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 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

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

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

Effect of MC1568 treatment on fibrogenesis in a CCl₄ induced fibrosis mouse model.

<p>Mice were CCl₄ treated twice a week for a total period of 4 weeks. After the second week, mice also received intraperitoneal injections with MC1568 (50 mg/kg) every two days for two more weeks. The day after the final CCl₄ injection, mice were sacrificed and liver tissue was extracted for analysis. (A) Sirius Red staining was performed to visualize deposited collagens. Image J software was used for quantification of the red surface area. Scale bar = 100 µm. (B) Serum levels of ALT and AST. (C) HDAC activity was measured in protein lysates of livers at the end of treatments using HDAC-Glo. (D) A mathematical correlation between the HDAC activity and the red stained area, after Sirius staining was determined using Pearson correlation method. The a and b refer to HDAC-activity values of the corresponding liver lysates of the images given for CCl₄ + MC1568 in A).</p

List of antibodies for immunocytochemistry and western blot.

<p>List of antibodies for immunocytochemistry and western blot.</p

Class IIa and class IIb HDACs are constantly expressed during <i>in vitro</i> mouse hepatic stellate cell activation.

<p>Mouse HSCs were isolated from healthy mice and cultured for the indicated time. (A) Shows morphological changes associated to the mHSC activation process <i>in vitro</i>. (B) mRNA levels of activation markers <i>Col1a1</i>, <i>Col3a1</i>, <i>Acta2 and Lox</i> were determined using qPCR to confirm <i>in vitro</i> activation. (C) mRNA expression of Class IIa and IIb <i>Hdac</i>s was measured by qPCR. Values represent 3 replicates, ***: p<0,001, **: p<0, 01, *: p<0, 05.</p

Effect of MC1568 treatment on stellate cell activation markers.

<p>(A) Freshly isolated HSCs were plated in presence or absence of 1 µM MC1568 for 10 days. The effect on HSC activation markers <i>Acta2</i>, <i>Lox</i>, <i>Col1a1</i> and <i>Col3a1</i> was evaluated by qPCR. (B) Mouse HSCs were cultured for 10 days in presence or absence of 1 µM MC1568. The effect on HSC activation markers Collagen I, Lysyl oxidase and α-Smooth muscle actin was investigated by western blot. β-actin was used as a loading control. (D) The effect of MC1568 treatment on HSC proliferation was investigated with an EdU incorporation assay. Cells were cultured for 48 hours in the presence or absence of MC1568. Nuclei were stained with 4′,6-Diamidino-2-phenylindole. The percentage of EdU-positive cells was determined from three independent experiments. (D) In order to test the reversibility of MC1568 treatment, freshly isolated mouse HSCs were treated for 7 days, after 7 days the inhibitor was washed out and cells were further cultured until day 10 (recovery). Then cells were collected and mRNA expression of HSC activation markers was determined. (E) Freshly isolated mouse HSCs were cultured for seven days. At day seven, cells were formalin fixed and stained with an antibody against the acetylated form of tubulin. Prior to fixation, cells were treated with 1 µM MC1568 for 24 hours. Nuclei were visualized with 4′,6-Diamidino-2-phenylindole. Scale bar = 100 µM. * p<0.05, ** p<0,01.</p

Role of class II HDACs during HSC activation by selective siRNA mediated knock-down.

<p>Freshly isolated HSCs were transfected with siRNA after 24 hours of culture and a second time on the fifth day of culture, RNA was collected 3 days after the final transfection (day 8). (A) <i>Hdac</i> knock-down was evaluated by qPCR. (B) In addition the effect on activation markers was determined by qPCR. The dashed line represents the gene expression level in cells transfected with non-specific siRNA. (C) At day 4 after the second transfection protein samples were harvested. The effect of HDAC knock-down on Lox and expression was confirmed by WB. β-actin was used as loading control. (D) The effect of HDAc inhibition by MC1568 treatment was investigated after 10 days of treatment in culture. Total RNA was isolated from cultured cells, a microRNA reverse transcription was performed followed by qPCR assay for selective amplification of miR-29a, miR-29b and miR-29c, and RNU6 was used as internal control. (E) Freshly isolated HSCs were transfected with siRNA after 24 hours of culture and a second time on the fifth day of culture, RNA was collected 3 days after the final transfection (day 8). Total RNA was isolated from cultured cells and a qPCR assay for selective amplification of miR-29a, miR-29b and miR-29c was performed, RNU6 was used as internal control. miR-29 expression in HDAC knock down samples was calculated relative to miR-29 expression in a sample transfected with non-targeting siRNA (represented by dashed line). Graphs are representative of at least three experiments. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p