Amelioration of Ductular Reaction by Stem Cell Derived Extracellular Vesicles in MDR2 Knockout Mice via Lethal‐7 microRNA

Abstract

Cholangiopathies are diseases that affect cholangiocytes, the cells lining the biliary tract. Liver stem cells (LSCs) are able to differentiate into all cells of the liver and possibly influence the surrounding liver tissue by secretion of signaling molecules. One way in which cells can interact is through secretion of extracellular vesicles (EVs), which are small membrane‐bound vesicles that contain proteins, microRNAs (miRNAs), and cytokines. We evaluated the contents of liver stem cell–derived EVs (LSCEVs), compared their miRNA contents to those of EVs isolated from hepatocytes, and evaluated the downstream targets of these miRNAs. We finally evaluated the crosstalk among LSCs, cholangiocytes, and human hepatic stellate cells (HSCs). We showed that LSCEVs were able to reduce ductular reaction and biliary fibrosis in multidrug resistance protein 2 (MDR2)‐/‐ mice. Additionally, we showed that cholangiocyte growth was reduced and HSCs were deactivated in LSCEV‐treated mice. Evaluation of LSCEV contents compared with EVs derived from hepatocytes showed a large increase in the miRNA, lethal‐7 (let‐7). Further evaluation of let‐7 in MDR2‐/‐ mice and human primary sclerosing cholangitis samples showed reduced levels of let‐7 compared with controls. In liver tissues and isolated cholangiocytes, downstream targets of let‐7 (identified by ingenuity pathway analysis), Lin28a (Lin28 homolog A), Lin28b (Lin28 homolog B), IL‐13 (interleukin 13), NR1H4 (nuclear receptor subfamily 1 group H member 4) and NF‐κB (nuclear factor kappa B), are elevated in MDR2‐/‐ mice, but treatment with LSCEVs reduced levels of these mediators of ductular reaction and biliary fibrosis through the inhibition of NF‐κB and IL‐13 signaling pathways. Evaluation of crosstalk using cholangiocyte supernatants from LSCEV‐treated cells on cultured HSCs showed that HSCs had reduced levels of fibrosis and increased senescence. Conclusion: Our studies indicate that LSCEVs could be a possible treatment for cholangiopathies or could be used for target validation for future therapies.

Melatonin and circadian rhythms in liver diseases: Functional roles and potential therapies

“…EVs mediate inflammatory or fibrogenic responses in hepatic cells by delivering cargo mediators, which means that EVs could be used as therapeutic tools or drug carriers to regulate functions of cells by delivering mediators to maintain homeostasis during liver diseases . For example, EVs secreted from liver stem cells contained elevated levels of miRNA let‐7a, and injection of stem cell‐derived EVs ameliorated ductular reaction and liver fibrosis in Mdr2 −/− mice by delivering cargo let‐7a . Tunicamycin is a drug that causes hepatic ER stress followed by steatosis in mice and used as a drug‐induced NAFLD model.…”

“…106 For example, EVs secreted from liver stem cells contained elevated levels of miRNA let-7a, and injection of stem cell-derived EVs ameliorated ductular reaction and liver fibrosis in Mdr2 −/− mice by delivering cargo let-7a. 107 Tunicamycin is a drug that causes hepatic ER stress followed by steatosis in mice and used as a drug-induced NAFLD model. Rong et al isolated EVs secreted from cultured primary adipocytes harvested from mice treated with melatonin (20 mg/kg daily for 2 weeks, i.p.…”

Concise Review: Functional Roles and Therapeutic Potentials of Long Non-coding RNAs in Cholangiopathies

“…H19 deficient mice represented attenuated liver damage and fibrosis compared to wild-type mice after BDL, indicating the association of expression levels of H19 and liver conditions during cholestatic liver injury (18). Multidrug resistance 2 knockout (Mdr2 −/− ) mice are the most common transgenic mice that are utilized as the animal model of human PSC (19). Mdr2 −/− mice represent liver damage and fibrosis as well as elevated H19 expression in the liver, especially in female mice (20).…”

“…This means that EVs could be utilized as a drug or therapeutic mediator carrier to manage liver conditions. A recent study has demonstrated that injection of EVs isolated from liver stem cells attenuates ductular reaction and liver fibrosis in Mdr2 −/− mice via delivering cargo let-7, indicating the potentials of EVs as a therapeutic tool and an miRNA carrier (19). Injection of EVs carrying mediators, such as small molecules or nucleotides targeting lncRNAs could be performed to regulate lncRNA functions in vivo and manage liver conditions.…”

Long Noncoding RNA H19 Contributes to Cholangiocyte Proliferation and Cholestatic Liver Fibrosis in Biliary Atresia

“…The miRNA, let‐7, belongs to a family of miRNAs required for development, growth, and metabolism regulation . A recent study demonstrated that dysregulation of let‐7 expression contributes to cholestatic liver diseases …”