RAGE impairs murine diabetic atherosclerosis regression and implicates IRF7 in macrophage inflammation and cholesterol metabolism
Laura Senatus, Raquel López-Díez, Lander Egaña-Gorroño, Jianhua Liu, Jiyuan Hu, Gurdip Daffu, Qing Li, Karishma Rahman, Yuliya Vengrenyuk, Tessa J. Barrett, M. Zahidunnabi Dewan, Liang Guo, Daniela Fuller, Aloke V. Finn, Renu Virmani, Huilin Li, Richard A. Friedman, Edward A. Fisher, Ravichandran Ramasamy, Ann Marie Schmidt
Laura Senatus, Raquel López-Díez, Lander Egaña-Gorroño, Jianhua Liu, Jiyuan Hu, Gurdip Daffu, Qing Li, Karishma Rahman, Yuliya Vengrenyuk, Tessa J. Barrett, M. Zahidunnabi Dewan, Liang Guo, Daniela Fuller, Aloke V. Finn, Renu Virmani, Huilin Li, Richard A. Friedman, Edward A. Fisher, Ravichandran Ramasamy, Ann Marie Schmidt
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Research Article Inflammation Vascular biology

RAGE impairs murine diabetic atherosclerosis regression and implicates IRF7 in macrophage inflammation and cholesterol metabolism

Abstract

Despite advances in lipid-lowering therapies, people with diabetes continue to experience more limited cardiovascular benefits. In diabetes, hyperglycemia sustains inflammation and preempts vascular repair. We tested the hypothesis that the receptor for advanced glycation end-products (RAGE) contributes to these maladaptive processes. We report that transplantation of aortic arches from diabetic, Western diet–fed Ldlr—/— mice into diabetic Ager—/— (Ager, the gene encoding RAGE) versus WT diabetic recipient mice accelerated regression of atherosclerosis. RNA-sequencing experiments traced RAGE-dependent mechanisms principally to the recipient macrophages and linked RAGE to interferon signaling. Specifically, deletion of Ager in the regressing diabetic plaques downregulated interferon regulatory factor 7 (Irf7) in macrophages. Immunohistochemistry studies colocalized IRF7 and macrophages in both murine and human atherosclerotic plaques. In bone marrow–derived macrophages (BMDMs), RAGE ligands upregulated expression of Irf7, and in BMDMs immersed in a cholesterol-rich environment, knockdown of Irf7 triggered a switch from pro- to antiinflammatory gene expression and regulated a host of genes linked to cholesterol efflux and homeostasis. Collectively, this work adds a new dimension to the immunometabolic sphere of perturbations that impair regression of established diabetic atherosclerosis and suggests that targeting RAGE and IRF7 may facilitate vascular repair in diabetes.

Authors

Laura Senatus, Raquel López-Díez, Lander Egaña-Gorroño, Jianhua Liu, Jiyuan Hu, Gurdip Daffu, Qing Li, Karishma Rahman, Yuliya Vengrenyuk, Tessa J. Barrett, M. Zahidunnabi Dewan, Liang Guo, Daniela Fuller, Aloke V. Finn, Renu Virmani, Huilin Li, Richard A. Friedman, Edward A. Fisher, Ravichandran Ramasamy, Ann Marie Schmidt

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Figure 6

RAGE regulates the expression of IRF7 in macrophages.

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RAGE regulates the expression of IRF7 in macrophages. (A) IRF7 and CD68 ...
(A) IRF7 and CD68 colocalization in diabetic Ager—/— and WT recipient mice atherosclerotic plaques at day 5 after aortic arch transplantation from Ldlr—/— diabetic mice (N = 4 mice/group). Scale bar: 250 μm. (B) IRF7 colocalizes with CD68 in diabetic human atherosclerotic plaques. Representative image of human coronary atherosclerotic lesions is shown with additional human subject data shown in Supplemental Figure 5 (diabetic subjects) and Supplemental Figure 6 (nondiabetic subjects). Scale bar: 1 mm. Quantification of immunohistochemical staining of human plaque area colocalization of CD68/IRF7 area over total CD68+ staining area calculated as a percentage. (C) Irf7 gene expression in Ager—/— and WT BMDMs exposed to a normolipidemic environment (2% serum from C57BL/6 mice fed a normal chow diet) for 48 hours (2% serum of C57BL/6 mice fed a normal chow diet). N = 4 independent mice/group. (D) Irf7 gene expression in WT BMDMs after 48 hours of Ager or scrambled control knockdown (75 nM) in a normolipidemic environment. Knockdown of Ager by siRNA is shown. N = 6 independent mice/group. (E) Irf7 gene expression in Ager—/— and WT BMDMs exposed to a hyperlipidemic environment for 48 hours (2% serum of Ldlr—/— mice fed a Western diet). N = 4 mice/group. (F) Irf7 gene expression levels in WT BMDMs after 48 hours of Ager or scrambled (75 nM) in a hyperlipidemic environment. Knockdown of Ager by siRNA is shown. N = 6 mice/group. (G) Irf7 gene expression in WT and Ager—/— BMDMs grown in the presence of 1% serum of Ldlr—/— mice fed Western diet for 48 hours, followed by an additional 16 hours in CML-AGE (100 μg/mL) versus vehicle. N = 4 mice/group. (H) Irf7 gene expression in WT BMDMs after 48 hours of Ager or scrambled (75 nM) in a normolipidemic environment in cells treated with CML-AGE (200 μg/mL) or vehicle for 16 hours. Knockdown of Ager is shown. N = 6 mice/group. Secondary antibody alone controls are shown in A and B. Mean ± SEM. Unpaired t test or Mann-Whitney U test was performed to assess the differences in A–F and H (for Ager endpoint) depending on the normality of data. One-way ANOVA with post hoc Holm-Šídák multiple comparisons test was used in G and H (for Irf7 endpoints). *P < 0.05, ***P < 0.001, and ****P < 0.0001.