Recent studies have identified a cholestatic variant of nonalcoholic fatty liver disease (NAFLD) with portal inflammation and ductular reaction. Based on reports of biliary damage, as well as increased circulating free fatty acids (FFAs) in NAFLD, we hypothesized the involvement of cholangiocyte lipoapoptosis as a mechanism of cellular injury. Here, we demonstrate that the saturated FFAs palmitate and stearate induced robust and rapid cell death in cholangiocytes. Palmitate and stearate induced cholangiocyte lipoapoptosis in a concentration-dependent manner in multiple cholangiocyte-derived cell lines. The mechanism of lipoapoptosis relied on the activation of caspase 3/7 activity. There was also a significant up-regulation of the proapoptotic BH3-containing protein, PUMA. In addition, palmitate-induced cholangiocyte lipoapoptosis involved a time-dependent increase in the nuclear localization of forkhead family of transcription factor 3 (FoxO3). We show evidence for posttranslational modification of FoxO3, including early (6 hours) deacetylation and dephosphorylation that coincide with localization of FoxO3 in the nuclear compartment. By 16 hours, nuclear FoxO3 is both phosphorylated and acetylated. Knockdown studies confirmed that FoxO3 and its downstream target, PUMA, were critical for palmitate- and stearate-induced cholangiocyte lipoapoptosis. Interestingly, cultured cholangiocyte-derived cells did not accumulate appreciable amounts of neutral lipid upon FFA treatment. Conclusion Our data show that the saturated FFAs palmitate and stearate induced cholangiocyte lipoapoptosis by way of caspase activation, nuclear translocation of FoxO3, and increased proapoptotic PUMA expression. These results suggest that cholangiocyte injury may occur through lipoapoptosis in NAFLD and nonalcoholic steatohepatitis patients.

Nonalcoholic steatohepatitis (NASH) patients have elevated plasma saturated free fatty acid levels. These toxic fatty acids can induce liver cell death and our recent results demonstrated that the biliary epithelium may be susceptible to lipotoxicity. Here, we explored the molecular mechanisms of cholangiocyte lipoapoptosis in cell culture and in an animal model of NASH. Treatment of cholangiocytes with palmitate (PA) showed increased caspase 3/7 activity and increased levels of cleaved poly (ADP-ribose) polymerase and cleaved caspase 3, demonstrating cholangiocyte lipoapoptosis. Interestingly, treatment with PA significantly increased the levels of microRNA miR-34a, a pro-apoptotic microRNA known to be elevated in NASH. PA induction of miR-34a was abolished in cholangiocytes transduced with forkhead family of transcription factor class O (FoxO)3 shRNA, demonstrating that FoxO3 activation is upstream of miR-34a and suggesting that FoxO3 is a novel transcriptional regulator of miR-34a. Further, anti-miR-34a protected cholangiocytes from PA-induced lipoapoptosis. Direct and indirect targets of miR-34a, such as SIRT1, receptor tyrosine kinase (MET), Kruppel-like factor 4, fibroblast growth factor receptor (FGFR)1, and FGFR4, were all decreased in PA-treated cholangiocytes. SIRT1 and MET were partially rescued by a miR-34a antagonist. Cholangiocyte apoptosis and miR-34a were dramatically increased in the liver of mice with early histologic features of NASH. Our study provides evidence for the pro-apoptotic role of miR-34a in PA-induced cholangiocyte lipoapoptosis in culture and in the liver.

Cholangiocarcinoma (CCA) is the second most common primary liver malignancy with extremely poor therapeutic outcome due to high drug resistance, widespread metastasis and lack of effective treatment options. CCA progression and metastasis are regulated by multiple biological factors including multiple miRNAs and chemokine receptor CXCR4. The goal of this study was to test if nanotherapeutic blockade of CXCR4 by polymeric CXCR4 antagonist (PCX) combined with inhibition of hypoxia-inducible miR-210 cooperatively enhances therapeutic efficacy in CCA through reducing invasiveness, inducing cell killing, and reversing drug resistance.Methods: We first tested the activity of PCX to inhibit migration of CCA cells. We then prepared PCX/anti-miRNA nanoparticles and analyzed their miRNA delivery efficacy and anticancer activity in vitro. Finally, in vivo biodistribution assay and anticancer activity study were performed in CCA tumor-bearing mice.Results: Our results show that PCX had a broad inhibitory effect on cell migration, effectively delivered anti-miR-210, and downregulated miR-210 expression in CCA cells. Combination PCX/anti-miR-210 nanoparticles showed cytotoxic activity towards CCA cells and reduced the number of cancer stem-like cells. The nanoparticles reversed hypoxia-induced drug resistance and sensitized CCA cells to standard gemcitabine and cisplatin combination treatment. Systemic intravenous treatment with the nanoparticles in a CCA xenograft model resulted in prominent combined antitumor activity.Conclusion: Our findings support PCX-based nanoparticles as a promising delivery platform of therapeutic miRNA in combination CCA therapies.

Cholangiocarcinoma is the second most common primary liver malignancy with extremely poor prognosis due to early invasion and widespread metastasis. The invasion and metastasis are regulated by multiple factors including CXCR4 chemokine receptor and multiple microRNAs. The goal of this study was to test the hypothesis that inhibition of CXCR4 combined with the action of miR-200c mimic will cooperatively enhance the inhibition of the invasion of human cholangiocarcinoma cells. The results show that CXCR4-inhibition polycation PCX can effectively deliver miR-200c mimic and that the combination treatment consisting of PCX and miR-200c results in cooperative antimigration activity, most likely by coupling the CXCR4 axis blockade with epithelial-to-mesenchymal transition inhibition in the cholangiocarcinoma cells. The ability of the combined PCX/miR-200c treatment to obstruct two migratory pathways represents a promising antimetastatic strategy in cholangiocarcinoma.

MicroRNAs are emerging as a hot topic in research, and rightfully so. They show great promise as targets of treatment and as markers for common human diseases, such as cancer and metabolic diseases. In this review, we address some of the basic questions regarding microRNA function in human disease and the clinical significance of microRNAs. Specifically, microRNAs in epigenetics, cancer, and metabolic diseases are discussed, with examples taken from cholangiocarcinoma and nonalcoholic fatty liver disease.

Cholangiocarcinoma cells are dependent on antiapoptotic signaling for survival and resistance to death stimuli. Recent mechanistic studies have revealed that increased cellular expression of the E3 ubiquitin-protein ligase X-linked inhibitor of apoptosis (XIAP) impairs TRAIL- and chemotherapy-induced cytotoxicity, promoting survival of cholangiocarcinoma cells. This study was undertaken to determine if pharmacologic antagonism of XIAP protein was sufficient to sensitize cholangiocarcinoma cells to cell death. We employed malignant cholangiocarcinoma cell lines and used embelin to antagonize XIAP protein. Embelin treatment resulted in decreased XIAP protein levels by 8 hours of treatment with maximal effect at 16 hours in KMCH and Mz-ChA-1 cells. Assessment of nuclear morphology demonstrated a concentration-dependent increase in nuclear staining. Interestingly, embelin induced nuclear morphology changes as a single agent, independent of the addition of TNF-related apoptosis inducing ligand (TRAIL). However, caspase activity assays revealed that increasing embelin concentrations resulted in slight inhibition of caspase activity, not activation. In addition, the use of a pan-caspase inhibitor did not prevent nuclear morphology changes. Finally, embelin treatment of cholangiocarcinoma cells did not induce DNA fragmentation or PARP cleavage. Apoptosis does not appear to contribute to the effects of embelin on cholangiocarcinoma cells. Instead, embelin caused inhibition of cell proliferation and cell cycle analysis indicated that embelin increased the number of cells in S and G2/M phase. Our results demonstrate that embelin decreased proliferation in cholangiocarcinoma cell lines. Embelin treatment resulted in decreased XIAP protein expression, but did not induce or enhance apoptosis. Thus, in cholangiocarcinoma cells the mechanism of action of embelin may not be dependent on apoptosis.

MicroRNA dysregulation is a common feature of cancer and due to the promiscuity of microRNA binding this can result in a wide array of genes whose expression is altered. miR-106b is an oncomiR overexpressed in cholangiocarcinoma and its upregulation in this and other cancers often leads to repression of anti-tumorigenic targets. The goal of this study was to identify the miR-106b-regulated gene landscape in cholangiocarcinoma cells using a genome-wide, unbiased mRNA analysis. Through RNA-Seq we found 112 mRNAs significantly repressed by miR-106b. The majority of these genes contain the specific miR-106b seed-binding site. We have validated 11 genes from this set at the mRNA level and demonstrated regulation by miR-106b of 7 proteins. Combined analysis of our miR-106b-regulated mRNA data set plus published reports indicate that miR-106b binding is anchored by G:C pairing in and near the seed. Novel targets Kruppel-like factor 2 (KLF2) and KLF6 were verified both at the mRNA and at the protein level. Further investigation showed regulation of four other KLF family members by miR-106b. We have discovered coordinated repression of multiple members of the KLF family by miR-106b that may play a role in cholangiocarcinoma tumor biology.

Extracellular vesicles (EVs) are a heterogeneous group of biological nano-sized vesicles that are released from cells and contribute to intercellular communication. Emerging knowledge about their biogenesis, composition, release, and uptake has resulted in broad interest in elucidating their potential roles in disease pathophysiology. The distinct biological properties of these biological nanoparticles emphasize several appealing advantages for potential therapeutic applications compared with the use of synthetic nanoparticles. When administered systemically, EVs are taken up and sequestered within the liver, further emphasizing opportunities for therapeutic use. Consequently, there is growing interest in their use for liver diseases. EVs can be used directly as therapeutics, and several studies have highlighted the intrinsic therapeutic properties of mesenchymal stem cell-derived EVs for chronic and acute liver diseases. Alternatively, EVs can be modified to facilitate their use for the delivery of therapeutic cargo. In this review, we discuss the cellular sources of EV, provide a concise overview of their potential use in diverse processes, and outline several promising applications for the use of EV-based therapeutics for liver diseases. The use of EV-based therapeutics provides a viable approach to target hepatic pathophysiology. (Hepatology 2021;74:2863-2875). Extracellular Vesicles as Biological NanotherapeuticsExtracellular vesicles (EVs) are lipid bilayer encapsulated biological nanoparticles that are released from mammalian cells. They can be taken up by and transfer their cargo into nearby cells, thereby contributing to intercellular communication. EVs are also present within body fluids and in blood and can be transported to distant tissues to exert biological effects. There is growing interest in elucidating the biological roles of EVs and their involvement in physiological or disease-relevant processes. There are several different types of EVs, such as exosomes or microvesicles (MVs). These differ in their modes of biogenesis, size, morphology, membrane composition, and cargo profiles. Their biological functions and biophysical properties support the development of EVs as a therapeutic platform for human diseases. Although the therapeutic use of different subtypes of vesicles has been reported, these cannot be readily distinguished due to their concomitant presence within the biological milieu, the overlap of size, composition and cargo, and the lack of subtype-specific markers. Thus, we collectively refer to all subtypes of cell-derived vesicles herein as EVs. In this overview, we outline the promise and potential use of EV-based therapeutics for liver diseases. SoURCeS oF tHeRapeUtIC eVsEVs are ubiquitously expressed by most if not all cell types, and are detectable in the tissue milieu,