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STAT1-dependent signal integration between IFNγ and TLR4 in vascular cells reflect pro-atherogenic responses in human atherosclerosis.

Chmielewski S, Olejnik A, Sikorski K, Pelisek J, Błaszczyk K, Aoqui C, Nowicka H, Zernecke A, Heemann U, Wesoly J, Baumann M, Bluyssen HA - PLoS ONE (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Nephrology, Klinikum rechts der Isar, Technical University Munich, Munich, Germany; Department of Human Molecular Genetics, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University Poznan, Poznan, Poland.

ABSTRACT
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 Cxcl9, Ccl12, Ccl8, Ccrl2, Cxcl10 and Ccl5, adhesion molecules Cd40, Cd74, and antiviral and antibacterial genes Rsad2, Mx1, Oasl1, Gbp5, Nos2, Batf2 and Tnfrsf11a. Among the amplified genes was also Irf8, of which Ccl5 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.

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STAT1-mediated abolished response to norepinephrine and sodium nitroprusside is associated with disturbed NO production.A, WT and STAT1−/− VSMCs were treated as described in Fig. 1. RNA was isolated and qRT-PCR for Nos2 using Gapdh as internal control was performed (upper panel) B, After stimulation as described in Fig. 1, 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.
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pone-0113318-g005: STAT1-mediated abolished response to norepinephrine and sodium nitroprusside is associated with disturbed NO production.A, WT and STAT1−/− VSMCs were treated as described in Fig. 1. RNA was isolated and qRT-PCR for Nos2 using Gapdh as internal control was performed (upper panel) B, After stimulation as described in Fig. 1, 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.

Mentions: Among the genes that were highly amplified upon treatment with IFNγ and LPS was inducible nitric oxide synthase (iNOS, Nos2). Indeed, treatment of WT-VSMCs but not STAT1−/− with IFNγ and LPS caused amplified expression of Nos2 as compared to stimulation with both factors alone (Fig. 5A). The RNA levels reflected nitrite accumulation in the medium (Fig. 5B). Since dysregulation of Nos2 expression and its activity affects vessel function, we evaluated the physiological ramifications of these experimental conditions using a wire myograph/organ chamber setting. Stimulation of the aortic rings isolated from WT animals with IFNγ and LPS resulted in drastic impairment of contractility after subjection to norepinephrine treatment (Fig. 5C, left panel). WT vessels treated with both IFNγ and LPS manifested also high loss of the sensitivity to sodium nitroprusside (Fig. 5D, left panel). In contrast to WT, aortic rings from STAT1-deficient mice did not reveal ameliorated response to noradrenaline and sodium nitroprusside as compared to LPS stimulated vessel (Fig. 5C, Fig. 5D, right panel).


STAT1-dependent signal integration between IFNγ and TLR4 in vascular cells reflect pro-atherogenic responses in human atherosclerosis.

Chmielewski S, Olejnik A, Sikorski K, Pelisek J, Błaszczyk K, Aoqui C, Nowicka H, Zernecke A, Heemann U, Wesoly J, Baumann M, Bluyssen HA - PLoS ONE (2014)

STAT1-mediated abolished response to norepinephrine and sodium nitroprusside is associated with disturbed NO production.A, WT and STAT1−/− VSMCs were treated as described in Fig. 1. RNA was isolated and qRT-PCR for Nos2 using Gapdh as internal control was performed (upper panel) B, After stimulation as described in Fig. 1, 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.
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Related In: Results  -  Collection

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pone-0113318-g005: STAT1-mediated abolished response to norepinephrine and sodium nitroprusside is associated with disturbed NO production.A, WT and STAT1−/− VSMCs were treated as described in Fig. 1. RNA was isolated and qRT-PCR for Nos2 using Gapdh as internal control was performed (upper panel) B, After stimulation as described in Fig. 1, 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.
Mentions: Among the genes that were highly amplified upon treatment with IFNγ and LPS was inducible nitric oxide synthase (iNOS, Nos2). Indeed, treatment of WT-VSMCs but not STAT1−/− with IFNγ and LPS caused amplified expression of Nos2 as compared to stimulation with both factors alone (Fig. 5A). The RNA levels reflected nitrite accumulation in the medium (Fig. 5B). Since dysregulation of Nos2 expression and its activity affects vessel function, we evaluated the physiological ramifications of these experimental conditions using a wire myograph/organ chamber setting. Stimulation of the aortic rings isolated from WT animals with IFNγ and LPS resulted in drastic impairment of contractility after subjection to norepinephrine treatment (Fig. 5C, left panel). WT vessels treated with both IFNγ and LPS manifested also high loss of the sensitivity to sodium nitroprusside (Fig. 5D, left panel). In contrast to WT, aortic rings from STAT1-deficient mice did not reveal ameliorated response to noradrenaline and sodium nitroprusside as compared to LPS stimulated vessel (Fig. 5C, Fig. 5D, right panel).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Nephrology, Klinikum rechts der Isar, Technical University Munich, Munich, Germany; Department of Human Molecular Genetics, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University Poznan, Poznan, Poland.

ABSTRACT
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 Cxcl9, Ccl12, Ccl8, Ccrl2, Cxcl10 and Ccl5, adhesion molecules Cd40, Cd74, and antiviral and antibacterial genes Rsad2, Mx1, Oasl1, Gbp5, Nos2, Batf2 and Tnfrsf11a. Among the amplified genes was also Irf8, of which Ccl5 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.

Show MeSH
Related in: MedlinePlus