bioengineered livers containing human induced pluripotent stem cell (iPSC)-derived liver cells are being utilized to study liver disease and to identify and test potential therapeutics. Moreover, bioengineered livers containing pig hepatocytes and endothelial cells have been shown to function and survive after transplantation into pig models of liver failure, providing preclinical evidence toward future clinical applications. Finally, bioengineered livers containing human iPSC-derived liver cells have been shown to function and survive after transplantation in rodents but require considerable optimization and testing prior to clinical use. In conclusion, bioengineered livers have emerged as a suitable tool for modeling liver diseases and as a promising alternative graft for clinical transplantation. The integration of novel technologies and techniques for the assembly and analysis of bioengineered livers will undoubtedly expand future applications in basic research and clinical transplantation.

Transmembrane channel activity in human hepatocytes and cholangiocytes derived from induced pluripotent stem cells.

The initial creation of human-induced pluripotent stem cells (iPSCs) set the foundation for the future of regenerative medicine. Human iPSCs can be differentiated into a variety of cell types in order to study normal and pathological molecular mechanisms. Currently, there are well-defined protocols for the differentiation, characterization, and establishment of functionality in human iPSC-derived hepatocytes (iHep) and iPSC-derived cholangiocytes (iCho). Electrophysiological study on chloride ion efflux channel activity in iHep and iCho cells has not been previously reported. We generated iHep and iCho cells and characterized them based on hepatocyte-specific and cholangiocyte-specific markers. The relevant transmembrane channels were selected: cystic fibrosis transmembrane conductance regulator, leucine rich repeat-containing 8 subunit A, and transmembrane member 16 subunit A. To measure the activity in these channels, we used whole-cell patch-clamp techniques with a standard intracellular and extracellular solution. Our iHep and iCho cells demonstrated definitive activity in the selected transmembrane channels, and this approach may become an important tool for investigating human liver biology of cholestatic diseases.

Hepatocyte Nuclear Factor 4 alpha 2 Messenger RNA Reprograms Liver-Enriched Transcription Factors and Functional Proteins in End-Stage Cirrhotic Human Hepatocytes.

The only definitive therapy for end-stage liver disease is whole-organ transplantation. The success of this intervention is severely limited by the complexity of the surgery, the cost of patient care, the need for long-term immunosuppression, and the shortage of donor organs. In rodents and humans, end-stage degeneration of hepatocyte function is associated with disruption of the liver-specific transcriptional network and a nearly complete loss of promoter P1-driven hepatocyte nuclear factor 4-alpha (P1-HNF4α) activity. Re-expression of HNF4α2, the predominant P1-HNF4α, reinstates the transcriptional network, normalizes the genes important for hepatocyte function, and reverses liver failure in rodents. In this study, we tested the effectiveness of supplementary expression of human HNF4α2 messenger RNA (mRNA) in primary human hepatocytes isolated from explanted livers of patients who underwent transplant for end-stage irreversibly decompensated liver failure (Child-Pugh B, C) resulting from alcohol-mediated cirrhosis and nonalcoholic steatohepatitis. Re-expression of HNF4α2 in decompensated cirrhotic human hepatocytes corrects the disrupted transcriptional network and normalizes the expression of genes important for hepatocyte function, improving liver-specific protein expression. End-stage liver disease in humans is associated with both loss of P1-HNF4α expression and failure of its localization to the nucleus. We found that while HNF4α2 re-expression increased the amount of P1-HNF4α protein in hepatocytes, it did not alter the ability of hepatocytes to localize P1-HNF4α to their nuclei. Conclusion: Re-expression of HNF4α2 mRNA in livers of patients with end-stage disease may be an effective therapy for terminal liver failure that would circumvent the need for organ transplantation. The efficacy of this strategy may be enhanced by discovering the cause for loss of nuclear P1-HNF4α localization in end-stage cirrhosis, a process not found in rodent studies.

Is HSD17B13 Genetic Variant a Protector for Liver Dysfunction? Future Perspective as a Potential Therapeutic Target.

As diet and lifestyle have changed, fatty liver disease (FLD) has become more and more prevalent. Many genetic risk factors, such as variants of PNPLA3, TM6SF2, GCKR, and MBOAT7, have previously been uncovered via genome wide association studies (GWAS) to be associated with FLD. In 2018, a genetic variant (rs72613567, T > TA) of hydroxysteroid 17-ß dehydrogenase family 13 (HSD17B13) was first associated with a lower risk of developing alcoholic liver disease and non-alcoholic fatty liver disease (NAFLD) in minor allele carriers. Other HSD17B13 variants were also later linked with either lower inflammation scores among NAFLD patients or protection against NAFLD (rs6834314, A > G and rs9992651, G > A) respectively. HSD17B13 is a lipid droplet-associated protein, but its function is still ambiguous. Compared to the other genetic variants that increase risk for FLD, HSD17B13 variants serve a protective role, making this gene a potential therapeutic target. However, the mechanism by which these variants reduce the risk of developing FLD is still unclear. Because studies in cell lines and mouse models have produced conflicting results, human liver tissue modeling using induced pluripotent stem cells may be the best way to move forward and solve this mystery.

Role of Src/FAK in migration and invasion mediated by extracellular vesicles from MDA-MB-231 cells stimulated with linoleic acid.

Linoleic acid (LA) is the most abundant polyunsaturated fatty acid in occidental diets, which mediate a variety of processes in human breast cancer cells, including migration and invasion. Extracellular vesicles (EVs) are vesicles released from endosomes and plasma membrane that are composed of a variety of molecules, including proteins, nucleic acids and lipids. EVs from cancer cells promote processes related with cancer progression. In the present study, we demonstrate that treatment of MDA-MB-231 cells with EVs from MDA-MB-231 cells stimulated with LA (LA EVs) promote migration and invasion via Src activity. LA EVs induce activation of FAK via Src activity and of Src and Akt2. LA EVs also induce the assembly of focal adhesions and MMP-9 secretion. These findings demonstrate that LA EVs mediate an autocrine and/or paracrine Src/FAK signaling pathway to promote migration and invasion.

Human Hepatocytes Isolated from Explanted Livers: A Powerful Tool to Understand End-stage Liver Disease and Drug Screening.

The use of primary human hepatocytes has been hampered by limited availability of adequate numbers of fresh and viable cells due to the ongoing shortage of liver donors. Thus, there is no surplus of healthy organs from which freshly isolated cells can be prepared when needed. However, primary hepatocytes can be successfully isolated from explanted liver specimens obtained from patients receiving orthotopic liver transplantation for decompensated liver cirrhosis or for metabolic liver disease without end-stage liver disease and are a valuable resource for the pharmaceutical industry research. This review focuses on the isolation, characterization and cryopreservation of hepatocytes derived from therapeutically resected livers with various hepatic diseases.

Therapeutic Potential of HNF4α in End-stage Liver Disease.

The prevalence of end-stage liver disease (ESLD) in the US is increasing at an alarming rate. It can be caused by several factors; however, one of the most common routes begins with nonalcoholic fatty liver disease (NAFLD). ESLD is diagnosed by the presence of irreversible damage to the liver. Currently, the only definitive treatment for ESLD is orthotopic liver transplantation (OLT). Nevertheless, OLT is limited due to a shortage of donor livers. Several promising alternative treatment options are under investigation. Researchers have focused on the effect of liver-enriched transcription factors (LETFs) on disease progression. Specifically, hepatocyte nuclear factor 4-alpha (HNF4α) has been reported to reset the liver transcription network and possibly play a role in the regression of fibrosis and cirrhosis. In this review, we describe the function of HNF4α, along with its regulation at various levels. In addition, we summarize the role of HNF4α in ESLD and its potential as a therapeutic target in the treatment of ESLD.

Role of phospholipase D in migration and invasion induced by linoleic acid in breast cancer cells.

Linoleic acid (LA) is an essential and omega-6 polyunsaturated fatty acid that mediates a variety of biological processes, including migration and invasion in breast cancer cells. Phospholipase D (PLD) catalyses the hydrolysis of phosphatidylcholine to produce phosphatidic acid and choline. Increases of expression and activity of PLD are reported in several human cancers, including gastric, colorectal, renal, stomach, lung and breast. In this article, we demonstrate that LA induces an increase of PLD activity in MDA-MB-231 breast cancer cells. Particularly, PLD1 and/or PLD2 mediate migration and invasion induced by LA. Moreover, LA induces increases in number and size of spheroids via PLD activity. FFAR1 also mediates migration and invasion, whereas PLD activation induced by LA requires the activities of FFAR1, FFAR4 and EGFR in MDA-MB-231 cells. In summary, PLD plays a pivotal role in migration and invasion induced by LA in MDA-MB-231 breast cancer cells.

Linoleic acid induces an increased response to insulin in MDA-MB-231 breast cancer cells.

Epidemiological studies and animal models suggest a link between high levels of dietary fat intake and an increased risk of developing breast cancer. Hyperinsulinemia is a feature of obesity, diabetes, and metabolic syndrome that is associated with an increased breast cancer risk. Insulin is a hormone involved in metabolic regulation of carbohydrate. However, it is also a growth factor that mediates proliferation and migration. Linoleic acid (LA) is a fatty acid that induces migration and invasion in breast cancer cells. In the present study, we demonstrate, for the first time, that treatment with LA increases IR and IGF1R expression through a Free Fatty Acid Receptor 4 (FFAR4)-, lipooxygenases (LOXs)-, and SRC-dependent pathway in MDA-MB-231 breast cancer cells, and similarly induces an increase of IR expression in MCF-7 breast cancer cells. In addition, insulin induces tyrosine phosphorylation of IR/IGF1R and migration in MDA-MB-231 cells pretreated with LA, whereas it augments the increase in migration in MCF-7 cells pretreated with LA. Pretreatment of MDA-MB-231 cells with LA induces invasion, proliferation, and increase the MMP-9 secretion induced by insulin. In summary, our findings demonstrate that treatment with LA induces a higher response to insulin in breast cancer cells.

Insulin induces an EMT-like process in mammary epithelial cells MCF10A.

Diabetes mellitus has been related with an increased risk of breast cancer, whereas it has been suggested that links between diabetes mellitus and cancer are hyperinsulinemia, insulin resistance, hyperglycemia, and chronic inflammation induced by adipose tissue. Contribution of hyperinsulinemia to carcinogenesis is mediated through resistance to endogenous insulin and by exogenous insulin used in treatment. Epithelial to mesenchymal transition (EMT) is a process by which epithelial cells are transdifferentiated to a mesenchymal state that has been implicated in cancer progression. However, the role of insulin in EMT process has not been studied in detail. In the present study, we demonstrate that insulin induces downregulation of E-cadherin expression, accompanied with an increase of N-cadherin and vimentin expression, and an increase of MMP-2 and -9 secretions. Insulin also induces FAK activation, an increase of NFκB DNA binding activity, migration, and invasion of mammary non-tumorigenic epithelial cells MCF10A. In addition, migration requires the activity of insulin receptors and insulin-like growth factor receptor 1 (IGF1R). In summary, our results demonstrate that insulin induces an EMT-like process in MCF10A cells.

Linoleic acid induces migration and invasion through FFAR4- and PI3K-/Akt-dependent pathway in MDA-MB-231 breast cancer cells.

An increased risk of developing breast cancer has been associated with high levels of dietary fat intake. Linoleic acid (LA) is an essential fatty acid and the major ω-6 polyunsaturated fatty acid in occidental diets, which is able to induce inappropriate inflammatory responses that contribute to several chronic diseases including cancer. In breast cancer cells, LA induces migration. However, the signal transduction pathways that mediate migration and whether LA induces invasion in MDA-MB-231 breast cancer cells have not been studied in detail. We demonstrate here that LA induces Akt2 activation, invasion, an increase in NFκB-DNA binding activity, miR34a upregulation and miR9 downregulation in MDA-MB-231 cells. Moreover, Akt2 activation requires EGFR and PI3K activity, whereas migration and invasion are dependent on FFAR4, EGFR and PI3K/Akt activity. Our findings demonstrate, for the first time, that LA induces migration and invasion through an EGFR-/PI3K-/Akt-dependent pathway in MDA-MB-231 breast cancer cells.