Discovery of σ₂R/TMEM97 as a Novel Biomarker for Atherosclerotic Plaques: A PET Imaging and Validation Study

The aims of this study were to evaluate σ₂R (sigma-2 receptor)/TMEM97 (transmembrane protein 97) expression in atherosclerotic plaques, and assess the feasibility of in vivo atherosclerotic plaques imaging using the σ₂R/TMEM97 targeting probe 1-(4-(5,6-dimethoxyisoindolin-2-yl)butyl)-3-(2-[¹⁸F]fluoroethyl)-1,3-dihydro-2H-benzo[d]imidazol-2-one ([¹⁸F]SYB-NF) developed in our laboratory.

Hematoxylin and eosin and immunohistochemical staining were performed on both human coronary endarterectomy specimens and mouse samples. The expression of σ₂R/TMEM97 in RAW 264.7 cells incubated with ox-LDL (oxidized low-density lipoprotein) was analyzed using western blot analysis. Positron emission tomography imaging with [¹⁸F]SYB-NF, [¹⁸F]NaF, and 2-[¹⁸F]fluoro-2-deoxy-d-glucose was conducted in wide-type C57BL/6 and ApoE^-/- mice. Specific binding was evaluated by coinjecting [¹⁸F]SYB-NF with the σ₂R/TMEM97 antagonist CM398 ([1-(4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)butyl)-3-methyl-1H-benzo[d]imidazol-2(3H)-one]). Autoradiography and Oil Red O staining were performed on harvested aortas and corresponding sections.

Staining results demonstrated significant upregulatiaon of σ₂R/TMEM97 expression at the early plaque formation and throughout the atherosclerosis progression. Western blot analysis indicated that incubation of macrophages with ox-LDL led to increased σ₂R/TMEM97 expression. [¹⁸F]SYB-NF specifically accumulated in the aortic arch of ApoE^-/- mice. Treatment with CM398 significantly reduced the standardized uptake value in the aortic arch of ApoE^-/- mice. [¹⁸F]SYB-NF exhibited a higher standardized uptake value in the aortic arch (0.67±0.09 versus 0.51±0.07) and higher aortic arch-to-heart ratio (2.58 versus 0.56) in ApoE^-/- mice compared with 2-[¹⁸F]fluoro-2-deoxy-d-glucose, and a higher aortic arch-to-bone ratio (2.24 versus 0.44) compared with [¹⁸F]NaF. Autoradiography analysis revealed a strong correlation between the positive area in Oil Red O staining and autoradiography (Pearson correlation coefficient=0.993; P=0.001), further supporting the association between elevated σ₂R/TMEM97 expression and plaque formation.

σ₂R/TMEM97 may serve as a potential biomarker for atherosclerotic plaques, and σ₂R/TMEM97 positron emission tomography imaging may be used to monitor plaque formation and progression, as well as the efficacy of emerging therapeutic strategies for atherosclerotic plaques.