Targeted Folate Receptor β Fluorescence Imaging as a Measure of Inflammation to Estimate Vulnerability Within Human Atherosclerotic Carotid Plaque

Abstract

The probability of atherosclerotic plaque rupture and its thrombotic sequelae are thought to be increased at sites of macrophage accumulation. Folate receptor β (FR-β) is present on activated macrophages but not on quiescent macrophages or other immune cells. By conjugating the ligand folate with a fluorescent contrast agent, fluorescein isothiocyanate (FITC), we aimed to explore the potential role of FR-β fluorescence imaging in the distinction of vulnerable sites from more stable regions. Carotid specimens were taken from 20 patients and incubated with folate-FITC for 30 min. Ex vivo fluorescence imaging was performed to determine the exact location of folate-FITC uptake. Sections displaying regions of high uptake (determined as hot spots) were compared with sections showing low uptake (cold spots) through immunohistochemistry and real-time quantitative reverse-transcription polymerase chain reaction for FR-β. Hot spots showed significantly higher folate-FITC uptake than cold spots (P < 0.001). Hot spots tended to contain more macrophages and areas of hypoxia than cold spots. A positive correlation between messenger RNA levels of CD68 (marker for macrophages), FR-β (r = 0.53, P = 0.045), and hypoxia-inducible factor-1α expression (marker for intraplaque hypoxia; r = 0.55, P = 0.034) was found. Compared with areas with low folate-FITC uptake, areas of high folate-FITC uptake within human atherosclerotic plaques had an increased number of activated macrophages and higher areas of hypoxia. These characteristics of vulnerability imply that molecular imaging of FR-β through folate conjugates might be a good indicator for plaque vulnerability in future noninvasive imaging studies.