Fluorescence spectroscopic and histochemical analysis using hematoporphyrin as a microenvironmental probe for atherosclerotic change in the human aorta

Abstract

Hematoporphyrin (HP) was used as a microenvironmental fluorescent probe to investigate the development of human atherosclerotic plaques. We compared the site of HP accumulation with changes in the HP fluorescence spectrum in atheromatous plaques and in a liposome control model, using a confocal laser scanning microscope equipped with a photonic multi-channel analyzer linked to a fluorescence spectrometer. Wavelength shifts of the two peaks of HP fluorescence (F1, 620 nm; and F2, 640-690 nm) were monitored, and the integrated F2/F1 ratio was calculated as a measure of HP fluorescence. The F1 peak is characteristic of a predominantly aqueous site. The ratio reflects selective changes in the distribution and overcrowding of HP molecules in the limited space of the artificial membrane model. Compared with a normal artery, the atherosclerotic lesions showed an increase in the area of HP fluorescence, increased fluorescence intensity, a red shift of the HP fluorescence spectrum, and an increased F2/F1 ratio. The F2/F1 ratio of the membranous structures was markedly greater in the cores of the fibrous plaques than in the other plaques. The F1 peak showed increased intensity in the atheromatous plaque core, whereas in hydrophilic fibrous regions, such as the cap of the plaque, the intensity of the F1 peak was lower than in the core region. HP aggregation was observed in damaged cells and in water surrounded by lipid in the atheromatous core. Using HP as a probe allowed us to determine not only the ionization or polarity of each region in the atherosclerotic plaques but also to detect the separation and fusion of the lipid bilayer or micelle lipids, as well as damage to the cellular membranes and cholesterol enrichment. These findings suggest that HP is useful for detecting clinically important changes in atherosclerotic lesions, to lipid-rich, unstable, and vulnerable plaques, which are closely associated with cardiovascular events.