Folate receptor-β imaging using ^99mTc-folate to explore distribution of polarized macrophage populations in human atherosclerotic plaque

In atherosclerotic plaques, the risk of rupture is increased at sites of macrophage accumulation. Activated macrophages express folate receptor-β (FR-β), which can be targeted by folate coupled to radioactive ligands to visualize vulnerability. The aim of this study was to explore the presence of activated macrophages in human atherosclerotic plaques by ^99mTc-folate imaging and to evaluate whether this technique can discriminate between an M1-like and M2-like macrophage phenotype. Methods: Carotid endarterectomy specimens of 20 patients were incubated with ^99mTc-folate, imaged using micro-SPECT, and divided into 3-mm slices. The mean accumulation was calculated per slice, and the distribution of M1-like and M2-like macrophages per slice was quantified by immunohistochemical staining for CD86 as well as inducible nitric oxide synthase (iNOS) for M1 and CD163 and FR-β for M2 macrophages. Monocytes from healthy donors were differentiated toward M1-like or M2-like phenotype by in vitro culturing. Messenger RNA levels of specific M1 and M2 markers were measured by reverse-transcription polymerase chain reaction and expression of FR-β, CD86, and CD163 by flow cytometry. Results: There was a heterogeneous accumulation of ^99mTc-folate in plaques (median, 2.45 [0.77-6.40] MBq/g). Slices with the highest ^99mTc-folate accumulation of each plaque showed significantly more expression of FR-β and CD163, compared with slices with the lowest ^99mTc-folate accumulation, which showed significantly more expression of iNOS. In in vitro polarized macrophages, messenger RNA expression of FR-β, mannose receptor, IL-10, and matrix metalloproteinase-9 was significantly increased in M2-like macrophages, compared with M1-like macrophages. On a receptor level, CD86 was shown to be overexpressed on M1-like macrophages whereas FR-β and CD163 were overexpressed on M2-like macrophages measured by flow cytometry. Conclusion: Higher numbers of M2-like macrophages were present in areas of high ^99mTc-folate accumulation than areas with low accumulation. It is anticipated that ^99mTc-folate imaging using SPECT as a marker for M2-like macrophages in atherosclerosis might be a good indicator for plaque vulnerability.