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      Cholangiocyte autophagy contributes to hepatic cystogenesis in polycystic liver disease and represents a potential therapeutic target

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          Abstract

          <p class="first" id="P1">Polycystic liver disease (PLD) is a group of genetic disorders with limited treatment and significant morbidity. Hepatic cysts arise from cholangiocytes exhibiting a hyperproliferative phenotype. Considering that hyperproliferation of many cell types is associated with alterations in autophagy, we hypothesized that autophagy is altered in PLD cholangiocytes, contributes to hepatic cystogenesis, and might represent a potential therapeutic target. We employed Functional Pathway Cluster Analysis (FPCA) and NextGen Sequencing (NGS), transmission electron microscopy, immunofluorescence confocal microscopy, and western blotting to assess autophagy in human and rodent PLD cholangiocytes. A 3-D culture model was utilized to study the effects of molecular and pharmacologic inhibition of autophagy on hepatic cystogenesis <i>in vitro</i>, and the PCK rat, an animal model of PLD, to study the effects of hydroxychloroquine (HCQ), a drug that interferes with the autophagy pathway, on disease progression <i>in vivo</i>. Assessment of the transcriptome of PLD cholangiocytes followed by FPCA revealed that the autophagy-lysosomal pathway is one of the most altered pathways in PLD. Direct evaluation of autophagy in PLD cholangiocytes both <i>in vitro</i> and <i>in vivo</i> showed increased number and size of autophagosomes, lysosomes and autolysosomes, overexpression of autophagy-related proteins (Atg5, Beclin1, Atg7, and LC3), and enhanced autophagic flux associated with activation of the cAMP-PKA-CREB signaling pathway. Molecular and pharmacologic intervention in autophagy with <i>ATG7</i> siRNA, Bafilomycin A <sub>1</sub> and HCQ reduced proliferation of PLD cholangiocytes <i>in vitro</i> and growth of hepatic cysts in 3-D cultures. HCQ also efficiently inhibited hepatic cystogenesis in the PCK rat. </p><div class="section"> <a class="named-anchor" id="S1"> <!-- named anchor --> </a> <h5 class="section-title" id="d7139971e164">Conclusion</h5> <p id="P2">Autophagy is increased in PLD cholangiocytes, contributes to hepatic cystogenesis, and represents a potential therapeutic target for disease treatment. </p> </div>

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          A critical role for the autophagy gene Atg5 in T cell survival and proliferation

          Macroautophagy (hereafter referred to as autophagy) is a well-conserved intracellular degradation process. Recent studies examining cells lacking the autophagy genes Atg5 and Atg7 have demonstrated that autophagy plays essential roles in cell survival during starvation, in innate cell clearance of microbial pathogens, and in neural cell maintenance. However, the role of autophagy in T lymphocyte development and survival is not known. Here, we demonstrate that autophagosomes form in primary mouse T lymphocytes. By generating Atg5−/− chimeric mice, we found that Atg5-deficient T lymphocytes underwent full maturation. However, the numbers of total thymocytes and peripheral T and B lymphocytes were reduced in Atg5 chimeras. In the periphery, Atg5−/− CD8+ T lymphocytes displayed dramatically increased cell death. Furthermore, Atg5−/− CD4+ and CD8+ T cells failed to undergo efficient proliferation after TCR stimulation. These results demonstrate a critical role for Atg5 in multiple aspects of lymphocyte development and function and suggest that autophagy may be essential for both T lymphocyte survival and proliferation.
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            Functions of autophagy in normal and diseased liver.

            Autophagy has emerged as a critical lysosomal pathway that maintains cell function and survival through the degradation of cellular components such as organelles and proteins. Investigations specifically employing the liver or hepatocytes as experimental models have contributed significantly to our current knowledge of autophagic regulation and function. The diverse cellular functions of autophagy, along with unique features of the liver and its principal cell type the hepatocyte, suggest that the liver is highly dependent on autophagy for both normal function and to prevent the development of disease states. However, instances have also been identified in which autophagy promotes pathological changes such as the development of hepatic fibrosis. Considerable evidence has accumulated that alterations in autophagy are an underlying mechanism of a number of common hepatic diseases including toxin-, drug- and ischemia/reperfusion-induced liver injury, fatty liver, viral hepatitis and hepatocellular carcinoma. This review summarizes recent advances in understanding the roles that autophagy plays in normal hepatic physiology and pathophysiology with the intent of furthering the development of autophagy-based therapies for human liver diseases.
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              Octreotide inhibits hepatic cystogenesis in a rodent model of polycystic liver disease by reducing cholangiocyte adenosine 3',5'-cyclic monophosphate.

              In polycystic liver diseases (PCLDs), increased cholangiocyte proliferation and fluid secretion are key features and cholangiocyte adenosine 3',5'-cyclic monophosphate (cAMP) is an important regulator of these processes. Thus, we assessed cAMP levels and evaluated octreotide (an analogue of somatostatin known to inhibit cAMP) in hepatic cyst growth using an in vitro model of cystogenesis and an in vivo animal model of autosomal recessive polycystic kidney disease (ARPKD), one of the PCLDs. Expression of somatostatin receptors (SSTRs) was assessed by reverse-transcription polymerase chain reaction and confocal microscopy in cholangiocytes from normal and polycystic kidney (PCK) rats, the ARPKD model of autosomal recessive polycystic kidney disease. Effects of octreotide on cAMP levels and cyst expansion were studied in vitro using PCK bile ducts grown in 3-dimensional culture. The effects of octreotide on hepatic and renal cystogenesis were investigated in PCK rats in vivo. In cholangiocytes and serum of PCK rats, cAMP concentrations were approximately 2 times higher than in normal rats. SSTR subtypes that bind octreotide (ie, SSTR2, SSTR3, and SSTR5) were expressed in both normal and PCK cholangiocytes. In vitro, octreotide inhibited cAMP levels by 35% and reduced cyst growth by 44%. In vivo, octreotide lowered cAMP content in cholangiocytes and serum by 32%-39% and inhibited hepatic disease progression, leading to 22%-60% reductions in liver weight, cyst volume, hepatic fibrosis, and mitotic indices. Similar effects were observed in kidneys of PCK rats. This preclinical study provides a strong rationale for assessing the potential value of octreotide in the treatment of PCLDs.
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                Author and article information

                Journal
                Hepatology
                Hepatology
                Wiley
                02709139
                March 2018
                March 2018
                February 01 2018
                : 67
                : 3
                : 1088-1108
                Affiliations
                [1 ]Division of Gastroenterology and Hepatology; Mayo Clinic College of Medicine and Science; Rochester MN
                Article
                10.1002/hep.29577
                5826832
                29023824
                8d4f14a6-8125-4002-bb7f-9f91de32af04
                © 2018

                http://doi.wiley.com/10.1002/tdm_license_1.1

                http://onlinelibrary.wiley.com/termsAndConditions#vor

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