Additional file 1: Tables S1–S4. of Histone acetylation and histone acetyltransferases show significant alterations in human abdominal aortic aneurysm

Table S1. Characteristics of the study subjects. Table S2. Nomenclature of histone acetyltransferases used in the study. Table S3. Summary of expression levels of KATs analyzed in this study normalized to the expression of GAPDH. Table S4. Correlation between KAT expression and clinical findings of AAA patients. (DOC 84 kb

Additional file 2: Figure S1. of Histone acetylation and histone acetyltransferases show significant alterations in human abdominal aortic aneurysm

Selected examples of scatter plot graphs from correlation analysis of inter-relationships between KAT2B and KAT3B (A), KAT2B and KAT6B (B), KAT3b and KAT6B (C) in AAA at mRNA level. Quantification was performed by SYBR green-based RT-PCR using KATs expression intensity normalized to GAPDH. AAA (n = 37). (PPTX 74 kb

Additional file 3: Figure S2. of Histone acetylation and histone acetyltransferases show significant alterations in human abdominal aortic aneurysm

Selected examples of scatter plot graphs from correlation analysis between expression of KATs and specific markers of cells in AAA at mRNA level. KAT2B (A), KAT3B) (B), KAT6B (C). Quantification was performed by SYBR green-based RT-PCR. The expression of all factors was normalized to GAPDH. AAA (n = 37). (PPTX 152 kb

STAT1-mediated abolished response to norepinephrine and sodium nitroprusside is associated with disturbed NO production.

<p>A, <i>WT</i> and <i>STAT1^−/− VSMCs</i> were treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113318#pone-0113318-g001" target="_blank">Fig. 1</a>. RNA was isolated and qRT-PCR for <i>Nos2</i> using <i>Gapdh</i> as internal control was performed (upper panel) B, After stimulation as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113318#pone-0113318-g001" target="_blank">Fig. 1</a>, medium was refreshed and left for 24 h. Next, 100 µl of the medium was taken and the product of Nos2- nitrite was measured. Data represent means of at least 3 independent biological experiments ±SEM and p<0.05 was considered as significant. Data were tested for significance by one-way ANOVA followed by post-hoc Tukey or unpaired two-tailed student T-test when appropriate. C, D Isolated aortic rings from WT and STAT^−/− mice were incubated with 10 ng/ml IFNγ for 8 h or with 1 ug/ml of LPS for 4 h or with IFNγ for 4 h followed by LPS for additional 4 h. Next, response to norepinephrine and sodium nitroprusside was tested on the wire myograph. C, Response to noradrenaline in WT and STAT1-deficient aortic rings presented as a percentage of maximal constriction to KPSS.*p<0.001 vs. WT control; •p<0.001 vs. WT LPS; ○p<0.001 vs. STAT1^−/− control. D, Response to stepwise increased concentration of sodium nitroprusside. xp<0.05 vs. WT control; ∞p<0.01 vs. WT LPS; □p<0.05 STAT1^−/−control. Aortas isolated from 3–4 animals per group were taken. Two-way ANOVA test with Bonferroni post hoc test was used. Statistical significance for the highest concentration is given.</p

STAT1-Dependent Signal Integration between IFNγ and TLR4 in Vascular Cells Reflect Pro-Atherogenic Responses in Human Atherosclerosis

<div><p>Signal integration between IFNγ and TLRs in immune cells has been associated with the host defense against pathogens and injury, with a predominant role of STAT1. We hypothesize that STAT1-dependent transcriptional changes in vascular cells involved in cross-talk between IFNγ and TLR4, reflect pro-atherogenic responses in human atherosclerosis. Genome-wide investigation identified a set of STAT1-dependent genes that were synergistically affected by interactions between IFNγ and TLR4 in VSMCs. These included the chemokines <i>Cxcl9</i>, <i>Ccl12</i>, <i>Ccl8</i>, <i>Ccrl2</i>, <i>Cxcl10</i> and <i>Ccl5</i>, adhesion molecules <i>Cd40</i>, <i>Cd74</i>, and antiviral and antibacterial genes <i>Rsad2</i>, <i>Mx1</i>, <i>Oasl1</i>, <i>Gbp5</i>, <i>Nos2</i>, <i>Batf2</i> and <i>Tnfrsf11a</i>. Among the amplified genes was also <i>Irf8</i>, of which <i>Ccl5</i> was subsequently identified as a new pro-inflammatory target in VSMCs and ECs. Promoter analysis predicted transcriptional cooperation between STAT1, IRF1, IRF8 and NFκB, with the novel role of IRF8 providing an additional layer to the overall complexity. The synergistic interactions between IFNγ and TLR4 also resulted in increased T-cell migration and impaired aortic contractility in a STAT1-dependent manner. Expression of the chemokines CXCL9 and CXCL10 correlated with STAT1 phosphorylation in vascular cells in plaques from human carotid arteries. Moreover, using data mining of human plaque transcriptomes, expression of a selection of these STAT1-dependent pro-atherogenic genes was found to be increased in coronary artery disease (CAD) and carotid atherosclerosis. Our study provides evidence to suggest that in ECs and VSMCs STAT1 orchestrates a platform for cross-talk between IFNγ and TLR4, and identifies a STAT1-dependent gene signature that reflects a pro-atherogenic state in human atherosclerosis.</p></div

Expression of synergistically amplified genes in atherosclerotic vessels.

<p>A, Venn diagram with analysis of microarray datasets obtained from human coronary plaques and human carotid plaques. B, Promoter analysis of the differentially expressed genes in carotid (left panel) and coronary plaques (right panel). For details see text.</p

STAT1-mediated abolished response to norepinephrine and sodium nitroprusside is associated with disturbed NO production.

<p>A, <i>WT</i> and <i>STAT1^−/− VSMCs</i> were treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113318#pone-0113318-g001" target="_blank">Fig. 1</a>. RNA was isolated and qRT-PCR for <i>Nos2</i> using <i>Gapdh</i> as internal control was performed (upper panel) B, After stimulation as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113318#pone-0113318-g001" target="_blank">Fig. 1</a>, medium was refreshed and left for 24 h. Next, 100 µl of the medium was taken and the product of Nos2- nitrite was measured. Data represent means of at least 3 independent biological experiments ±SEM and p<0.05 was considered as significant. Data were tested for significance by one-way ANOVA followed by post-hoc Tukey or unpaired two-tailed student T-test when appropriate. C, D Isolated aortic rings from WT and STAT^−/− mice were incubated with 10 ng/ml IFNγ for 8 h or with 1 ug/ml of LPS for 4 h or with IFNγ for 4 h followed by LPS for additional 4 h. Next, response to norepinephrine and sodium nitroprusside was tested on the wire myograph. C, Response to noradrenaline in WT and STAT1-deficient aortic rings presented as a percentage of maximal constriction to KPSS.*p<0.001 vs. WT control; •p<0.001 vs. WT LPS; ○p<0.001 vs. STAT1^−/− control. D, Response to stepwise increased concentration of sodium nitroprusside. xp<0.05 vs. WT control; ∞p<0.01 vs. WT LPS; □p<0.05 STAT1^−/−control. Aortas isolated from 3–4 animals per group were taken. Two-way ANOVA test with Bonferroni post hoc test was used. Statistical significance for the highest concentration is given.</p

IRF8 mediated cross-talk and functional activity of synergistically amplified chemokines.

<p><i>WT, STAT1^−/− and IRF8^−/−</i> VSMCs and HMECs were treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113318#pone-0113318-g001" target="_blank">Fig. 1</a>. A, RNA was isolated and qRT-PCR for <i>IRF8</i> using <i>GAPDH</i> as internal control was performed in VSMCs (left panel) and ECs (right panel). B, Protein extracts were analyzed for IRF8, tyrosine-phosphorylated STAT1, total STAT1 and GAPDH. C, <i>CCL5</i> mRNA expression (left panel) and protein presence in the medium (right panel) was measured. D, Expression profiles of <i>Cxcl9</i> (left panel) and <i>Cxcl10</i> (right panel) between VSMCs <i>WT</i>, and <i>IRF8^−/−</i> were compared. E, Migration assay of CD45⁺/CD3⁺ performed on conditioned medium remained after treatment of VSMCs <i>WT</i> and <i>STAT1^−/</i>⁻. Data represent means of at least 3 independent biological experiments ±SEM and p<0.05 was considered as significant. Data were tested for significance by one-way ANOVA followed by post-hoc Tukey or unpaired two-tailed student T-test when appropriate.</p

Effect of STAT1 dependent signal integration on chemokine expression.

<p><i>WT</i> and <i>STAT1^−/− VSMCs</i>, HMECs or <i>WT</i> aortic ring segments were treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113318#pone-0113318-g001" target="_blank">Fig. 1</a>. A, RNA from VSMCs was isolated and qRT-PCR for <i>Ccl5</i>, <i>Cxcl9</i> using <i>Gapdh</i> as internal control was performed. B, On the medium remained after treatment of VSMCs ELISA for Ccl5 and Cxcl9 was performed. C, Expression of <i>CXCL10, CXCL9 and CCL5</i> upon stimulation in ECs. D, RNA from incubated aortic rings was isolated and qRT-PCR for <i>Cxcl10</i>, <i>Cxcl9</i> using <i>Gapdh</i> as internal control was performed. Data represent means of at least 3 independent biological experiments ±SEM and p<0.05 was considered as significant. Data were tested for significance by one-way ANOVA followed by post-hoc Tukey or unpaired two-tailed student T-test when appropriate. E, ChIP-qPCR analysis of the <i>Cxcl10</i> promoter region containing NFκB and ISRE binding sites show the enrichment with STAT1, NFκB and IRF1 antibodies compared with IgG control in an IFNγ, LPS or IFNy+LPS-dependent manner in <i>WT VSMCs</i>. Immunoprecipitated DNA was quantified by qPCR and normalized to values obtained after amplification of unprecipitated (input) DNA. A representative experiment is shown.</p

CXCL10 amplified by IFNγ and LPS in VSMCs is STAT1 dependent.

<p>A, <i>WT</i> and <i>STAT1^−/−</i> VSMCs were treated with 10 ng/ml IFNγ for 8 h or with 1 ug/ml of LPS for 4 h or with IFNγ for 4 h followed by LPS for additional 4 h. RNA was isolated and qRT-PCR for <i>Cxcl10</i> using <i>Gapdh</i> as internal control was performed. B, Cells were treated as in A. On the medium remained after treatment ELISA for CXCL10 was performed. Data represent means of at least 3 independent biological experiments ±SEM and p<0.05 was considered as significant. Data were tested for significance by one-way ANOVA followed by post-hoc Tukey or unpaired two-tailed student T-test when appropriate.</p