Mdr2 Knockout Mice Research Articles

Oral N-acetylcysteine ameliorates liver fibrosis and enhances regenerative responses in Mdr2 knockout mice

Cholangiopathies are poorly understood disorders with no effective therapy. The extrahepatic biliary tree phenotype is less studied compared to the intrahepatic biliary injury in both human disease and Mdr2−/− mice, the established cholestatic mouse model. This study aimed to characterize the extra hepatic biliary tree of Mdr2−/− mice at various ages and to determine if injury can be repaired with the antioxidant and glutathione precursor N-acetyl-L-Cysteine treatment (NAC). We characterized extra hepatic bile ducts (EHBD)s at various ages from 2 to 40 weeks old FVB/N and Mdr2−/− mice. We examined the therapeutic potential of local NAC ex vivo using EHBD explants at early and late stages of injury; and systematic therapy by in vivo oral administration for 3 weeks. EHBD and liver sections were assessed by histology and immunofluorescent stains. Serum liver enzyme activities were analyzed, and liver spatial protein expression analysis was performed. Mdr2−/− mice developed progressive EHBD injury, similar to extrahepatic PSC. NAC treatment of ex vivo EHBD explants led to improved duct morphology. In vivo, oral administration of NAC improved liver fibrosis, and decreased liver enzyme activities. Spatial protein analysis revealed cell-type specific differential response to NAC, collectively indicating a transition from pro-apoptotic into proliferative state. NAC treatment should be further investigated as a potential therapeutic option for human cholangiopathies.

Evolution and Discovery of Matrine Derivatives as a New Class of Anti-Hepatic Fibrosis Agents Targeting Ewing Sarcoma Breakpoint Region 1 (EWSR1).

A series of new tricyclic matrinane derivatives were continuously synthesized and evaluated for their inhibitory effects on genes and proteins related to hepatic fibrosis at the cellular level, including collagen type I α1 chain (COL1A1), α smooth muscle actin (α-SMA), connective tissue growth factor (CTGF), and matrix metalloprotein 2 (MMP-2). Among them, compound 6k exerted an appealing potency and significantly reduced liver injury and fibrosis in both bile duct ligation (BDL) rats and Mdr2 knockout mice. An activity-based protein profiling (ABPP) assay indicated that 6k might directly bind to Ewing sarcoma breakpoint region 1 (EWSR1) to inhibit its function and affect the expression of downstream liver fibrosis-related genes and thus regulate liver fibrosis. These results provided a potential novel target for the treatment of liver fibrosis and powerful information for the development of tricyclic matrinanes into promising anti-hepatic fibrosis agents.

FRI-369 - CD44 reduces fibrosis development in chronic cholestatic liver injury in Mdr2 knock-out mice

FRI-369 - CD44 reduces fibrosis development in chronic cholestatic liver injury in Mdr2 knock-out mice

Gypenosides ameliorate ductular reaction and liver fibrosis via inhibition of hedgehog signaling.

Backgroud and aims: Ductular reaction (DR) is a common pathological change and thought to have a key role in the pathogenesis and progression of liver fibrosis. Our previous study reported Gypenosides (GPs) ameliorated liver fibrosis, however, the anti-fibrotic mechanisms of GPs are still unclear. Methods: Liver fibrosis was induced in rats by carbon tetrachloride combining with 2-acerylaminofluorene (CCl4/2-AAF), and Mdr2 knockout (Mdr2 -/-) mice to evaluate the anti-fibrotic role of GPs. In vitro, WB-F344 cells, a hepatic progenitor cells (HPCs) line, with or without Gli1 overexpressing lentiviral vectors, were induced by sodium butyrate (SB) to validate the mechanism of GPs and NPLC0393, the main ingredient of GPs. Results: Both in CCl4/2-AAF-treated rats and Mdr2 -/- mice, GPs obviously reduced the deposition of collagen and hydroxyproline content, inhibited the activation of hepatic stellate cells and inflammatory cell infiltration. Notably, GPs reduced the expressions of Epcam, CK19, CK7, Dhh, Smo, Ptch2, Gli1 and Gli2. Furthermore, CK19+ cells co-expressed Gli1, while the number of CK19+/Gli1+ cells was decreased by GPs. In vitro, GPs and NPLC0393 inhibited the differentiation of WB-F344 cells toward a biliary phenotype. Mechanistically, GPs and NPLC0393 protected against DR by inhibiting hedgehog signaling, which was supported by the results that DR, triggered directly by Gli1 overexpressing lentiviral vector was blocked by administration with GPs or NPLC0393. Conclusion: GPs attenuated DR and liver fibrosis by inhibiting hedgehog signaling, which provided more evidences and a novel mechanism of anti-fibrotic effect of GPs.

Loss of bile salt export pump aggravates lipopolysaccharide-induced liver injury in mice due to impaired hepatic endotoxin clearance.

Background and AimsLipopolysaccharide (LPS) clearance is delayed in cholestatic liver diseases. While compromised clearance by Kupffer cells (KCs) is involved, the role of LPS uptake into hepatocytes and canalicular excretion remains unclear.Approach and ResultsWild‐type (WT) and bile salt export pump (Bsep) knockout (KO) mice were challenged i.p. with LPS. Liver injury was assessed by serum biochemistry, histology, molecular inflammation markers, and immune cell infiltration. LPS concentrations were determined in liver tissue and bile. Subcellular kinetics of fluorescently labeled LPS was visualized by intravital two‐photon microscopy, and the findings in Bsep KO mice were compared to common bile duct–ligated (BDL) and multidrug resistance protein 2 (Mdr2) KO mice. Changes in gut microbiota composition were evaluated by 16S ribosomal RNA gene amplicon sequencing analysis. Bsep KO mice developed more pronounced LPS‐induced liver injury and inflammatory signaling, with subsequently enhanced production of proinflammatory cytokines and aggravated hepatic immune cell infiltration. After LPS administration, its concentrations were higher in liver but lower in bile of Bsep KO compared to WT mice. Intravital imaging of LPS showed a delayed clearance from sinusoidal blood with a basolateral uptake block into hepatocytes and reduced canalicular secretion. Moreover, LPS uptake into KCs was reduced. Similar findings with respect to hepatic LPS clearance were obtained in BDL and Mdr2 KO mice. Pretreatment with the microtubule inhibitor colchicine inhibited biliary excretion of LPS in WT mice, indicating that LPS clearance is microtubule‐dependent. Microbiota analysis showed no change of the gut microbiome between WT and Bsep KO mice at baseline but major changes upon LPS challenge in WT mice.ConclusionsAbsence of Bsep and cholestasis in general impair LPS clearance by a basolateral uptake block into hepatocytes and consequently less secretion into canaliculi. Impaired LPS removal aggravates hepatic inflammation in cholestasis.

Application of Mdr2 gene knockout mice in liver disease research

Mdr2 knockout mice is a liver disease model, which causes cholestasis due to the lack of phospholipids in the bile. At present, it is not only used for the study of human homologous MDR3 gene, but also widely used as an animal model of liver diseases such as primary sclerosing cholangitis, liver fibrosis, progressive familial intrahepatic cholestasis, liver cancer. Herein, we review the Mdr2 knockout mice physiological characteristics and its application in liver disease research.

Fibrotic Events in the Progression of Cholestatic Liver Disease.

Cholestatic liver diseases including primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC) are associated with active hepatic fibrogenesis, which can ultimately lead to the development of cirrhosis. However, the exact relationship between the development of liver fibrosis and the progression of cholestatic liver disease remains elusive. Periductular fibroblasts located around the bile ducts seem biologically different from hepatic stellate cells (HSCs). The fibrotic events in these clinical conditions appear to be related to complex crosstalk between immune/inflammatory mechanisms, cytokine signalling, and perturbed homeostasis between cholangiocytes and mesenchymal cells. Several animal models including bile duct ligation (BDL) and the Mdr2-knockout mice have improved our understanding of mechanisms underlying chronic cholestasis. In the present review, we aim to elucidate the mechanisms of fibrosis in order to help to identify potential diagnostic and therapeutic targets.

Administration of Human MSC-Derived Extracellular Vesicles for the Treatment of Primary Sclerosing Cholangitis: Preclinical Data in MDR2 Knockout Mice.

Primary Sclerosing Cholangitis (PSC) is a progressive liver disease for which there is no effective medical therapy. PSC belongs to the family of immune-mediated biliary disorders and it is characterized by persistent biliary inflammation and fibrosis. Here, we explored the possibility of using extracellular vesicles (EVs) derived from human, bone marrow mesenchymal stromal cells (MSCs) to target liver inflammation and reduce fibrosis in a mouse model of PSC. Five-week-old male FVB.129P2-Abcb4tm1Bor mice were intraperitoneally injected with either 100 µL of EVs (± 9.1 × 109 particles/mL) or PBS, once a week, for three consecutive weeks. One week after the last injection, mice were sacrificed and liver and blood collected for flow cytometry analysis and transaminase quantification. In FVB.129P2-Abcb4tm1Bor mice, EV administration resulted in reduced serum levels of alkaline phosphatase (ALP), bile acid (BA), and alanine aminotransferase (ALT), as well as in decreased liver fibrosis. Mechanistically, we observed that EVs reduce liver accumulation of both granulocytes and T cells and dampen VCAM-1 expression. Further analysis revealed that the therapeutic effect of EVs is accompanied by the inhibition of NFkB activation in proximity of the portal triad. Our pre-clinical experiments suggest that EVs isolated from MSCs may represent an effective therapeutic strategy to treat patients suffering from PSC.

Ghrelin reverses ductular reaction and hepatic fibrosis in a rodent model of cholestasis

The orexigenic peptide ghrelin (Ghr) stimulates hunger signals in the hypothalamus via growth hormone secretagogue receptor (GHS-R1a). Gastric Ghr is synthetized as a preprohormone which is proteolytically cleaved, and acylated by a membrane-bound acyl transferase (MBOAT). Circulating Ghr is reduced in cholestatic injuries, however Ghr’s role in cholestasis is poorly understood. We investigated Ghr’s effects on biliary hyperplasia and hepatic fibrosis in Mdr2-knockout (Mdr2KO) mice, a recognized model of cholestasis. Serum, stomach and liver were collected from Mdr2KO and FVBN control mice treated with Ghr, des-octanoyl-ghrelin (DG) or vehicle. Mdr2KO mice had lower expression of Ghr and MBOAT in the stomach, and lower levels of circulating Ghr compared to WT-controls. Treatment of Mdr2KO mice with Ghr improved plasma transaminases, reduced biliary and fibrosis markers. In the liver, GHS-R1a mRNA was expressed predominantly in cholangiocytes. Ghr but not DG, decreased cell proliferation via AMPK activation in cholangiocytes in vitro. AMPK inhibitors prevented Ghr-induced FOXO1 nuclear translocation and negative regulation of cell proliferation. Ghr treatment reduced ductular reaction and hepatic fibrosis in Mdr2KO mice, regulating cholangiocyte proliferation via GHS-R1a, a G-protein coupled receptor which causes increased intracellular Ca2+ and activation of AMPK and FOXO1, maintaining a low rate of cholangiocyte proliferation.

Bile Acids Modulate Colonic MAdCAM‐1 Expression in a Murine Model of PSC‐IBD

Background & ObjectivePrimary Sclerosing Cholangitis (PSC) is a progressive cholestatic liver disease of unknown etiology that carries a strong association with inflammatory bowel disease (IBD). PSC‐associated IBD (PSC‐IBD) displays a unique colitis phenotype characterized by right side predominant inflammation and increased risk of colorectal cancer compared to non‐PSC‐IBD. Although the initiation/progression PSC‐IBD remains unclear, some evidence supports an integral role for the gut‐specific mucosal vascular addressin cell adhesion molecule (MAdCAM‐1) in modulating both hepatic and intestinal inflammation. Multidrug resistance protein 2 (Mdr2) knockout mice develop spontaneous cholestatic liver injury and fibrosis mirroring human PSC. To further elucidate disease mechanisms of PSC‐IBD, we combined Mdr2−/− mice with the chemically inducible model of DSS colitis (Mdr2−/−/DSS). We hypothesize that alterations in bile acids promote mucosal inflammation in this murine model of combined cholestasis/colitis. There are currently no good models for PSC‐IBD, so our objective is to develop a murine model mimicking PSC‐IBD for studying the gut‐liver axis and proinflammatory crosstalk.Methods & ResultsMice were fed 1.5% DSS for 5 days and recovered for 4 days before being euthanized. Transformed sinusoidal endothelial cells (TSECs) were treated with 5 ng/ml of TNF‐αand/or 100μM of ursodeoxycholic acid (UDCA). UDCA treated mice were fed 0.5% UDCA fortified chow for 2 weeks and throughout the previously described DSS course.In this model, Mdr2−/− mice displayed marked alterations in intestinal bile acid composition and increased susceptibility to DSS colitis based on weight loss, disease activity index, histology, and inflammatory cytokines, etc. Mdr2−/− mice also displayed an upregulation of colonic MAdCAM‐1 expression as compared to C57BL/6 colitis controls. In vitro work utilizing transformed sinusoidal endothelial cells (TSECs) and bEnd.3 cells (brain endothelial cells) revealed that MAdCAM‐1 was potently induced by TNF‐α and that this induction was significantly attenuated by co‐treatment with ursodeoxycholic acid (UDCA). In vivo, DSS‐treated Mdr2−/− mice fed with UCDA fortified chow demonstrated significant attenuation of colitis susceptibility and decreased colonic MAdCAM‐1 expression as compared to standard chow controls.ConclusionsThese findings suggest a unique interplay between alterations in bile acid homeostasis and intestinal MAdCAM‐1 expression in modulating pro‐inflammatory cross‐talk along the gut‐liver axis and identify intestinal bile acid signaling as a potential therapeutic target for the treatment of PSC‐IBD.Support or Funding InformationUniversity of Colorado GI and Liver Innate Immune Program (GALIIP)

TGF-β2 silencing to target biliary-derived liver diseases

ObjectiveTGF-β2 (TGF-β, transforming growth factor beta), the less-investigated sibling of TGF-β1, is deregulated in rodent and human liver diseases. Former data from bile duct ligated and MDR2 knockout (KO) mouse...

Wnt/β-Catenin Signaling Plays a Protective Role in the Mdr2 Knockout Murine Model of Cholestatic Liver Disease.

The Wnt/β-catenin signaling pathway has a well-described role in liver pathobiology. Its suppression was recently shown to decrease bile acid (BA) synthesis, thus preventing the development of cholestatic liver injury and fibrosis after bile duct ligation (BDL). To generalize these observations, we suppressed β-catenin in Mdr2 knockout (KO) mice, which develop sclerosing cholangitis due to regurgitation of BA from leaky ducts. When β-catenin was knocked down (KD) in KO for 2 weeks, hepatic and biliary injury were exacerbated in comparison to KO given placebo, as shown by serum biochemistry, ductular reaction, inflammation, and fibrosis. Simultaneously, KO/KD livers displayed increased oxidative stress and senescence and an impaired regenerative response. Although the total liver BA levels were similar between KO/KD and KO, there was significant dysregulation of BA transporters and BA detoxification/synthesis enzymes in KO/KD compared with KO alone. Multiphoton intravital microscopy revealed a mixing of blood and bile in the sinusoids, and validated the presence of increased serum BA in KO/KD mice. Although hepatocyte junctions were intact, KO/KD livers had significant canalicular defects, which resulted from loss of hepatocyte polarity. Thus, in contrast to the protective effect of β-catenin KD in BDL model, β-catenin KD in Mdr2 KO aggravated rather than alleviated injury by interfering with expression of BA transporters, hepatocyte polarity, canalicular structure, and the regenerative response. The resulting imbalance between ongoing injury and restitution led to worsening of the Mdr2 KO phenotype, suggesting caution in targeting β-catenin globally for all cholestatic conditions.

From the Editor’s Desk: September 2019

From the Editor’s Desk: September 2019

Cholangiocyte-Derived Exosomal Long Noncoding RNA H19 Promotes Hepatic Stellate Cell Activation and Cholestatic Liver Fibrosis.

Activation of hepatic stellate cells (HSCs) represents the primary driving force to promote the progression of chronic cholestatic liver diseases. We previously reported that cholangiocyte-derived exosomal long noncoding RNA-H19 (lncRNA-H19) plays a critical role in promoting cholestatic liver injury. However, it remains unclear whether cholangiocyte-derived lncRNA-H19 regulates HSC activation, which is the major focus of this study. Both bile duct ligation (BDL) and Mdr2 knockout (Mdr2-/- ) mouse models were used. Wild-type and H19maternalΔExon1/+ (H19KO) mice were subjected to BDL. Mdr2-/- H19maternalΔExon1/+ (DKO) mice were generated. Exosomes isolated from cultured mouse and human cholangiocytes or mouse serum were used for in vivo transplantation and in vitro studies. Fluorescence-labeled exosomes and flow cytometry were used to monitor exosome uptake by hepatic cells. Collagen gel contraction and bromodeoxyuridine assays were used to determine the effect of exosomal-H19 on HSC activation and proliferation. Mouse and human primary sclerosing cholangitis (PSC)/primary biliary cholangitis (PBC) liver samples were analyzed by real-time PCR, western blot analysis, histology, and immunohistochemistry. The results demonstrated that hepatic H19 level was closely correlated with the severity of liver fibrosis in both mouse models and human patients with PSC and PBC.H19 deficiency significantly protected mice from liver fibrosis in BDL and Mdr2-/- mice.Transplanted cholangiocyte-derived H19-enriched exosomes were rapidly and preferentially taken up by HSCs and HSC-derived fibroblasts, and promoted liver fibrosis in BDL-H19KO mice and DKO mice. H19-enriched exosomes enhanced transdifferentiation of cultured mouse primary HSCs and promoted proliferation and matrix formation in HSC-derived fibroblasts. Conclusion: Cholangiocyte-derived exosomal H19 plays a critical role in the progression of cholestatic liver fibrosis by promoting HSC differentiation and activation and represents a potential diagnostic biomarker and therapeutic target for cholangiopathies.

Activated hepatic stellate cells and portal fibroblasts contribute to cholestatic liver fibrosis in MDR2 knockout mice

Chronic liver injury often results in the activation of hepatic myofibroblasts and the development of liver fibrosis. Hepatic myofibroblasts may originate from 3 major sources: hepatic stellate cells (HSCs), portal fibroblasts (PFs), and fibrocytes, with varying contributions depending on the etiology of liver injury. Here, we assessed the composition of hepatic myofibroblasts in multidrug resistance gene 2 knockout (Mdr2-/-) mice, a genetic model that resembles primary sclerosing cholangitis in patients. Mdr2-/- mice expressing a collagen-GFP reporter were analyzed at different ages. Hepatic non-parenchymal cells isolated from collagen-GFP Mdr2-/- mice were sorted based on collagen-GFP and vitamin A. An NADPH oxidase (NOX) 1/4 inhibitor was administrated to Mdr2-/- mice aged 12-16 weeks old to assess the therapeutic approach of targeting oxidative stress in cholestatic injury. Thy1+ activated PFs accounted for 26%, 51%, and 54% of collagen-GFP+ myofibroblasts in Mdr2-/- mice at 4, 8, and 16 weeks of age, respectively. The remaining collagen-GFP+ myofibroblasts were composed of activated HSCs, suggesting that PFs and HSCs are both activated in Mdr2-/- mice. Bone-marrow-derived fibrocytes minimally contributed to liver fibrosis in Mdr2-/- mice. The development of cholestatic liver fibrosis in Mdr2-/- mice was associated with early recruitment of Gr1+ myeloid cells and upregulation of pro-inflammatory cytokines (4 weeks). Administration of a NOX inhibitor to 12-week-old Mdr2-/- mice suppressed the activation of myofibroblasts and attenuated the development of cholestatic fibrosis. Activated PFs and activated HSCs contribute to cholestatic fibrosis in Mdr2-/- mice, and serve as targets for antifibrotic therapy. Activated portal fibroblasts and hepatic stellate cells, but not fibrocytes, contributed to the production of the fibrous scar in livers of Mdr2-/- mice, and these cells can serve as targets for antifibrotic therapy in cholestatic injury. Therapeutic inhibition of the enzyme NADPH oxidase (NOX) in Mdr2-/- mice reversed cholestatic fibrosis, suggesting that targeting NOXs may be an effective strategy for the treatment of cholestatic fibrosis.

Sa1522 – Galanin Enhances Hepatic Fibrosis in Mdr2 Knockout Mice Via Co-Ordinate Activation of Galanin Receptors on Cholangiocytes and Hepatic Stellate Cells

Sa1522 – Galanin Enhances Hepatic Fibrosis in Mdr2 Knockout Mice Via Co-Ordinate Activation of Galanin Receptors on Cholangiocytes and Hepatic Stellate Cells

Treatment of a Mouse Model of Cholestasis with a Thyromimetic Improves Biliary Injury But Exacerbates Hepatocyte Injury

Chronic cholestasis results from bile secretory defects or impairment of bile flow, and there are few effective medical therapies available. Thyroid hormone T3 and synthetic thyroid hormone receptor agonists are known to cause induction of hepatocyte proliferation during liver regeneration. However, whether these drugs have therapeutic benefits in cholestatic liver disease is unknown. In this study, we administered GC‐1, a thyromimetic that preferentially acts through the TRb receptor found predominantly in liver, to Mdr2 knockout (KO) mice, which is a commonly used model of sclerosing cholangitis characterized by bile acid (BA) regurgitation, periductular inflammation, and fibrosis. We determined Mdr2 KO mice fed 5mg/kg GC‐1 diet had decreased bilirubin, liver to body weight ratios, serum alkaline phosphatase, but increased serum alanine aminotransferase and aspartate aminotransferase compared to KO mice fed normal diet as early as 1 week on diet. Histologically, KO mice on GC‐1 diet had decreased ductular response, less bridging fibrosis, and fewer SOX9 positive hepatocytes compared to KO on normal diet. Although total liver BA were higher in KO mice fed GC‐1 diet for 2 weeks compared to normal diet, they normalized to KO levels at 4 weeks of diet. To elucidate the mechanism of increased BA accumulation and liver injury, we examined expression of BA transporters and detoxification enzymes. KO mice on GC‐1 diet had decreased bilirubin transport and detoxification genes, such as Mrp3, Cyp2b10, and Oatp4, compared to KO mice on normal diet, with the net result being retention of BA in the hepatocyte. Affymetrix gene array data also indicates that KO mice on GC‐1 have normalized bile acid synthesis compared to WT expression levels. Thus, GC‐1 reduces cholangiocyte injury during cholestasis by inducing retention of BA in hepatocytes, causing injury to the hepatocytes; this occurs through as‐yet unknown mechanisms that are upstream of BA transporters and biosynthesis enzymes and appears to be b‐catenin independent.Support or Funding InformationNational Institute of Biomedical Imaging and Bioengineering of the National Institute of Health under Award Number T32EB0010216UPP Academic Foundation Award 1R01DK103775This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Fine-Tuning of Sirtuin 1 Expression Is Essential to Protect the Liver From Cholestatic Liver Disease.

Cholestasis comprises aetiologically heterogeneous conditions characterized by accumulation of bile acids in the liver that actively contribute to liver damage. Sirtuin 1 (SIRT1) regulates liver regeneration and bile acid metabolism by modulating farnesoid X receptor (FXR); we here investigate its role in cholestatic liver disease. We determined SIRT1 expression in livers from patients with cholestatic disease, in two experimental models of cholestasis, as well as in human and murine liver cells in response to bile acid loading. SIRT1‐overexpressing (SIRToe) and hepatocyte‐specific SIRT1‐KO (knockout) mice (SIRThep–/–) were subjected to bile duct ligation (BDL) and were fed with a 0.1% DDC (3,5‐diethoxycarbonyl‐1,4‐dihydrocollidine) diet to determine the biological relevance of SIRT1 during cholestasis. The effect of NorUDCA (24‐norursodeoxycholic acid) was tested in BDL/SIRToe mice. We found that SIRT1 was highly expressed in livers from cholestatic patients, mice after BDL, and Mdr2 knockout mice (Mdr2–/–) animals. The detrimental effects of SIRT1 during cholestasis were validated in vivo and in vitro. SIRToe mice showed exacerbated parenchymal injury whereas SIRThep–/– mice evidenced a moderate improvement after BDL and 0.1% DDC feeding. Likewise, hepatocytes isolated from SIRToe mice showed increased apoptosis in response to bile acids, whereas a significant reduction was observed in SIRThep–/– hepatocytes. Importantly, the decrease, but not complete inhibition, of SIRT1 exerted by norUDCA treatment correlated with pronounced improvement in liver parenchyma in BDL/SIRToe mice. Interestingly, both SIRT1 overexpression and hepatocyte‐specific SIRT1 depletion correlated with inhibition of FXR, whereas modulation of SIRT1 by NorUDCA associated with restored FXR signaling. Conclusion: SIRT1 expression is increased during human and murine cholestasis. Fine‐tuning expression of SIRT1 is essential to protect the liver from cholestatic liver damage.

SAT-171 - Depletion of the HDC/histamine axis ablates tumor formation, angiogenesis, EMT and inflammation in Mdr2 knockout mice

SAT-171 - Depletion of the HDC/histamine axis ablates tumor formation, angiogenesis, EMT and inflammation in Mdr2 knockout mice

Deletion of microRNA‐34a alleviates endothelial dysfunction and inflammatory response during experimental cholestasis

BackgroundSustained cholestasis results in injury to the biliary epithelium with subsequent ductular reaction, angiogenesis, pericholangitis, and fibrogenesis. The early event in chronic cholestatic liver disease, portal hypertension, is induced by an increased intrahepatic resistance, a major consequence of liver cirrhosis. Endothelial dysfunction in liver sinusoidal endothelial cells (LSECs) decreases the production of vasodilators, such as nitric oxide (NO) and favors vasoconstriction. We aimed to characterize microRNA regulated sinusoidal endothelial dysfunction (SED) in the mouse model of cholestatic liver injury.MethodsBile duct ligation (BDL) and MDR2 knockout mice (MDR2−/−) were used as animal models of cholestatic liver injury (CLI). The upstream modulators and downstream mediators of sinusoidal injury were defined in TLR‐4, constitutive and liver specific miR‐34a knockout mice in vivo and cultured human hepatic sinusoidal endothelial cells with miR‐34a modifications in vitro by real‐time PCR array/assay, immunohistochemistry and Western blot analysis.ResultsUsing BDL and MDR2−/− mouse model of cholestatic liver injury, results showed the significant increase in serum ALT and hepatic miR‐34a, severe inflammation, pericellular fibrosis, and intensified nitrosative stress induced by a 24‐ or 31‐fold induction of nitric oxide synthase (NOS2), respectively. The upregulation of miR‐34a in human sinusoidal endothelial cells led to a time‐dependent repression of its target protein Sirt1 levels as shown by western blot analyses, and a significant increase of the expression of NOS2. SED markers ICAM‐1, VEGFR‐2, and E‐selectin were significantly upregulated in the progressive phases of CLI. Lack of miR‐34a in vivo, reversed the serum ALT level, and restored the levels of Sirt1 coupled with decreased NOS2 mRNA expression as well as SED dysfunction markers. Depletion of miR‐34a in vivo also decreased overall vessel formation and neutrophil infiltration. This was significant in portal areas of CLI mice and was associated with a significant down‐regulation of profibrogenic genes and matrix metalloproteinases. Interestingly, in mice lacking TLR‐4 with CLI, significantly reduced miR‐34a levels, increased Sirt1 repression and decreased NOS2 mRNA expression were observed. In isolated sinusoidal endothelial cells by laser capture microdissection (LCM) from CLI mice, enhanced expression of miR‐34a was observed and SED was discovered. Finally liver specific knockout of miR‐34a in CLI mice also showed reduced NOS2 mRNA level and reversed SED.ConclusionThe discovery that the activation of miR‐34a plays a significant role in the process of cholestatic liver injury through sinusoidal endothelial dysfunction for an exciting field in which the epigenomic microRNAs of hepatic endothelial cells may be manipulated with potential therapeutic benefits to liver inflammation and fibrosis.Support or Funding InformationThis work was supported in part by the Dr. Nicholas C. Hightower Centennial Chair of Gastroenterology from Scott & White, a VA Research Career Scientist Award, a VA Merit award to Dr. Alpini (5I01BX000574), a VA Merit Award (5I01BX002192) to Dr. Glaser, a VA Merit Award (1I01BX001724) to Dr. Meng from the United States (U.S.) Department of Veterans Affairs Biomedical Laboratory Research and Development Service, and the NIH grants DK058411, DK076898, DK107310 and DK062975 to Drs. Alpini, Meng and Glaser. This material is the result of work supported by resources at the Central Texas Veterans Health Care System. The views expressed in this article are those of the authors and do not necessarily represent the views of the Department of Veterans Affairs.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.