RAGE: A key pattern recognition receptor in tissue injury and its therapeutic intervention

• Gene Information: Receptor for Advanced Glycation End-products (RAGE), also known as AGER, is a protein-coding gene located on chromosome 6p21.32. It encodes a multiligand transmembrane receptor belonging to the immunoglobulin superfamily.
• Protein Expression: RAGE is constitutively expressed at exceptionally high levels on lung alveolar type I epithelial cells. Its expression is dramatically upregulated across endothelial cells, vascular smooth muscle cells, and infiltrating leukocytes (macrophages, neutrophils) during inflammation and cellular stress.
• Signaling Pathway: RAGE exerts its biological effects by recognizing and binding a diverse array of damage-associated molecular patterns (DAMPs)—including advanced glycation end-products (AGEs), HMGB1, and S100 protein family members. Upon ligand binding, it triggers robust downstream activation of the canonical NF-κB, MAPK, and JAK/STAT pathways, setting off a chronic, self-sustaining inflammatory loop.
• Therapeutic Inhibition: By blocking RAGE or neutralizing its interactions with extracellular DAMPs, candidate therapeutics (such as azeliragon or sRAGE decoy analogs) inhibit downstream inflammatory amplification, tissue remodeling, and endothelial dysfunction, aiming to improve clinical outcomes in diabetic complications, acute lung injury, and chronic inflammatory diseases.

RAGE

• Exons 1-11, including 3’UTR of the mouse RAGE gene that encodes the full-length protein, were replaced by human RAGE exons 1-11 including human 3’UTR in B-hRAGE mice plus.
• The endogenous mouse promoter and 5′ UTR regions are retained, allowing human RAGE expression to be driven by the native mouse Rage promoter, while endogenous mouse Rage transcription and translation are abolished.

• Human RAGE mRNA was exclusively detectable in B-hRAGE mice plus.

Strain specific analysis of RAGE mRNA expression in wild-type C57BL/6 mice and B-hRAGE mice plus by RT-PCR. Lung RNA were isolated from wild-type C57BL/6 mice (+/+) and homozygous B-hRAGE mice plus (H/H), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse or human RAGE primers. Mouse RAGE mRNA was detectable only in wild-type C57BL/6 mice. Human RAGE mRNA was detectable only in homozygous B-hRAGE mice plus but not in wild-type mice.

• Human RAGE mRNA was exclusively detectable in B-hRAGE mice plus.

Strain specific analysis of RAGE expression in wild-type C57BL/6N mice and heterozygous humanized B-hRAGE mice plus by RT-qPCR. Lung and blood RNA were isolated from wild-type C57BL/6N mice (+/+) (n=3, 9-week-old) and B-hRAGE mice plus (n=5, 9-week-old), then cDNA libraries were synthesized by reverse transcription, followed by real-time quantitative PCR with RAGE primers. The mRNA levels of RAGE were in detectable in B-hRAGE mice plus with Applied Biosystems SYBR, but not wild-type C57BL/6N mice. The mRNA levels of RAGE in lung from B-hRAGE mice plus were higher than those in blood from B-hRAGE mice plus.

• Human RAGE protein was exclusively detectable in B-hRAGE mice plus.

Western blot analysis of RAGE protein expression in homozygous B-hRAGE mice plus. Lung tissue lysates were collected from wild-type C57BL/6N mice (+/+) and homozygous B-hRAGE mice plus (H/H), and then analyzed by western blot with species-specific anti-RAGE antibody (Mouse/Rat RAGE Antibody, R&D, MAB1179-SP; Human RAGE Antibody, R&D, MAB11451-SP). 40 μg total proteins were loaded for western blotting analysis. RAGE was detected in lung.

• Human RAGE protein was exclusively detectable in B-hRAGE mice plus.

Strain specific RAGE expression analysis in wild-type C57BL/6N mice and heterozygous humanized B-hRAGE mice plus by ELISA. Serum was collected from wild-type C57BL/6N mice (+/+) (male, n=3, 7-week-old) and heterozygous B-hRAGE mice plus (H/+)  (male, n=3, 15-week-old). Expression level of mouse and human RAGE were analyzed by ELISA (Human RAGE Quantikine ELISA Kit: R&D, DRG00). Human RAGE was exclusively detectable in heterozygous B-hRAGE mice plus (n=3). Values are expressed as mean ± SEM.

• Human RAGE protein was exclusively detectable in B-hRAGE mice plus.

Strain specific RAGE expression analysis in wild-type C57BL/6N mice and heterozygous humanized B-hRAGE mice plus by ELISA. Lung homogenates were collected from wild-type C57BL/6N mice (+/+) (male, n=3, 7-week-old) and heterozygous B-hRAGE mice plus (H/+)  (male, n=3, 15-week-old). Expression level of mouse and human RAGE were analyzed by ELISA (Human RAGE Quantikine ELISA Kit: R&D, DRG00). Human RAGE was exclusively detectable in heterozygous B-hRAGE mice plus (n=3). Values are expressed as mean ± SEM.

The inhibitory efficiency of the RAGE targeted nucleic acid drugs in homozygous B-hRAGE mice plus. B-hRAGE mice plus were randomly divided into 6 groups (5-6 weeks old, female). The human RAGE-targeted nucleic acid drug (provided by a client) and PBS were administered to the mice individually. The nucleic acid drug was administered in the form of PBS aqueous solution. The mice were sacrificed on day 7 and day 28.

The inhibitory efficiency of the RAGE targeted nucleic acid drugs in homozygous B-hRAGE mice plus. (A) The changes in RAGE mRNA expression levels in lung tissue on day 7 after administration, compared to the levels before administration. (B) The changes in RAGE protein expression in lung tissue on day 28 after administration. Values are expressed as mean ± SEM. Analyzed by one way-ANOVA, *P<0.05.