Micro ATR-FTIR spectroscopic imaging of atherosclerosis: an investigation of the contribution of inducible nitric oxide synthase to lesion composition in ApoE-null mice.

Inducible nitric oxide synthase (iNOS) has previously been shown to contribute to atherosclerotic lesion formation and protein nitration. Micro attenuated total reflection (ATR)-Fourier transform infrared (FTIR) spectroscopic imaging was applied ex vivo to analyse lesions in atherosclerotic (ApoE-/-) mice. Histologies of cardiovascular tissue of ApoE-/- mice that contain the gene for iNOS and ApoE-/- mice without iNOS (ApoE-/-iNOS-/- mice) were examined. Spectroscopic imaging of the aortic root revealed that iNOS did not affect the composition of the tunica media; furthermore, irrespective of iNOS presence, lipid esters were found to form the atherosclerotic plaque. ApoE-/- mouse aortic root lesions exhibited a more bulky atheroma that extended into the medial layer; signals characteristic of triglycerides and free fatty acids were apparent here. In ApoE-/-iNOS-/- mouse specimens, lesions composed of free cholesterol were revealed. ATR-FTIR spectra of the intimal plaque from the two mouse strains showed higher lipid concentrations in ApoE-/- mice, indicating that iNOS contributes to lesion formation. The reduction of lesion prevalence in ApoE-/-iNOS-/- mice compared with ApoE-/- mice is consistent with previous data. Moreover, the analysis of the plaque region revealed a change in the spectral position of the amide I band, which may be indicative of protein nitration in the ApoE-/- mouse, correlating with a more ordered (beta-sheet) structure, while a less ordered structure was apparent for the ApoE-/-iNOS-/- mouse, in which protein nitration is attenuated. These results indicate that micro ATR-FTIR spectroscopic imaging with high spatial resolution is a valuable tool for investigating differences in the structure and chemical composition of atherosclerotic lesions of ApoE-/- and ApoE-/-iNOS-/- mice fed a high-fat Western diet and can therefore be applied successfully to the study of mouse models of atherosclerosis.