RAGE silencing deters CML-AGE induced inflammation and TLR4 expression in endothelial cells

Area of Science:

• Endocrinology and Metabolism
• Cell Biology
• Vascular Biology

Background:

• Nε-(carboxymethyl)lysine (CML) is a major advanced glycation end product (AGE) implicated in diabetic complications.
• While CML-induced Receptor for AGEs (RAGE) signaling is known, CML's effect on multiple AGEs receptor expression and RAGE's role in modulating these pathways remain unclear.

Purpose of the Study:

• To investigate CML-mediated expression of various AGEs receptors and signaling molecules in microvascular and macrovascular endothelial cells.
• To determine the role of RAGE silencing in modulating CML-induced pro-inflammatory cytokine release and associated signaling pathways.

Main Methods:

• Human umbilical vein endothelial cells (HUVEC) and human retinal endothelial cells (HREC) were treated with CML.
• Expression of RAGE, AGER1, AGER2, Gal-3, TLR4, TLR2, CD36, FEEL-1, FEEL-2, and HMGB1 was quantified using qPCR and western blotting.
• RAGE was silenced using siRNA (Si-RAGE) or neutralizing antibody, and downstream effects on inflammatory markers, ROS, and mitochondrial potential were assessed.

Main Results:

• CML significantly upregulated RAGE, TLR4, HMGB1, and Gal-3 expression in endothelial cells and in diabetic retinal tissues.
• RAGE silencing markedly reduced TLR4 and HMGB1 expression, inhibited NFκB and ERK1/2 phosphorylation, and downregulated TLR4 adaptors MyD88 and TIRAP.
• CML exposure led to increased pro-inflammatory gene expression, ROS production, and altered mitochondrial membrane potential.

Conclusions:

• CML induces the expression of AGEs receptors, mirroring changes seen in diabetic retinas.
• RAGE silencing mitigates CML-induced inflammation by modulating TLR4 signaling pathways, particularly through its adaptors MyD88 and TIRAP.
• Targeting TLR4 adaptors like MyD88 and TIRAP presents a potential therapeutic strategy for diabetic vascular complications.