Molecular sieving and mass spectroscopy reveal enhanced collagen degradation in rabbit atheroma

Background: Collagen degradation is the major mechanism of atherosclerotic plaque destabilization. It is unknown whether collagen breakdown is involved into formation of early atherosclerotic lesions. Methods: Current paper describes a novel collagen degradation assay based on a combination of molecular sieving and mass spectroscopy. The first step of the assay consists of the extraction of total collagen from tissue. This extract includes both intact collagen and its breakdown products. Molecular sieving is used to isolate low molecular weight collagen fragments. Since the low molecular weight fraction of the extract may contain some non-collagenous molecular species, the collagen-specific amino acid hydroxyproline is quantified using mass spectroscopy. Results: This assay was validated in various experimental systems with known/predictable level of collagen breakdown in vitro, ex vivo and in vivo. When applied to cholesterol-fed rabbit aorta, it revealed enhanced collagen degradation in rabbit atheromas compared to unaffected aortic regions. Conclusion: A novel assay has been developed to demonstrate enhanced collagen degradation in rabbit atherosclerotic plaques. Accurate quantification of collagen breakdown products should provide a new relevant end point in the analysis of plaque development and stability.