RAGE/DIAPH1 and atherosclerosis through an evolving lens: Viewing the cell from the "Inside - Out"

Abstract

Background and aims: In hyperglycemia, inflammation, oxidative stress and aging, Damage Associated Molecular Patterns (DAMPs) accumulate in conditions such as atherosclerosis. Binding of DAMPs to receptors such as the receptor for advanced glycation end products (RAGE) activates signal transduction cascades that contribute to cellular stress. The cytoplasmic domain (tail) of RAGE (ctRAGE) binds to the formin Diaphanous1 (DIAPH1), which is important for RAGE signaling. This Review will detail the evidence linking the RAGE/DIAPH1 signaling pathway to atherosclerosis and envisages future therapeutic opportunities from the "inside-out" point of view in affected cells.

Methods: PubMed was searched using a variety of search terms, including "receptor for advanced glycation end products" along with various combinations including "and atherosclerosis," "soluble RAGE and atherosclerosis," "statins and RAGE," "PPAR and RAGE" and "SGLT2 inhibitor and RAGE."

Results: In non-diabetic and diabetic mice, antagonism or global deletion of Ager (the gene encoding RAGE) retards progression and accelerates regression of atherosclerosis. Global deletion of Diaph1 in mice devoid of the low density lipoprotein receptor (Ldlr) significantly attenuates atherosclerosis; mice devoid of both Diaph1 and Ldlr display significantly lower plasma and liver concentrations of cholesterol and triglyceride compared to mice devoid of Ldlr. Associations between RAGE pathway and human atherosclerosis have been identified based on relationships between plasma/serum concentrations of RAGE ligands, soluble RAGEs and atherosclerosis.

Conclusions: Efforts to target RAGE/DIAPH1 signaling through a small molecule antagonist therapeutic strategy hold promise to quell accelerated atherosclerosis in diabetes and in other forms of cardiovascular disease.

Keywords: Atherosclerosis; Diabetes; Receptor for AGE; Soluble RAGEs.

Fig. 1.. Blockade of RAGE suppresses progression of established atherosclerosis in Apoe null mice.

Male Apoe null mice were rendered diabetic with STZ or control at age 6 weeks; at age 14 weeks, mice were sacrificed or treated with sRAGE or murine serum albumin (MSA) vehicle. At age 20 weeks, animals were sacrificed and the aortic tree dissected photographed (a through e) and sections from the aortic arch subjected to staining with Oil Red O (f through j). Quantification of mean atherosclerotic lesion area (k) was determined. The following numbers of mice were used for each condition. At 14 weeks, citrate, n = 29 mice; and diabetes, n = 42 mice. At 20 weeks, citrate, n = 29 mice; diabetes/MSA, n = 38 mice; diabetes/sRAGE, n = 21 mice; and citrate/sRAGE, n = 20 mice. Scale bar: a through e, 0.3 cm; f through j, 125 μm. Adapted from Fig. 1 in Ref. [6].

Fig. 2.. Assessment of atherosclerosis regression in mice.

Model schematic: atherosclerotic aortic arches from Ldlr^−/− mice were transplanted into WT, Ager null, Diaph1 null, or Transgenic (Tg) glyoxalase-1 (Glo1) recipient mice and harvested at the indicated times after aortic arch transplantation. Adapted from Fig. 1A in Ref. [20].

Fig. 3.. RAGE, GLO1, DIAPH1, and regression of atherosclerosis.

Representative images of H&E (A), CD68 (B), Oil red O (C), and Picrosirius red staining (D) of aortic arch donor and transplant sections and their quantification in diabetic Ldlr null (donor) and WT, Ager null, Tg Glo1, and Diaph1 null diabetic recipient mice 5 days posttransplantation (N = 10 mice/group). Scale bar in A–D: 100 μm. In B, the secondary antibody–alone control is shown. Mean ± SEM; 1-way ANOVA with post hoc Tukey’s test was used. **p < 0.01, ***p < 0.001, and ****p < 0.0001. Adapted from Fig. 2 in Ref. [20].

Fig. 4.. ctRAGE interacts with mDia1 (DIAPH1) FH1.

Shown is the mDia1 (DIAPH1) FH1-ctRAGE interaction map. Residues broadened during the NMR titration experiment are indicated in red. Adapted from Fig. 3C in Ref. [22],

Fig. 5.. RNA-sequencing reveals roles for DIAPH1 in regulation of hepatic lipid metabolism.

Ldlr null and Ldlr null Diaph1null male mice were fed WD for 16 weeks. (A) Hierarchical clustering of differentially expressed genes in liver tissue from the indicated N = 4 independent mice/group. (B) The expression of the indicated genes identified as differentially expressed in the RNAseq data between Ldlr null mice vs. Ldlr null Diaph1 null mice was determined by RT-qPCR. (C) The expression of the indicated genes identified as not differentially expressed in the RNAseq among these groups of mice was confirmed by RT-qPCR. The number of independent mice/group is indicated in the figure as individual data points. Statistical analyses regarding testing for the normality of data followed by appropriate statistical analyses were described in Materials and Methods. p-values (in B and C) were determined by unpaired T test or Wilcoxon rank-sum test depending on if the data passed the Shapiro-Wilk normality test. Adapted from Fig. 4 in Reference [30].

Fig. 6.. Graphical abstract.

The receptor for advanced glycation end products and its multiple ligands, many of which are damage associated molecular patterns (DAMPs) form and accumulate in multiple settings of stress such as hyperglycemia, oxidative stress (ROS, reactive oxygen species) and ischemia. In the vasculature (endothelial cells (ECs) and smooth muscle cells (SMCs)), ligand binding to these RAGE-expressing cells triggers immune cell recruitment and upregulation of pro-inflammatory pathways. These pathways, driven by AGEs and DAMPs, may synergize with oxidized lipids, especially in diabetes, to accelerate atherosclerosis. Studies have demonstrated roles for RAGE expression in atherosclerosis emerging from the bone marrow compartment, as well. The cytoplasmic tail of RAGE (ctRAGE) binds to the formin, Diaphanous 1 (DIAPH1), which is important for RAGE signaling. Recent studies have uncovered that DIAPH1 contributes to the regulation of lipid metabolism. Experimental evidence to roles for DIAPH1 in hepatocytes in these mechanisms. Together, these considerations point to RAGE/DIAPH1 as a target for therapeutic intervention in diabetes, atherosclerosis and cardiovascular disease. Created with BioRender.com.