Biochemistry & Molecular Biology Department Theses and Dissertations

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Browsing Biochemistry & Molecular Biology Department Theses and Dissertations by Author "Alpini, Gianfranco"

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    Elucidating the Role of Biliary Senescence and Mast Cell-Mediated Therapy in Non-Alcoholic Fatty Liver Disease
    (2023-05) Kundu, Debjyoti; Francis, Heather; Dong, Charlie X.; Alpini, Gianfranco; Linnemann, Amelia; Ekser, Burcin
    Non-alcoholic fatty liver disease, or NAFLD, is characterized by excess fat deposition in the liver. Cellular senescence is a critical hallmark of NAFLD. Cholangiocytes in the liver plays a significant role in the progression of fatty liver by contributing to senescence. p16 is the main senescent protein expressed by cholangiocytes in primary sclerosing cholangitis (PSC). Thus, we aimed to downregulate p16 by vivo-morpholino and evaluate the disease phenotypes and signaling mechanisms in a murine model of NAFLD. We found that downregulation of p16 reduced i) steatosis), ii) inflammation, iii) fibrosis, and cholangiocyte proliferation in HFD mice compared to the HFD-fed, control vivo-morpholino injected mice. Moreover, the downregulation of p16 reduced insulin-like growth factor-1 (IGF-1) in cholangiocytes, previously identified by our laboratory as a principal SASP factor secreted from cholangiocytes during NAFLD. By ingenuity pathway analysis, we found that p16 might regulates IGF-1 expression via the E2F1/FOXO1axis. Further analyses indicate that p16 downregulation reduces E2F1 mRNA transcription, inhibiting FOXO1 and subsequent IGF-1 expression in cholangiocytes. The presence of mast cells in the liver has been implicated in multiple cholangiopathies. Our lab demonstrated that mast cell stabilization by cromolyn sodium treatment reduced histamine secretion, fibrosis, and biliary proliferation in Mdr2-/- mice, a model of PSC. Thus, we aimed to determine mast cell stabilization as a therapeutic approach to managing NAFLD and its more advanced form, NASH. We found that cromolyn sodium ameliorated i) serum histamine levels, ii) intrahepatic mast cells, iii) inflammation, iv) fibrosis, v) steatosis, and cholangiocyte proliferation in methionine choline deficient diet-fed mice compared to the saline controls. Overall, we report that amelioration of senescence is a critical factor in improving the disease phenotypes in NAFLD. Biliary senescence plays a crucial role in modulating the disease progression in NAFLD, and mast cell stabilization can be used as a therapeutic approach to reduce pathological hallmarks of fatty liver.
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    Mast Cells Regulate Bile Acid Signaling and Cholestasis via Alteration of Farnesoid X Receptor/Fibroblast Growth Factor 15 Axis in Mice
    (2022-03) Meadows, Victoria E.; Francis, Heather; Alpini, Gianfranco; Dong, X Charlie; Esker, Burcin; Ren, Hongxia
    Primary Sclerosing Cholangitis (PSC) is a rare and slow progressing cholangiopathy characterized by hepatic inflammation, fibrosis and ductular reaction with liver transplantation as the sole therapeutic option. PSC patients are at high risk of auto-immune comorbidities like irritable bowel disease (IBD), found in up to 80% of PSC patients (PSC-IBD). There are indications of genetic and environmental components for auto-immune development in IBD; however, its etiology remains unclear. Mast cells (MCs) infiltrate the liver and can become activated leading to degranulation and release of mediators, like histamine (HA), which result in increased intrahepatic bile duct mass, biliary senescence, hepatic inflammation, and hepatic stellate cell activation. Similarly, MCs infiltrate the intestine and increase inflammation which alters host-microbiome communication. MCs are necessary for successful liver regeneration and the combat of intestinal pathogens; however, chronic HA signaling exacerbates damage in cholangiopathies and IBD. Bile acid synthesis is tightly regulated by Farnesoid X Receptor (FXR) and its downstream mediator, fibroblast growth factor 15 (FGF15, -19 in humans). Cholangiocytes (i) are the target of cholangiopathies, (ii) modify and recycle bile acids through Apical Sodium Bile Acid Transporter (ASBT)-mediated cholehepatic shunting, which functions outside of enterohepatic circulation of bile acids and (iii) are capable of autocrine HA signaling. The complex relationship between hepatic and intestinal MC infiltration and bile acid signaling has not been established; therefore, identifying MC regulation of bile acid pool and FXR/FGF15 signaling pathway will provide insight into therapeutic treatment of PSC-IBD. Under the rationale that (i) cholestatic liver diseases are positively correlated with auto-immune comorbidities like IBD, (ii) during disease, MCs infiltrate the liver and intestine and release signaling factors like HA, and (iii) MCs express FXR and secrete FGF15/19; we propose the central hypothesis that MC activation regulates bile acid signaling and PSC progression through paracrine crosstalk with cholangiocytes in the liver and intestinal inflammation.
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    Melatonin-MT1 Signaling Axis Ameliorates the Phenotypes of Primary Sclerosing Cholangitis
    (2023-03) Ceci, Ludovica; Alpini, Gianfranco; Francis, Heather; Ekser, Burcin; Dong, Charlie X.; Maiers, Jessica L.
    Background: Primary Sclerosing Cholangitis (PSC) is characterized by hepatic fibrosis and portal inflammation. Melatonin is synthesized by arylalkylamine N-acetyltransferase (AANAT) in cholangiocytes. We found that: (i) the MT1 receptor is primarily expressed in cholangiocytes; (ii) melatonin reduces biliary proliferation via MT1 receptor signaling; and (iii) melatonin treatment for 1 wk decreases ductular reaction (DR) and liver fibrosis in cholestatic rats by downregulation of MT1 and clock genes. Melatonin administration to male Mdr2-/- mice (PSC model) reduces angiogenesis and portal inflammation via decreased miR-200b. Downregulation of maspin triggers angiogenesis during tumorigenesis by interaction with glutathione S-transferase (GST). We aimed to evaluate the effects of long-term melatonin treatment and MT1 signaling on PSC phenotypes in Mdr2-/- mice. Methods: Male FVB/NJ and Mdr2-/- mice had access ad libitum to drinking water with/without melatonin for 3 months. Immortilized-SV40-cholangiocytes isolated from human liver samples (control and PSC) were treated with melatonin (10-3 mol/L) for 24 hr. Male C3H-Hej (WT for MT1-/-), FVB/NJ (WT for Mdr2-/-), MT1-/-, Mdr2-/- mice and MT1-/-/Mdr2-/- mice were euthanized at 12 wk. We analyzed liver damage, PSC phenotypes, angiogenesis and AANAT, melatonin receptors and clock genes by immunohistochemistry, immunofluorescence, ELISA and western blots in liver samples and isolated cholangiocytes. Melatonin signaling was evaluated in human control and PSC samples. Results: Long-term melatonin treatment and inhibition of MT1 receptor ameliorates cholestatic liver phenotypes in Mdr2-/-mice by decreasing the immunoreactivity of melatonin enzymes and clock genes. GST activity and maspin expression decreased in Mdr2-/- mice and human PSC samples compared to controls; the phenotypes were reversed by melatonin. Conclusion: Chronic melatonin treatment improves liver histology and restores biliary circadian rhythm by interaction with MT1. Suppression of MT1 ameliorates biliary/liver phenotypes through changes in clock genes and melatonin enzymes. Restoration of the circadian rhythm by modulation of melatonin/MT1 signaling may be key for PSC management.