Detection of coronary atherosclerotic plaques with superficial proteoglycans and foam cells using real-time intrinsic fluorescence spectroscopy

Abstract

Objectives: The protein components of low-density lipoprotein (LDL), oxidized LDL and proteoglycans such as versican contain tryptophan, an amino acid with characteristic fluorescence features at 308 nm excitation wavelength. We hypothesize that intrinsic fluorescence spectroscopy at 308 nm excitation wavelength IFS308, a method suitable for clinical use, can identify coronary artery lesions with superficial foam cells (SFCs) and/or proteoglycans.

Methods: We subjected 119 human coronary artery specimens to in vitro fluorescence and reflectance spectroscopy. We used 5 basis spectra to model IFS308, and extracted their contributions to each individual IFS308 spectrum. A diagnostic algorithm using the contributions of Total Tryptophan and fibrous cap to IFS308 was built to identify specimens with SFCs and/or proteoglycans in their top 50 μm.

Results: We detected SFCs and/or proteoglycans, such as versican or the glycosaminoglycan hyaluronan, in 24 fibrous cap atheromas or pathologic intimal thickening (PIT) lesions. An algorithm using the contributions of Total Tryptophan and fibrous cap to IFS308 was able to identify these segments with 92% sensitivity and 80% specificity.

Conclusion: We were able to establish a set of characteristic LDL, oxidized LDL, versican and hyaluronan fluorescence spectra, ready to be used for real-time diagnosis. The IFS(308) technique detects SFCs and/or proteoglycans in fibrous cap atheromas and PIT lesions. SFCs and proteoglycans are histological markers of vulnerable plaques, and this study is a step further in developing an invasive clinical tool to detect the vulnerable atherosclerotic plaque.

Figure 1

Intrinsic fluorescence basis spectra at 308 nm excitation (a). Comparison between FCAP (fibrous cap spectrum) and MED (media spectrum) versus collagen and elastin spectra (b). Intrinsic fluorescence spectra at 308 nm excitation wavelength of low-density lipoprotein (LDL), tryptophan, and oxidized low-density lipoprotein (OxdLDL) (c).

Figure 2

Spectra and fit models of oxidized low-density lipoprotein (OxdLDL), decorin-biglycan, versican and hyaluronan.

Figure 3

Spectra of osteocalcin and osteopontin compared to the tryptophan spectrum.

Figure 4

Transversal sections of five human coronary specimens and their corresponding intrinsic fluorescence spectra and fit models at 308 nm: a) fibrous cap atheroma with high content of SFCs (superficial foam cells) and lymphocytic infiltration of intima and media (hematoxylin-eosin, x10), accompanied by proteoglycan accumulation in the superficial layers (Movat’s pentachrome, not shown); b) fibrous cap atheroma with SFCs accompanied by proteoglycans in the top 50 µm (Movat’s pentachrome, blue coloration for proteoglycans, x10, with a zoom-in in the left panel for better illustration of SFCs); c) pathological intimal thickening with SFCs accompanied by proteoglycans (Movat’s pentachrome, not shown) and versican accumulations (anti-versican antisera staining, red-brown coloration for versican, x20, with a zoom-out in the left panel for better illustration of anti-versican staining); d) calcified fibrous cap atheroma with intense accumulations of proteoglycans (Movat’s pentachrome, not shown), versican (anti-versican antisera, not shown) and hyaluronan in the top 50 µm, accompanied by lymphocytic infiltration, in the absence of SFCs (hyaluronan binding protein staining, red-brown coloration for hyaluronan, x10, with a zoom-in in the left panel for better illustration of anti-hyaluronan staining); e) atherosclerotic specimen with no SFCs or proteoglycan accumulations (Movat’s pentachrome, x10). Solid bars indicate 50 µm length.

Figure 5

Binary diagnostic algorithm for identification of specimens with SFCs (superficial foam cells) and/or proteoglycans.