Non-Parenchymal Cell Biology
Article first published online: 15 OCT 2013
Copyright © 2013 American Association for the Study of Liver Diseases
Special Issue: The 64th Annual Meeting of the American Association for the Study of Liver Diseases: The Liver Meeting 2013
Volume 58, Issue S1, pages 455A–459A, October 2013
How to Cite
(2013), Non-Parenchymal Cell Biology. Hepatology, 58: 455A–459A. doi: 10.1002/hep.26847
- Issue published online: 1 OCT 2013
- Article first published online: 15 OCT 2013
Human amniotic epithelial cell and conditioned media has both anti-fibrotic and pro-regenerative effects on fibrotic liver disease
Alexander Hodge1, 2, Dinushka Lourensz1, 2, Jorge Tchongue1, 2, George Yeoh3, Rebecca Lim4, William Sievert1, 2; 1Gastroenterology and Hepatology, Monash Medical Centre, Monash University, Melbourne, VIC, Australia; 2Centre for Inflammatory Disease, Monash University, Melbourne, VIC, Australia; 3Western Australian Institute for Medical Research, Perth, WA, Australia; 4The Ritchie Centre for Translational Research, Monash University, Melbourne, VIC, Australia
Recovery from cirrhosis requires extra-cellular matrix degradation plus hepatocyte regeneration. Decreased hepatic stellate cell (HSC) activation and expression of matrix degrading enzymes by both macrophages (MP) and HSC create a fibrolytic environment. Hepatocyte regeneration by liver progenitor cells (LPC) requires interactions among LPC, HSC and MP. We have shown that cell therapy with human amniotic epithelial cells (hAEC) ameliorates hepatic fibrosis in a murine model (1)by decreasing HSC activation and inducing MPderived matrix metalloproteinases (MMP). This study investigated whether factors secreted by hAEC contained in conditioned media (CM) (i) reduce hepatic fibrosis in vivo, (ii) exert an anti-fibrotic effect on HSC and MP and (iii) promote LPC proliferation. Methods: Mice given carbon tetrachloride for 12 weeks (wk) were administered intravenous hAEC CM for the last 4 wk of the model. To compare CM to cell therapy, hAEC were injected at 8 wk. Liver fibrosis area was determined by Sirius red staining. The effect of hAEC CM in vitro was explored using human HSC (LX2 cells) and murine bone marrow derived MP exposed to CM for 24 or 48 hour, or mouse LPC (BMOL cell line) given CM given for 4 or 8 days. Differentiation media was applied to LPC as a positive control. MMP9, alpha-fetoprotein (AFP), albumin and cytokeratin-19 (CK-19) gene expression was assessed by RT-PCR. Intracellular collagen was determined by 3H-proIine incorporation. Ly6Chi MP were identified by FACS. Proliferation with determined by Brd-U incorporation. Results: Hepatic fibrosis area was significantly reduced in CM treated animals, equivalent to hAEC cell therapy. hAEC CM treatment reduced MP and HSC numbers but LPC persisted showing that the anti-inflammatory effect of hAEC CM did not impair LPC proliferation. Treatment of HSC by CM resulted in significant reduction in collagen production and increased MMP9 expression. CM treated MP showed a significant reduction in Ly6Chi expression and increase in MMP9 expression. CM treated LPC demonstrated increased proliferation and expression of albumin and AFP but not CK-19.Conclusions: hAEC CM therapy significantly reduced liver fibrosis in vivo and induced a fibrolytic phenotype in HSC and MP. hAEC CM treatment of LPC resulted in proliferation and differentiation down the hepatocyte lineage while markers of cholangiocyte regeneration remained unchanged. hAEC-based therapy appears promising for patients with chronic liver disease. 1.Human Amniotic Epithelial Cell Transplantation Induces Markers of Alternative Macrophage Activation and Reduces Established Hepatic Fibrosis. PLoS ONE. 2012; 7: e38631
William Sievert - Speaking and Teaching: Gilead Sciences, Bristol Myers Squibb, Merck, Roche, Gilead Sciences, Bristol Myers Squibb, Merck, Roche, Gilead Sciences, Bristol Myers Squibb, Merck, Roche, Gilead Sciences, Bristol Myers Squibb, Merck, Roche
The following people have nothing to disclose: Alexander Hodge, Dinushka Lourensz, Jorge Tchongue, George Yeoh, Rebecca Lim
A Specific and Facile Method for Isolating Murine Liver Sinusoidal Endothelial Cells using a CD32B Monoclonal Antibody
Barbara Donofrio, Takhar Kasumov, srinivasan Dasarathy, Arthur J. McCullough; Gastroenterology, Cleveland Clinic, Cleveland, OH
Background: Liver sinusoidal endothelial cells (LSEC) comprise 20% of all liver cells and are now recognized to be both a target and mediator of injury in a number of liver diseases. Despite their emerging importance, current methods of LSEC isolation (centrificugal elutriation or percoll adherence) are cumbersome, produce variable yields and have limited utility as a practical laboratory method. The proposed technique is based on the differential staining characteristics between CD31 (a marker of all endothelial cells) and CD32B (a marker specific for LSEC). Aim: To develop an isolation method for LSEC that would reliably obtain high yield with a specificity and reproducibility that would be easily available for wide spread use. Methods: We used an immunomagnetic method with the monoclonal antibodies: Rat Anti-Mouse F4/80 and Rat Anti-Mouse Fc gamma RIIB/CD32B. Non-Parenchymal cells were obtained by 0.05% Collagenase liver digestion and a 17% OptiPrep (iodixanol) density gradient. Cells were then subjected to Rat Anti-Mouse F4/80-conjugated Dynabeads to remove Kupffer cells and blood Monocytes. The remaining cells were subjected to magnetic bead cell isolation using Rat Anti-Mouse Fc gamma RIIB/CD32B. The attached LSEC were released from the beads with a specific decoupling buffer. Cells were then plated on a fibronectin matrix. Ninety percent of bead isolated cells were recovered with an average yield of 5.5 x10exp6 ±2 LSEC per mouse liver. Both mouse liver tissue and CD32B isolated cells were stained with immune labeled LSEC markers; CD31, CD32B and StabiIin-2 (Stab-2). Isotype controls were run for CD31, CD32B and a blocking peptide control was run for Stab-2.Results: All three markers stained positive in both mouse liver tissue and CD32B isolated cells. A co-stain with CD32B and Stab-2 showed co-localization in isolated LSEC and in liver tissue in a sinusoidal distribution. The CD32B isolated cells also showed typical LSEC fenestrae and sieve plates on scanning electron microscopy. Importantly, 99% of the isolated cells took up formaldehyde-treated serum albumin and oxidized low-density lipoprotein (oxLDL), which are functions and markers specific for LSEC. Conclusion: This straight forward isolation method allows for the culture of LSEC with high purity and reproducibility with a potential for more wide spread use than currently available methods.
The following people have nothing to disclose: Barbara Donofrio, Takhar Kasumov, Srinivasan Dasarathy, Arthur J. McCullough
hepatic progenitors is crucial for liver regeneration and contributes to the regression of liver fibrosis. Methods: The oval-cell
Role of Sphingosine 1-Phosphate in Bone MarrowDerived Monocyte/Macrophages Motility in Chronic Liver Injury in Mice
Le Yang, Zhen Han, Yuanyuan Zhang, Liying Li; Department of Cell Biology, Capital Medical University, Beijing, China
Background/Aims: sphingosine 1-phosphate (S1P) system has been implicated in the pathological process of liver injury in multiple models. This study was designed to evaluate the effects of S1P on bone marrow-derived monocyte/macrophages (BMM) migration in mouse models of chronic liver injury, and identify the signaling pathway underlying this process. Methods: Expression of S1P receptors type 1-3 (S1PR1-3) in BMM was characterized by immunofluorescent staining, real-time RTPCR and Western blot analysis. Cell migration was determined in Boyden chambers by using the selective S1PR1-3 agonist/antagonists or specific siRNAs. Moreover, PTX and LY-294002 were used to define the roles for Gi/o and PI3K pathways in S1P-induced BMM migration, respectively. In vivo, mice were lethally irradiated and received bone marrow transplants from enhanced green fluorescent protein transgenic mice. Bile duct ligation (BDL) was used to induce liver fibrosis with the administration of S1PR2 antagonist, JTE-013 or S1PR3 antagonist, CAY-10444.Liver tissue was examined by immunofluorescent staining and flow cytometric analysis to identify BMM, and was collected for real-time RT-PCR, H&E and Sirius red staining to evaluate the degree of inflammation and fibrosis after chronic liver injury. Results: S1PR1, S1PR2 and S1PR3 were all expressed in BMM. S1P exerted a powerful migratory action on BMM, and the pro-migratory effects were markedly blocked by S1PR2/3 antagonists or correspondent siRNAs. Furthermore, PTX and LY-294002 prevented the S1P-induced BMM migration, and Rac1 activation by S1P was inhibited by JTE-013 or CAY-10444.In vivo, JTE-013 or CAY-10444 significantly reduced BMM recruitment in BDL-induced liver injury in mice, as the proportion of BMM decreased markedly compared with that without JTE-013 or CAY-10444 treatment. In addition, JTE-013 or CAY-10444 administration attenuated BDL-induced hepatic inflammation and fibrosis. Conclusion: S1P/S1PR2/3 system mediates BMM motility by PTX-PI3K-Rac1 signaling pathway, which provides new compelling information on the role of S1P in liver injury and opens new perspectives for the pharmacological treatment of hepatic fibrosis.
The following people have nothing to disclose: Le Yang, Zhen Han, Yuanyuan Zhang, Liying Li
EGF, an Antagonist Cytokine, Reverts and Blocks TGFβ1-Induced Myofibroblast Phenotype and Dysfunction of Hepatic Oval Cells mainly through EGFR-Dependent ERK1/2 Phosphorylation
Ping Wang, Min Cong, Tianhui Liu, Hong Ma, Jidong Jia, Hong You; Liver Resear Cencer, Capital Medical University, Bejing, China
Background: Hepatic progenitors possess proliferation and differentiation capacity to replenish damaged liver in cases of chronic liver disease or acute necrosis. Yet, through exposure to the fibrogenic cytokine, transforming growth factor-β1 (TGF-β1), these cells may undergo epithelial-mesenchymal transition to express extracellular matrix, thereby enhancing the progression of liver fibrosis and even initiating hepatocellular carcinoma. How to revert and block TGF-β1-induced transition of hepatic progenitors is crucial for liver regeneration and contributes to the regression of liver fibrosis. Methods: The oval-cell culture medium was used to reverse the myofibroblast phenotype in 16-day TGF-β1-pretreated rat hepatic oval cells. The specific component responsible for reverting was figured out in the oval-cell culture medium, and the specific component was further used to blockTGF-β1-induced transition. Signaling array and western blot was carried out to find out the signal molecules in charge of reversing and blocking TGF-β1-induced transition. Results: In the presence of TGF-β1 for 16 days, rat hepatic oval cells obtained myofibroblast phenotypes with onehundred times increase of a-smooth muscle actin to control cells, along with losing their proliferation capacity and differentiation potential. First, removing TGF-β1 and adding oval-cell culture medium reversed TGF-β1-induced myofibroblast phenotype and the alteration of hepatic progenitor cell functions to the level of control oval cells. Second, detailed analysis of the components in oval-cell culture medium, including fetal bovine serum, epidermal growth factor (EGF) and stem cell factor, showed that EGF was the cytokine reversed TGF-β1-induced phenotype transition and rescued the proliferation capacity and differentiation potential. Third, if EGF and TGF-β1 were co-administrated to the culture, EGF dose-dependently suppressed TGF-β1-induced phenotype transition and the alteration of hepatic progenitor cell functions, with 1ng/ml EGF totally blocking the transition induced by 1ng/ml TGF-β1.More importantly, using a specific inhibitor for eGf receptor (EGFR), 4-(3'-chloroanilino)−6, 7dimethoxyquinazoline (Tyrphostin AG1478), we not only confirmed the reversing and blocking effects of EGF on TGF-β1, but also found that EGF-regulated suppression and reversal was mediated mainly by EGFR-dependent phosphorylation of extracellular signal-regulated kinases (ERK) 1/2.Conclusion: EGF is the cytokine reversing and blocking TGF-β1-induced phenotype transition and dysfunction of hepatic progenitors, revealing the plasticity of hepatic progenitors and shedding light on the regression of liver fibrosis.
Jidong Jia - Consulting: BMS, GSK, MSD, Novartis, Roche
The following people have nothing to disclose: Ping Wang, Min Cong, Tianhui Liu, Hong Ma, Hong You
Postnatal Vegfr2 ablation in mice results in hepatic microvascular changes including decreased arteries, sinusoidal endothelial degeneration, infarcts, hepatic vein remodeling, and nodular regenerative hyperplasia
Vera Eremina1,Ian R. Wanless2, Marna Lunyova1, M. Hirashima3, Jordan J. Feld5, Susan Quaggin6,1; 1Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, ON, Canada; 2Pathology, QEII HSC, Halifax, NS, Canada; 3Physiology and Cell Biology, Kobe University, Kobe, Japan; 4Research Institute, Hospital for Sick Children, Toronto, ON, Canada; 5Medicine, University of Toronto, Toronto, ON, Canada; 6Nephrology, Northwestern University, Chicago, IL
Agents that block VEGF signaling are used clinically to reduce injurious angiogenic events. However, these drugs are associated with microvascular degeneration, especially in the kidney (Eremina 2008). Hepatic abnormalities have been described but not in detail. Nodular regenerative hyperplasia (NRH), defined histologically by nodules of hepatocytes separated by regions of atrophy with minimal fibrosis, is caused by microvascular obstruction, especially of portal veins (PV) (Wanless 1980). NRH, in the absence of PV obstruction has been described in an animal model with knockout of Notch1 which is involved in VEGF expression (Dill 2012). Another mouse model with disruption of RBP-J in the VEGF pathway was associated with veno-occlusive disease (Wang 2009). Here we describe the development of hepatic microvascular degenerative changes, including NRH, in mice following postnatal deletion of the major VEGF signaling receptor, Flk1/Kdr/Vegfr2.Methods. VEGFR2 knock-out mice were bred, as described (Eremina 2008 and Sison 2010). 20 mutant and 20 controls were sacrificed at 12 weeks of age. Formalin-fixed liver was cut and stained with H&E and glutamine synthetase. Electron microscopy (TEM and SEM) was performed on liver of 4 mutants and 3 controls. Morphometric analysis was performed on 4 mutant and 4 control livers; the number and dimensions of veins and arteries were recorded. Results. Mutant mice all had grossly nodular livers, associated with marked ascites in 50%. Light microscopy demonstrated hepatic micro-infarcts, zone 3 hepatocellular atrophy, and NRH. EM showed degenerative changes in sinusoidal endothelium with large gaps in place of fenestrated sieve plates. Morphometry demonstrated PV were normal in size and number. In contrast, HVs <2000 µm2 were increased 2.2 fold in number (p<0.01) with total cross-sectional area increased 2.0 fold (p<0.002), while larger HVs were unchanged. Recognizable arteries <10 µm diameter were decreased in number (52% of control, p<0.01) while larger arteries were normal. Conclusions. Normal hepatic microvasculature is dependent on intact VEGF signaling. In this model of VEGFR2 deficiency, we believe decreased development of arteries leads to sluggish sinusoidal flow with secondary infarcts, injury and remodeling of hepatic veins, and focal atrophy leading to the pattern of NRH. NRH in human liver occurs in 4 distinct patterns, explained by congestion and arterialization in various anatomic distributions and severity (Wanless 2013, unpublished observation). This mouse model demonstrates a unique pattern we call NRH-5.The microvascular changes in human liver after drugs affecting VEGF require further study.
Jordan J. Feld - Advisory Committees or Review Panels: Roche, Merck, Vertex, Gilead, Abbott, Tibotec, Theravance, Achillion; Speaking and Teaching: Merck, Roche, Abbott
The following people have nothing to disclose: Vera Eremina, Ian R. Wanless, Marina Lunyova, M. Hirashima, Susan Quaggin
Focal Nodular Hyperplasia is a response to artery-toportal vein shunt causing arterialization and congestive injury that explains the map-like pattern of glutamine synthetase
Ian R. Wanless1, Akiko Takayama2; 1 Pathology, QEII HSC, Halifax, NS, Canada; 2Division of Gastroenterology, Takayama Hospital, Imabari, Japan
Focal nodular hyperplasia (FNH) is a benign hepatocellular nodule that typically occurs in a normal liver and shows a central scar containing large vessels surrounded by multiple component nodules. Other elements include capillarized (arterialized) sinusoids, ductular reaction, cholestatic features, and absence of portal vein and duct. Glutamine synthetase (GS) expression shows a diagnostic map-like pattern (BioulacSage 2008). The pathogenesis of FNH and these multiple features have not been explained. In this study we review 30 resected FNH lesions, focusing on the mechanisms of these features. Hepatocellular GS expression develops when liver tissue is perfused by hypervascular tumor invading the portal vein in peritumoral hyperplasia (PTH) (Arnason 2013) and lost in congested and fibrotic states, apparently when endothelial-hepatocyte contact is lost (Fleming 2013). Methods. All resected liver specimens containing FNH at VGH from 2005-2013 (N=30) were stained with H&E, trichrome, CD34, CK7, and glutamine synthetase. Results. The large central vessels are portal veins modified by arterial ingrowth, with fibromuscular thickening and prominent internal elastic lamina, occasionally with residual organized portal vein thrombus. Sinusoidal endothelial cells are CD34+ with two patterns: faint diffuse staining with large lumina (PV pattern), and intense staining with small calibre lumina (arterial pattern). When the latter was seen there was often actin staining of the zone 1 subendothelial space. Congestive features were seen in central fibrous regions, portal zones, and in zone 3.Duct loss was seen in congested portal tracts. Component nodules have a variable appearance allowing classification into early and late forms. GS is diffuse with zone 1 to zone 3 expression in early nodules that have the PV pattern of CD34.GS is absent in zone 1 when the nodule becomes arterialized (arterial pattern of CD34). Thus, the maplike pattern of GS develops as zone 1 hepatocytes become GS negative. Further evolution occurs when congestive features become prominent in zone 3 followed by fibrosis. Discussion. We propose that FNH is a response to the formation of an arterio-portal shunt occurring in previously normal liver, usually as a result of local portal vein thrombosis. Thus, by definition, FNH is a region of arterialized liver and is useful to indicate how various tissue components respond to arterial flow. Diffuse GS expression occurs early, as seen in PTH, and becomes map-like after arterialization. Other features, including duct loss and central fibrosis occur when the outflow tract becomes narrowed with fibrosis and congestion becomes severe.
The following people have nothing to disclose: Ian R. Wanless, Akiko Takayama
Levistilide A inhibits liver fibrosis and angiogenesis
Zhimin Zhao1, Ye Tan1, Tao Guo1, Chenghai Liu1 , 2
1Institute of Liver Disease, ShuGuang Hospital, Shanghai University of Traditional Chinese Medicine, of Shanghai Academy of Traditional Chinese Medicine, 201203, China;,Shanghai, China; 2E-Institute of Traditional Chinese Medicine Internal Medicine, Shanghai, 201203, China;, Shanghai, China
Background and Aim: Levistilide A (the molecular weight is 380.48 and the formula is C24H28O4) is derived from Angelica Sinensis which has action of protecting liver injury in our previous works. In this study, we investigated its effects on liver fibrosis and angiogenesis in vivo and in vitro. Methods: Liver fibrosis was induced by hypodermic injection of CCl4 for 6 weeks in rats, and treated with 3 mg/kg and 6 mg/kg of Levistilide A from the 4th week of CCI4 intoxication to the end of experiment. Liver inflammation and fibrosis were observed by H&E and Sirius red staining, liver microvasculature was examined by synchrotron radiation X-ray two-dimensional imaging. Sinusoidal fenestration was detected by scanning electron microscope. SK-HEP-1 cells, a hepatic endothelial cell line, was hypoxia-induced by CoCl2. Cell viability was analyzed by MTT assay. vWF expression was examined by immunofluorescent staining. The levels of NO and NOS were observed by fluorescence probe. Tube formation assay and transgenic Zebrafish model were also performed. Results: Levistilide A attenuated liver inflammation and fibrosis in CCl4 rats. It could reduce hepatic microvessel texture and improve sinusoidal capillarization in vivo. It also could inhibit tube formation and the formation of functional vessels in transgenic zebrafish. It could protect SK-HEP-1 cells from the hypoxia injury, and decrease vWF protein expression but promote N〇 production in vitro. Conclusion: Levistilide A had potent effects against liver fibrosis and angiogenesis in vivo and in vitro. Keywords: Levistilide A; fibrosis; angiogenesis
Effects of Levistilide A on Liver fibrosis and angiogenesis. (A) Sirius red staining. (B) Synchrotron radiation 2-d imaging. (C) Scanning electron microscope. (D) Transgenic Zebrafish. (E) The structure of Levistilide A.
The following people have nothing to disclose: Zhimin Zhao, Ye Tan, Tao Guo, Chenghai Liu
The Portal Inflammatory Infiltrate and Ductular Niche in Non-Alcoholic Fatty Liver Disease
Victoria L. Gadd1, Richard Skoien1, Kevin Fagan1, Katharine Irvine1, Elizabeth E. Powell1 , 2 Andrew D. Clouston1
1Centre for Liver Disease Research, University of Queensland, Brisbane, QLD, Australia; 2Department of Gastroenterology and Hepatology, Princess Alexandra Hospital, Brisbane, QLD, Australia
Introduction: Progressive non-alcoholic fatty liver disease (NAFLD) is characterised by portal fibrosis, which is associated with a ductular reaction (DR). Growing evidence implicates portal inflammation as a key predictor of histological progression of NAFLD. We hypothesise that the manifestation of portal fibrosis and a pro-fibrogenic DR is driven by cross-talk between diverse infiltrating immune cells. Although numerous cytokines and multiple individual cell types have been implicated in NAFLD pathogenesis, the composition of the portal and periductal inflammatory infiltrate has not been systematically investigated. Methods: Liver biopsy sections from 32 NAFLD patients were immunostained for keratin-7 to highlight the DR, and costained for markers of candidate cellular components of the portal inflammatory infiltrate, including CD3, CD8, CD20, CD68, neutrophil elastase (NE), IL-17 and MMP-9. Fresh-frozen liver biopsy samples were available from 23 NAFLD patients for analysis of Collagen-1A and inflammatory gene expression (TGFβ, IL-1β, TNFα, IL-6, IFN-۷，IL-4 and IL-10). Results: Inflammatory cells within the portal tracts were more numerous than centrilobular regions. The portal inflammatory compartment was comprised of a mix of inflammatory cells, including T cells, B cells, macrophages and neutrophils. Significant portal infiltration was not observed until the presence of progressive NASH (fibrosis stage 2-4) (all P<0.05) with the exception of macrophage numbers, which increased with the appearance of simple steatosis (P<0.01)as well as progressive NASH (P<0.001) when compared with non-diseased control livers. Portal inflammatory cells were present in the peri-ductular inflammatory niche and cell numbers correlated with increasing grade of DR (all P<0.05) and stage of fibrosis (all P<0.001). Collagen-1A mRNA was elevated in patients with simple steatosis and highest in progressive NASH (P<0.001). TNFα, IL-1 p and IL-6 were highly expressed with progressive NASH (P<0.05) whereas TGFβ, IFN-۷, IL-4 and IL-10 showed no association with disease state and progression. Elevated levels of pro-inflammatory cytokines TNFα and IL-1 p occurred before significant portal inflammatory infiltration (P<0.05). Functional markers highlighted IL-17 production by a subset of neutrophils and MMP-9 by a subset of portal and lobular macrophages. Conclusion: Cell phenotypes and subsequent signalling are greatly influenced by their microenvironments. A modified hepatic inflammatory environment likely influences resident inflammatory cell phenotypes, and may promote a profibrogenic DR and further infiltration of inflamed portal tracts.
The following people have nothing to disclose: Victoria L. Gadd, Richard Skoien, Kevin Fagan, Katharine Irvine, Elizabeth E. Powell, Andrew D. Clouston
Liver Sinusoidal Endothelial Cells (LSECs) in Non-alcoholic Fatty Liver Disease (NAFLD) demonstrate compensatory transcriptional changes
Rachel McMahan1, Silvia Giugiiano1, Cara Porsche1, Lucy GoldenMason1, Hugo R. Rosen1 , 2
1Gastroenterology and Hepatology, University of Colorado, Aurora, CO; 2Integmted Program in Immunology, University of Colorado & National Jewish Hospital, Denver, CO
Background: NAFLD is a prevalent problem throughout the Western world. LSECs have been shown to play important roles in liver injury and repair but their role in the underlying pathogenetic mechanisms of NAFLD remains undefined. Here we evaluated the effects of steatosis on LSEC expression of genes involved in inflammation, apoptosis, angiogenesis and cell adhesion in a murine model of NAFLD and an immortalized human LSEC line. Methods: To evaluate gene expression changes in LSEC during NAFLD C56BL/6 mice were fed a western diet (21% anhydrous milkfat (butterfat), 34% sucrose, and a total of 0.2% cholesterol) or regular chow for 3 months. LSECs (CD45negCD31 +) were purified on a FACSAria cell sorter followed by RNA isolation. The Mouse Endothelial Cell Biology RT2 Profiler PCR Array (SA Biosciences) was used to evaluate gene expression changes in LSEC from lean or obese mice. The immortalized human-liver endothelial cell line TMNK1 was incubated with oliec acid (0.66mM) and palmitic acid (0.33mM) for 24 hours followed by RNA isolation and quantitative PCR. Results: Using PCR array technology we identified distinct gene expression profiles in LSECs from mice with NAFLD. Gene pathway analysis in LSECs from mice fed a high fat or low fat diet showed a number of differentially expressed genes including those involved in apoptosis (12), angiogenesis (20), cell adhesion (13) and inflammation (16). Interestingly, feeding a high fat diet led to an increase in the expression of anti-inflammatory molecules, including the chemokine CX3CL1 (Fraktaline), in LSECs. The high fat diet also led to a decrease in proinflammatory chemokines including CXCL2 (Mip-2) and CCL2 (MCP-1). In vitro incubation of the human LSEC line, TMNK-1, with palmitic acid and oliec acid also led to a significant increase in the expression of CXC3CL1 and a corresponding decrease in CCL2. Conclusions: LSECs isolated from steatotic livers demonstrated a shift towards a pro-apoptotic, anti-inflammatory phenotype. These pathways may represent a compensatory mechanism to reverse the liver damage associated with NAFLD.
The following people have nothing to disclose: Rachel McMahan, Silvia Giugliano, Cara Porsche, Lucy Golden-Mason, Hugo R. Rosen