Detection of altered extracellular matrix in surface layers of unstable carotid plaque: An optical spectroscopy, birefringence and microarray genetic analysis

Erosion and rupture of surface layers in atherosclerotic plaque can cause heart attack and stroke; however, changes in luminal surface composition are incompletely defined. Laser-induced fluorescence spectroscopy (LIFS), with limited tissue penetration, was used to investigate the surface of unstable carotid plaque and correlated with microscopy, birefringence and gene expression. Arterial matrix collagens I, III and elastin were assessed in unstable plaques (n = 25) and reference left internal mammary arteries (LIMA, n = 10). LIFS in addition to selective histological staining with picrosirius red, Movat pentachrome and immunostaining revealed decreased elastin and increased collagen I and III (P < 0.05) in carotid plaque when compared with LIMA. Within plaque, collagen I was elevated in the internal carotid region versus the common carotid region. Polarized light microscopy detected layers of aligned collagen and associated mechanical rigidity of the fibrous cap. Microarray analysis of three carotid and three LIMA specimens confirmed up-regulation of collagen I, III and IV, lysyl oxidase and MMP-12. In conclusion, LIFS analysis coupled with microscopy revealed marked regional differences in collagen I, III and elastin in surface layers of carotid plaque; indicative of plaque instability. Birefringence measurements demonstrated mechanical rigidity and weakening of the fibrous cap with complementary changes in ECM gene expression. Laser-induced fluorescence spectroscopy provides an effective nondestructive method of supplying information on connective tissue protein composition, specifically collagen and elastin. Optical spectroscopy was used to investigate the surface of human unstable carotid plaque and correlated with histology, birefringence and gene expression. Optical spectroscopy is an effective method for identifying clinically relevant compositional changes in connective tissue proteins associated with vascular remodeling despite the variability in plaque structure. Consistent regional differences were detected in the carotid specimens. In addition, birefringence measurements demonstrated mechanical rigidity and weakening of the fibrous cap with complementary changes in connective tissue gene expression.