Lindsey Kennedy

Regulation of the Extrinsic Apoptotic Pathway by MicroRNA-21 in Alcoholic Liver Injury

Background: miR-21 is an anti-apoptotic microRNA, and its role in alcoholic liver disease (ALD) is unknown. Results: miR-21, increased in ALD and regulated by IL-6/Stat3, is essential for transformation, survival, and liver fibrosis. Conclusion: miR-21 plays a protective role against alcoholic hepatitis through the anti-extrinsic apoptotic pathway. Significance: Understanding the recovery function of miR-21 may have important implications for patients with ALD. IL-6/Stat3 is associated with the regulation of transcription of key cellular regulatory genes (microRNAs) during different types of liver injury. This study evaluated the role of IL-6/Stat3 in regulating miRNA and miR-21 in alcoholic liver disease. By microarray, we identified that ethanol feeding significantly up-regulated 0.8% of known microRNAs in mouse liver compared with controls, including miR-21. Similarly, the treatment of normal human hepatocytes (N-Heps) and hepatic stellate cells (HSCs) with ethanol and IL-6 significantly increased miR-21 expression. Overexpression of miR-21 decreased ethanol-induced apoptosis in both N-Heps and HSCs. The expression level of miR-21 was significantly increased after Stat3 activation in N-Heps and HSCs, in support of the concept that the 5′-promoter region of miR-21 is regulated by Stat3. Using real time PCR, we confirmed that miR-21 activation is associated with ethanol-linked Stat3 binding of the miR-21 promoter. A combination of bioinformatics, PCR array, dual-luciferase reporter assay, and Western blot analysis revealed that Fas ligand (TNF superfamily, member 6) (FASLG) and death receptor 5 (DR5) are the direct targets of miR-21. Furthermore, inhibition of miR-21 by specific Vivo-Morpholino and knock-out of IL-6 in ethanol-treated mice also increased the expression of DR5 and FASLG in vivo during alcoholic liver injury. The identification of miR-21 as an important regulator of hepatic cell survival, transformation, and remodeling in vitro, as well as its upstream modulators and downstream targets, will provide insight into the involvement of altered miRNA expression in contributing to alcoholic liver disease progression and testing novel therapeutic approaches for human alcoholic liver diseases.

Inhibition of mast cell-derived histamine secretion by cromolyn sodium treatment decreases biliary hyperplasia in cholestatic rodents.

Cholangiopathies are characterized by dysregulation of the balance between biliary growth and loss. We have shown that histamine (HA) stimulates biliary growth via autocrine mechanisms. To evaluate the paracrine effects of mast cell (MC) stabilization on biliary proliferation, sham or BDL rats were treated by IP-implanted osmotic pumps filled with saline or cromolyn sodium (24 mg/kg BW/day (inhibits MC histamine release)) for 1 week. Serum, liver blocks and cholangiocytes were collected. Histidine decarboxylase (HDC) expression was measured using real-time PCR in cholangiocytes. Intrahepatic bile duct mass (IBDM) was evaluated by IHC for CK-19. MC number was determined using toluidine blue staining and correlated to IBDM. Proliferation was evaluated by PCNA expression in liver sections and purified cholangiocytes. We assessed apoptosis using real-time PCR and IHC for BAX. Expression of MC stem factor receptor, c-kit, and the proteases chymase and tryptase were measured by real-time PCR. HA levels were measured in serum by EIA. In vitro, MCs and cholangiocytes were treated with 0.1% BSA (basal) or cromolyn (25 μM) for up to 48 h prior to assessing HDC expression, HA levels and chymase and tryptase expression. Supernatants from MCs treated with or without cromolyn were added to cholangiocytes before measuring (i) proliferation by MTT assays, (ii) HDC gene expression by real-time PCR and (iii) HA release by EIA. In vivo, cromolyn treatment decreased BDL-induced: (i) IBDM, MC number, and biliary proliferation; (ii) HDC and MC marker expression; and (iii) HA levels. Cromolyn treatment increased cholangiocyte apoptosis. In vitro, cromolyn decreased HA release and chymase and tryptase expression in MCs but not in cholangiocytes. Cromolyn-treated MC supernatants decreased biliary proliferation and HA release. These studies provide evidence that MC histamine is key to biliary proliferation and may be a therapeutic target for the treatment of cholangiopathies.

Knockout of microRNA-21 reduces biliary hyperplasia and liver fibrosis in cholestatic bile duct ligated mice

Cholestasis is a condition that leads to chronic hepatobiliary inflammation, fibrosis, and eventually cirrhosis. Many microRNAs (miRs) are known to have a role in fibrosis progression; however, the role of miR-21 during cholestasis remains unknown. Therefore, the aim of this study was to elucidate the role of miR-21 during cholestasis-induced biliary hyperplasia and hepatic fibrosis. Wild-type (WT) and miR-21−/− mice underwent Sham or bile duct ligation (BDL) for 1 week, before evaluating liver histology, biliary proliferation, hepatic stellate cell (HSC) activation, fibrotic response, and small mothers against decapentaplegic 7 (Smad-7) expression. In vitro, immortalized murine biliary cell lines (IMCLs) and human hepatic stellate cell line (hHSC) were treated with either miR-21 inhibitor or control before analyzing proliferation, apoptosis, and fibrotic responses. In vivo, the levels of miR-21 were increased in total liver and cholangiocytes after BDL, and loss of miR-21 decreased the amount of BDL-induced biliary proliferation and intrahepatic biliary mass. In addition, loss of miR-21 decreased BDL-induced HSC activation, collagen deposition, and expression of the fibrotic markers transforming growth factor-β1 and α-smooth muscle actin. In vitro, IMCL and hHSCs treated with miR-21 inhibitor displayed decreased proliferation and expression of fibrotic markers and enhanced apoptosis when compared with control treated cells. Furthermore, mice lacking miR-21 show increased Smad-7 expression, which may be driving the decrease in biliary hyperplasia and hepatic fibrosis. During cholestatic injury, miR-21 is increased and leads to increased biliary proliferation and hepatic fibrosis. Local modulation of miR-21 may be a therapeutic option for patients with cholestasis.

Inhibition of mast cell‐secreted histamine decreases biliary proliferation and fibrosis in primary sclerosing cholangitis Mdr2−/− mice

Hepatic fibrosis is marked by activation of hepatic stellate cells (HSCs). Cholestatic injury precedes liver fibrosis, and cholangiocytes interact with HSCs promoting fibrosis. Mast cells (MCs) infiltrate following liver injury and release histamine, increasing biliary proliferation. We evaluated if inhibition of MC‐derived histamine decreases biliary proliferation and fibrosis. Wild‐type and multidrug resistance 2 knockout mice (9‐11 weeks) were treated with cromolyn sodium for 1 week to block MC‐derived histamine. Biliary mass and proliferation were evaluated by immunohistochemistry for cytokeratin 19 and Ki‐67. Bile flow, bicarbonate excretion, and total bile acids were measured in all mice. Fibrosis was evaluated by sirius red/fast green staining and by quantitative polymerase chain reaction for alpha‐smooth muscle actin, fibronectin, collagen type 1a, and transforming growth factor‐beta 1. HSC activation was evaluated by quantitative polymerase chain reaction in total liver and immunofluorescent staining in tissues for synaptophysin 9. Histamine serum secretion was measured by enzymatic immunoassay. Mouse liver and human liver samples from control or primary sclerosing cholangitis patients were evaluated for MC markers by quantitative polymerase chain reaction and immunohistochemistry. In vitro, cultured MCs were transfected with histidine decarboxylase short hairpin RNA to decrease histamine secretion and subsequently cocultured with cholangiocytes or HSCs prior to measuring fibrosis markers, proliferation, and transforming growth factor‐beta 1 secretion. Treatment with cromolyn sodium decreased biliary proliferation, fibrosis, histamine secretion, and bile flow in multidrug resistance 2 knockout mice. Primary sclerosing cholangitis mice and patients have increased MCs. Knockdown of MC histidine decarboxylase decreased cholangiocyte and HSC proliferation/activation. Conclusion: MCs are recruited to proliferating cholangiocytes and promote fibrosis. Inhibition of MC‐derived histamine decreases fibrosis, and regulation of MC mediators may be therapeutic for primary sclerosing cholangitis. (Hepatology 2016;64:1202‐1216)

Prolonged exposure of cholestatic rats to complete dark inhibits biliary hyperplasia and liver fibrosis

Biliary hyperplasia and liver fibrosis are common features in cholestatic liver disease. Melatonin is synthesized by the pineal gland as well as the liver. Melatonin inhibits biliary hyperplasia of bile duct-ligated (BDL) rats. Since melatonin synthesis (by the enzyme serotonin N-acetyltransferase, AANAT) from the pineal gland increases after dark exposure, we hypothesized that biliary hyperplasia and liver fibrosis are diminished by continuous darkness via increased melatonin synthesis from the pineal gland. Normal or BDL rats (immediately after surgery) were housed with light-dark cycles or complete dark for 1 wk before evaluation of 1) the expression of AANAT in the pineal gland and melatonin levels in pineal gland tissue supernatants and serum; 2) biliary proliferation and intrahepatic bile duct mass, liver histology, and serum chemistry; 3) secretin-stimulated ductal secretion (functional index of biliary growth); 4) collagen deposition, liver fibrosis markers in liver sections, total liver, and cholangiocytes; and 5) expression of clock genes in cholangiocytes. In BDL rats exposed to dark there was 1) enhanced AANAT expression/melatonin secretion in pineal gland and melatonin serum levels; 2) improved liver morphology, serum chemistry and decreased biliary proliferation and secretin-stimulated choleresis; and 4) decreased fibrosis and expression of fibrosis markers in liver sections, total liver and cholangiocytes and reduced biliary expression of the clock genes PER1, BMAL1, CLOCK, and Cry1. Thus prolonged dark exposure may be a beneficial noninvasive therapeutic approach for the management of biliary disorders.

Substance P increases liver fibrosis by differential changes in senescence of cholangiocytes and hepatic stellate cells.

Substance P (SP) is involved in the proliferation of cholangiocytes in bile duct–ligated (BDL) mice and human cholangiocarcinoma growth by interacting with the neurokinin‐1 receptor (NK‐1R). To identify whether SP regulates liver fibrosis during cholestasis, wild‐type or NK‐1R knockout (NK‐1R–/–) mice that received BDL or sham surgery and multidrug resistance protein 2 knockout (Mdr2–/–) mice treated with either an NK‐1R antagonist (L‐733,060) or saline were used. Additionally, wild‐type mice were treated with SP or saline intraperitoneally. In vivo, there was increased expression of tachykinin precursor 1 (coding SP) and NK‐1R in both BDL and Mdr2–/– mice compared to wild‐type mice. Expression of tachykinin precursor 1 and NK‐1R was significantly higher in liver samples from primary sclerosing cholangitis patients compared to healthy controls. Knockout of NK‐1R decreased BDL‐induced liver fibrosis, and treatment with L‐733,060 resulted in decreased liver fibrosis in Mdr2–/– mice, which was shown by decreased sirius red staining, fibrosis gene and protein expression, and reduced transforming growth factor‐β1 levels in serum and cholangiocyte supernatants. Furthermore, we observed that reduced liver fibrosis in NK‐1R–/– mice with BDL surgery or Mdr2–/– mice treated with L‐733,060 was associated with enhanced cellular senescence of hepatic stellate cells and decreased senescence of cholangiocytes. In vitro, L‐733,060 inhibited SP‐induced expression of fibrotic genes in hepatic stellate cells and cholangiocytes; treatment with L‐733,060 partially reversed the SP‐induced decrease of senescence gene expression in cultured hepatic stellate cells and the SP‐induced increase of senescence‐related gene expression in cultured cholangiocytes. Conclusion: Collectively, our results demonstrate the regulatory effects of the SP/NK‐1R axis on liver fibrosis through changes in cellular senescence during cholestatic liver injury. (Hepatology 2017;66:528–541).

Knockout of histidine decarboxylase decreases bile duct ligation-induced biliary hyperplasia via downregulation of the histidine decarboxylase/VEGF axis through PKA-ERK1/2 signaling

Histidine is converted to histamine by histidine decarboxylase (HDC). We have shown that cholangiocytes 1) express HDC, 2) secrete histamine, and 3) proliferate after histamine treatment via ERK1/2 signaling. In bile duct-ligated (BDL) rodents, there is enhanced biliary hyperplasia, HDC expression, and histamine secretion. This studied aimed to demonstrate that knockdown of HDC inhibits biliary proliferation via downregulation of PKA/ERK1/2 signaling. HDC(-/-) mice and matching wild-type (WT) were subjected to sham or BDL. After 1 wk, serum, liver blocks, and cholangiocytes were collected. Immunohistochemistry was performed for 1) hematoxylin and eosin, 2) intrahepatic bile duct mass (IBDM) by cytokeratin-19, and 3) HDC biliary expression. We measured serum and cholangiocyte histamine levels by enzyme immunoassay. In total liver or cholangiocytes, we studied: 1) HDC and VEGF/HIF-1α expression and 2) PCNA and PKA/ERK1/2 protein expression. In vitro, cholangiocytes were stably transfected with shRNA-HDC plasmids (or control). After transfection we evaluated pPKA, pERK1/2, and cholangiocyte proliferation by immunoblots and MTT assay. In BDL HDC(-/-) mice, there was decreased IBDM, PCNA, VEGF, and HDC expression compared with BDL WT mice. Histamine levels were decreased in BDL HDC(-/-). BDL HDC(-/-) livers were void of necrosis and inflammation compared with BDL WT. PKA/ERK1/2 protein expression (increased in WT BDL) was lower in BDL HDC(-/-) cholangiocytes. In vitro, knockdown of HDC decreased proliferation and protein expression of PKA/ERK1/2 compared with control. In conclusion, loss of HDC decreases BDL-induced biliary mass and VEGF/HIF-1α expression via PKA/ERK1/2 signaling. Our data suggest that HDC is a key regulator of biliary proliferation.

Gonadotropin-Releasing Hormone Stimulates Biliary Proliferation by Paracrine/Autocrine Mechanisms

During cholestatic liver disease, there is dysregulation in the balance between biliary growth and loss in bile duct-ligated (BDL) rats modulated by neuroendocrine peptides via autocrine/paracrine pathways. Gonadotropin-releasing hormone (GnRH) is a trophic peptide hormone that modulates reproductive function and proliferation in many cell types. We evaluated the autocrine role of GnRH in the regulation of cholangiocyte proliferation. The expression of GnRH receptors was assessed in a normal mouse cholangiocyte cell line (NMC), sham, and BDL rats. The effect of GnRH administration was evaluated in normal rats and in NMC. GnRH-induced biliary proliferation was evaluated by changes in intrahepatic bile duct mass and the expression of proliferation and function markers. The expression and secretion of GnRH in NMC and isolated cholangiocytes was assessed. GnRH receptor subtypes GnRHR1 and GnRHR2 were expressed in cholangiocytes. Treatment with GnRH increased intrahepatic bile duct mass as well as proliferation and function markers in cholangiocytes. Transient knockdown and pharmacologic inhibition of GnRHR1 in NMC decreased proliferation. BDL cholangiocytes had increased expression of GnRH compared with normal rats, accompanied by increased GnRH secretion. In vivo and in vitro knockdown of GnRH decreased intrahepatic bile duct mass/cholangiocyte proliferation and fibrosis. GnRH secreted by cholangiocytes promotes biliary proliferation via an autocrine pathway. Disruption of GnRH/GnRHR signaling may be important for the management of cholestatic liver diseases.

Molecular mechanisms of stem cell therapy in alcoholic liver disease

Alcoholic liver disease affects a great number of people worldwide. With limited therapeutic options, stem cell therapy offers significant potential for these patients. To date, a limited number of clinical trials have produced transient clinical responses to cell therapy in patients with alcoholic liver disease. Stem cell therapy to reorganize the postnatal liver is an important theme and mission for patients with chronic liver disorders including alcoholic liver injury. We therefore should redevelop the evidence of cell-based liver regeneration therapy, focusing on targets (disease, patients status and hepatic function), materials (cells, cytokines and genes), and methodology (stem cell types and their derived microparticles, transplantation route, implantation technology and tissue engineering). In this review, we summarize the recent findings regarding the experimental and clinical use of mesenchymal and liver stem cells, focusing mainly on the treatment of alcoholic liver disorders and their relevance in the field of regenerative medicine, and advances on the role of microvesicles and exosomes in this process. We discuss new advances in stem cell therapy from liver regeneration to liver re-organization, which is involved in the recent progress of on-going clinical trials, basic research in stem cell therapy and liver regeneration, and updated exosomes/microvesicles recovery/repairing technology.

Histamine regulation of pancreatitis and pancreatic cancer: a review of recent findings

The pancreas is a dynamic organ that performs a multitude of functions within the body. Diseases that target the pancreas, like pancreatitis and pancreatic cancer, are devastating and often fatal to the suffering patient. Histamine and histamine receptors (H1-H4HRs) have been found to play a critical role in biliary diseases. Accordingly, the biliary tract and the pancreas share similarities with regards to morphological, phenotypical and functional features and disease progression, studies related the role of H1-H4HRs in pancreatic diseases are important. In this review, we have highlighted the role that histamine, histidine decarboxylase (HDC), histamine receptors and mast cells (the main source of histamine in the body) play during both pancreatitis and pancreatic cancer. The objective of the review is to demonstrate that histamine and histamine signaling may be a potential therapeutic avenue towards treatment strategies for pancreatic diseases.