Gene expression changes by VPA treatment.

<p>(<b>A</b>) Hierarchical clustering analysis on genes with statistical significance (one-way anova, P < 0.05 followed by Benjamini-Hochberg correction), with at least a 2-fold down regulation by valproic acid (VPA) treatment (Ctrl64h vs. VPA64h) and with an up regulated/unchanged expression during hepatic stellate cell (HSC) activation. (<b>B</b>) Hierarchical clustering analysis on genes with statistically significance (one-way anova, P < 0.05 followed by Benjamini-Hochberg correction), with at least a 2-fold up regulation by VPA treatment (Ctrl64h vs. VPA64h) and with a down regulated/unchanged expression during HSC activation. Gene selection used for clustering analysis is highlighted in yellow in both schemes and the 5 top genes from this group are extracted from the clustering tables. Green color indicates a low expression value, black color indicates an intermediate expression value and red color indicates a high expression value. </p

Gene expression pattern changes during early culture induced mHSC activation.

<p>Hepatic stellate cells were isolated from normal mice and cultured in 10% fetal bovine serum. 4 hours, 16 hours and 64 hours after washing of the cells mRNA was extracted, cRNA was generated and a microarray analysis was performed. (<b>A</b>) Shown is a representative graph of 2 or 3 separate HSC isolations per group (“1,” “2,” and “3”) containing all genes that were more than 2-fold up regulated (shown in red) or down regulated (shown in green) in at least 1 of the time points in comparison with quiescent HSCs (4h) (one-way anova, P < 0.05 followed by Benjamini-Hochberg correction). (<b>B</b>) Graphic representation of observed trends in gene expression changes during early HSC activation. Normalized intensities are shown and a known HSC associated gene is given that follows the trend. A representative gene for each trend is shown above each graph. (<b>C</b>) Table representing fold changes of known HSC activation/quiescence markers. (<b>D</b>) The Venn diagram shows overlapping patterns of probe sets that were significantly (P < 0.05) and at least 2-fold up regulated and down regulated between 2 time points. Probe sets that were 2-fold up regulated or down regulated in one group of which the trend was continued with a 2-fold up or down regulation between 16-64h are in the intersection of the Venn diagram. (<b>E</b>) Top 5 “molecular and cellular functions” identified by Ingenuity Pathway Analysis performed with the genes from the Venn diagram intersection.</p

Gene Expression Profiling of Early Hepatic Stellate Cell Activation Reveals a Role for Igfbp3 in Cell Migration

<div><p>Background</p><p>Scarring of the liver is the result of prolonged exposure to exogenous or endogenous stimuli. At the onset of fibrosis, quiescent hepatic stellate cells (HSCs) activate and transdifferentiate into matrix producing, myofibroblast-like cells. </p> <p>Aim and methods</p><p>To identify key players during early HSC activation, gene expression profiling was performed on primary mouse HSCs cultured for 4, 16 and 64 hours. Since valproic acid (VPA) can partly inhibit HSC activation, we included VPA-treated cells in the profiling experiments to facilitate this search. </p> <p>Results</p><p>Gene expression profiling confirmed early changes for known genes related to HSC activation such as <i>alpha</i><i>smooth</i><i>muscle</i><i>actin</i> (<i>Acta2</i>)<i>, lysyl</i><i>oxidase</i> (<i>Lox</i>) and <i>collagen</i>, type <i>I</i>, alpha <i>1</i> (<i>Col1a1</i>). In addition we noticed that, although genes which are related to fibrosis change between 4 and 16 hours in culture, most gene expression changes occur between 16 and 64 hours. <i>Insulin-like</i><i>growth</i><i>factor</i><i>binding</i> protein <i>3</i> (<i>Igfbp3</i>) was identified as a gene strongly affected by VPA treatment. During normal HSC activation <i>Igfbp3</i> is up regulated and this can thus be prevented by VPA treatment <i>in</i><i>vitro</i> and <i>in</i><i>vivo</i>. siRNA-mediated silencing of <i>Igfbp3</i> in primary mouse HSCs induced matrix metalloproteinase (Mmp) <i>9</i> mRNA expression and strongly reduced cell migration. The reduced cell migration after <i>Igfbp3</i> knock-down could be overcome by tissue inhibitor of metalloproteinase (TIMP) 1 treatment. </p> <p>Conclusion</p><p>Igfbp3 is a marker for culture-activated HSCs and plays a role in HSC migration. VPA treatment prevents <i>Igfbp3</i> transcription during activation of HSCs <i>in</i><i>vitro</i> and <i>in</i><i>vivo</i>.</p> </div

Validation of VPA-dependent genes during HSC activation <i>in</i><i>vitro</i> and <i>in</i><i>vivo</i>.

<p>(<b>A</b>, <b>B</b>) mRNA levels of <i>Uchl1</i>, <i>Aplp1</i>, <i>Prtpn, Plat</i> and <i>Igfbp3</i> in <i>in </i><i>vitro</i> activating HSCs cultured for 4 or 64 hours with or without VPA supplementation determined by qPCR. n=3 (<b>C</b>) mRNA expression of HSC activation markers <i>Acta2</i> and Lox in HSCs isolated from CCl₄ treated mice , (<b>D</b>) <i>Uchl1</i>, <i>Aplp1</i> and <i>Igfbp3</i> mRNA levels in <i>in </i><i>vivo</i> activated hepatic stellate cells (HSCs). The cells were isolated from Balb/C jicco CBY mice that were treated for 2 weeks with CCl₄ with or without VPA supplementation to the drinking water. (<b>E</b>) Different liver cell types were isolated from healthy mouse livers; hepatocytes were obtained with Percoll gradients, HSCs with Nycodenz gradients and KC and LSECs were isolated using respectively FACS based F4/80- and CD146-FITC-positivity. QPCR analysis was performed for <i>Igfbp3</i> mRNA in the different liver cell types. Experiments were repeated at least 2 times. In the graphs, the results are displayed as means ± SEM. ns = not significant p ≥ 0.05, * p < 0.05, ** p < 0.01.</p

Effect of Igfbp3 knock-down on gene expression, migration and proliferation in activating HSCs (A)

<p>mRNA levels of <i>Igfbp3</i> in activating day 5 HSCs (HSC D5, once transfected at day 1) and day 9 HSCs (HSC D9, twice transfected at day 5/day7) transfected with a control siRNA (siCtrl) or with an siRNA for <i>Igfbp3</i> (siIgfbp3). (<b>B</b>) <i>Acta2</i>, Lox and <i>Col1a1</i> mRNA levels in activated day 9 HSCs transfected with siCtrl or with siIgfbp3 (<b>C</b>) Influence of <i>Igfbp3</i> knock-down on the proliferation of hepatic stellate cells (HSCs). At day 2, siIgfbp3-transfected (at day 1) and siCtrl-transfected HSCs were exposed to EdU, fixed 2 days later and stained for DNA-incorporated EdU. The percentage of EdU-positive cells is given. (<b>D</b>) Principle of PDGFbb-induced transwell migration (left). siIgfbp3 transfected HSCs were plated at day 9 in transwell inserts. Migration was determined 18 hours (h) later and compared to HSCs transfected with siCtrl. Results are relative to siCtrl/siIgfbp3 transfected HSCs without PDGFbb supplementation (right). (<b>E</b>) Vinculin (Vcl), Paxillin (Pxn), myosin heavy chain 9, 10, 11 (Myh9, -10, -11) and matrix metalloproteinase 2, 9 (Mmp2, -9) mRNA levels in activated day 9 HSCs transfected with siCtrl or with siIgfbp3 (<b>F</b>) Protein levels for MMP9 and GAPDH on day 9 HSCs transfected twice with siCtrl or siIgfbp3. Densitometry values (dens) for MMP9 relative to the GAPDH loading control are displayed below the Western blot. (<b>G</b>) Representative images of HSCs at 0 and 48h after wounding (left), and quantification of migration (right). HSCs were transfected with siCtrl, siIgfbp3 or siIgfbp3 in combination with a supplementation of 100 ng/mL recombinant murine TIMP-1 (siIgfbp3 + TIMP-1). Experiments were repeated at least 3 times. Results in the graphs are expressed as means ± SEM. (<b>H</b>) The contribution of MMP9 to HSC migration was evaluated by treatment of activated HSCs with an MMP9 selective inhibitor, using different concentrations, n=2. Migration is expressed as relative to the width of the wound at the start of the scratch. ns = non-significant p ≥ 0.05, * p < 0.05, ** p < 0.01, *** p < 0,001.</p

DDC diet induces biliary damage in mice.

<p>(A) Bilirubin and ALT serum levels were measured from control animals and after 7 and 14 days of DDC-diet with a clinical chemistry module. (B, C) Liver sections of control animals and mice subjected to DDC-Diet for 7 and 14 days were stained with HE, anti-F4/80 Ab, anti-CK19 Ab, Sirius Red and anti- Laminin Ab as described in material and methods. All images were taken in 40x original magnification. The percentage of F4/80-, CK19-, PicroSirius- and Laminin -positive area per field was assessed as described in detail in materials and methods section. All data are presented as mean ± SEM for n = 5/group, *p<0.05 and ***p<0.001.</p

Under macrophage depletion proliferation of CK19 positive ductules in the portal region is inhibited.

<p>(A) Liver sections of the same mouse collective were co-stained with CK19/Ki67 to visualize the proliferation index. All images were taken in 40x original magnification. PV = Portal vein. White arrow indicating porphyrin deposition. (B) To quantify the proliferation index, the quantity of Ki 67-positive cells/field was determined. (C) To obtain the extent of apoptosis, TUNEL stainings were carried out (<b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162286#pone.0162286.s008" target="_blank">S8 Fig</a>)</b> and the amount of TUNEL pos. cells per field was quantified. All stainings and quantifications were performed as described in materials and methods. All data are presented as mean ± SEM for n = 5/group, ***p<0.001.</p

Macrophage depletion attenuates activation of myofibroblast like cells and preserves a type I ductular reaction in DDC fed mice.

<p>(A) Bilirubin and ALT serum levels were measured from control animals and mice subjected to a 14 day DDC diet in co-treatment with either CLO^Lipo or PBS^Lipo at experimental day 7. (B) Liver sections were stained with HE, anti-F4/80 Ab, anti-CK19 Ab, Sirius Red, anti-Laminin Ab and anti-alpha-Sma Ab as described in material and methods. All single stained images were taken in 20x, original magnification. Orange arrow indicate irregular CK19⁺ cell clusters migrating from the portal tract into parenchyma or porto-portal (Type II DR). The percentage of F4/80-, CK19-, Sirius red-, Laminin- and αSma- positive area per field was assessed as described in materials and methods. All data are presented as mean ± SEM for n = 5/group. *p<0.05, **p<0.01, ***p<0.001.</p

Overall amount of laminin matrix deposition and maximum migration distance of CK19 positive cell clusters is positively correlated.

<p>(A) Liver sections of control animals and mice subjected to a 14 day DDC-Diet in co-treatment with either CLO^Lipo or PBS^Lipo at experimental day 7, were co-stained with CK19/Laminin to assess the correlation between CK19⁺ cell clusters and ECM. (B) Positive correlation between overall amount of laminin matrix deposition. (C) Maximum migration distance of CK19⁺ cell clusters from the portal vicinity into the lobule. All stainings and quantifications were performed as described in materials and methods. All data are presented as mean ± SEM for n = 5/group, *p<0.05 and ***p<0.001.</p

Schematic representation of the effect of macrophage depletion during DDC diet administration.

<p>DDC diet promotes a ductular reaction (DR) budding from resident bile ducts (rBD) chaperoned by laminin rich matrix. In PBS^Lipo co-treated mice (from day 7 onwards), a DR type II can be observed with CK19⁺ cells either forming amorphous bile ducts, clusters with pseudoluminae or rosettes, sprouting into the lobule. CLO^Lipo co-treatment (from day 7 onwards) preserves a type I DR, comprising confined ductular proliferates in proximity to the portal vein (PV). The newly budding CK19⁺ cells create more differentiated, bile ductular structures with regular concentric luminae.</p