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

How stable is repression of disallowed genes in pancreatic islets in response to metabolic stress?

<div><p>The specific phenotype of mature differentiated beta cells not only depends on the specific presence of genes that allow beta cell function but also on the selective absence of housekeeping genes (“disallowed genes”) that would interfere with this function. Recent studies have shown that both histone modifications and DNA methylation via the de novo methyltransferase DNMT3A are involved in repression of disallowed genes in neonatal beta cells when these cells acquire their mature phenotype. It is unknown, however, if the environmental influence of advanced age, pregnancy and the metabolic stress of high fat diet or diabetes could alter the repression of disallowed genes in beta cells. In the present study, we show that islet disallowed genes—which are also deeply repressed in FACS-purified beta cells—remain deeply repressed in animals of advanced age and in pregnant females. Moreover, the stability of this repression was correlated with strong and stable histone repression marks that persisted in islets isolated from 2 year old mice and with overall high expression of <i>Dnmt3a</i> in islets. Furthermore, repression of disallowed genes was unaffected by the metabolic stress of high fat diet. However, repression of about half of the disallowed genes was weakened in 16 week-old diabetic db/db mice. In conclusion, we show that the disallowed status of islet genes is stable under physiological challenging conditions (advanced age, pregnancy, high fat diet) but partially lost in islets from diabetic animals.</p></div

Epigenetic modifications in islets from ageing mice.

<p><b>(A)</b> Histone H3 modifications in promoter region of islet specific disallowed genes. Data sets include IgG immunoprecipitation controls (black bars); the epigenetic activation mark, histone H3 lysine 9 acetylation (H3K9ac) (blue bars) and the repression mark, histone H3 lysine 27 trimethylation (H3K27me3) (orange bars). Pancreatic islet chromatin was isolated from mice aged 1, 5.5 and 26 months; chromatin from liver and diaphragm of mice aged 5.5 and 26 months or from placenta of 15.5 days pregnant female mice age 3 months was taken as reference tissue. For all genes, high H3K27me3 islet signals and low H3K9ac signals were observed at all ages, while in the reference tissues the opposite is seen. For all of the tested genes except <i>Pdgfra</i> the H3K27me3 signal in islets is not significantly influenced by age. As a control housekeeping gene we investigated the beta actin promoter, which is active in all tissues: high H3K9ac signals and low H3K27m3 signals were measured in all tested conditions (bottom right panel). Data represent mean±SEM, N = 3. Statistical analysis: <i>student t</i>-test with multiple comparison correction (Bonferroni). <b>(B)</b> Upper panel, mRNA expression of the de novo methyltransferase <i>Dnmt3a</i> in different mouse tissues (12 week old mice) measured via Affymetrix, 430 2.0 arrays. Lower panel, DNA methylation measured via total 5-methylcytosine (5mC) levels in different male mouse tissues (12 week old mice); <b>(C)</b> mRNA expression of <i>Dnmt3a</i> during ageing, measured via quantitative RT-PCR. Data represent mean±SEM, N = 4. Statistical analysis: <i>student t</i>-test with multiple comparison correction (Bonferroni), compared to expression at 1 month of age. Data are normalized for <i>ActB</i> expression.</p

Effect of age on the expression of disallowed islet genes.

<p><b>(A)</b> Upper panel heat map of the expression signals of the signature of 14 islet specifically repressed genes in islets isolated from mice of different ages (1, 2, 6, 16 and 26 months) using Multiexperiment Viewer [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181651#pone.0181651.ref019" target="_blank">19</a>]. Group 1 has six genes in which repression matures between months 1 and 6 of life and is then maintained; group 2 has eight genes which repression is independent of age. Lower panel, mRNA expression levels of the different disallowed genes of group1 and group2. Quantitative RT-PCR data are normalized for beta actin, and expression level in liver (from 12 weeks old mice) is set as 1; <b>(B)</b> mRNA expression of key genes involved in proliferation/maturation and beta cell function. Quantitative RT-PCR, normalized for beta actin and expression at 1 month is set as 1. Data represent mean±SEM, N = 4. Statistical analysis: <i>student t</i>-test with multiple comparison correction (Bonferroni), compared to expression at 1 month of age.</p

Analysis of mRNA expression of islet disallowed genes in freshly collagenase-isolated islets, compared with FACS purified alpha and beta cells.

<p>Data are normalized for <i>ActB</i> and expression in liver (from 12 weeks old mice) is set as 1, since liver has a high and robust expression of the islet disallowed genes, making it immediately clear that these genes are very low expressed in islets. Data represent mean±SEM, N≥3, Statistical analysis: <i>student t</i>-test with multiple comparison correction (Bonferroni). UD, under detection limit.</p

Effect of high fat diet feeding and pregnancy on the expression of disallowed islet genes.

<p><b>(A)</b> mRNA expression level of the disallowed genes in islets of male mice (22 weeks old mice) fed a high fat diet for 16 weeks or <b>(B)</b> of female pregnant mice (pregnancy day 0–9.5–15.5) (12 weeks of age) measured via quantitative RT-PCR. Data are normalized for beta actin, and expression level in liver (from 12 weeks old mice) is set as 1; <b>(C)</b> mRNA expression of key genes involved in beta cell function, measured via quantitative RT-PCR and normalized for beta actin. Data represent mean±SEM, N = 4. Statistical analysis: <i>student t</i>-test with multiple comparison correction (Bonferroni).</p