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.