Characterization of the Pharmaceutical Effect of Drugs on Atherosclerotic Lesions in Vivo Using Integrated Fluorescence Imaging and Raman Spectral Measurements

Abstract

Direct assessment of the vascular lesions of model animals in vivo is important for the development of new antiatherosclerotic drugs. Nevertheless, biochemical analysis of the lipid profile in blood in vitro remains the most common way to evaluate the therapeutic effect of drugs targeting atherosclerosis because of an inherent difficulty to access the vascular wall. Using hypercholesterolemic zebrafish, we present an orchestrated application of Raman spectral measurements and confocal fluorescence imaging to interrogate the pharmacological response of atherosclerotic lesions in situ and in vivo. For demonstration, we investigated two commonly prescribed antihyperlipidemic drugs, ezetimibe and atorvastatin. The treatment of ezetimibe or atorvastatin alone decreased effectively the deposition of lipids in the vascular wall, and a combined dose showed a synergistic effect. Atorvastatin exerted a profound antioxidative effect on vascular fatty lesions. Analysis of individual lesions shows further that these lesions exhibited a heterogeneous response to the treatment of atorvastatin; a significant fraction of, but not all, the lesions became nonoxidized after the intervention. Beyond its efficacies in suppressing both the accumulation and oxidation of vascular lipids, atorvastatin expedited the clearance of vascular lipids. The possession of pleotropic (multiple) therapeutic effects on vascular fatty lesions of hypercholesterolemic zebrafish by atorvastatin is notably consistent with the known pharmaceutical effects of this drug on human beings. These results improve our understanding of the antiatherosclerotic effect of drugs. We envisage that our approach has the potential to become a platform to predict the pharmaceutical effects of new drugs aiming to cure human atherosclerotic diseases.

Raman Scattering: From Structural Biology to Medical Applications

“…RS also allows for one to study the mechanisms of treatment different diseases in vivo and in situ. The effect of two different drugs (ezetimibe and atorvastatin) on atherosclerotic plaques using fluorescent and RM was investigated [225]. The study confirmed the pharmaceutical effect of both drugs and showed that the drug ezetimibe reduces the deposition of lipids in the vascular walls.…”

Zebrafish larvae as a model system for systematic characterization of drugs and genes in dyslipidemia and atherosclerosis

“…Results from small-scale studies in samples of 20 to 100 pooled larvae suggest that treating larvae fed on a cholesterol-supplemented diet with statins and/or ezetimibe may prevent the elevated whole-body LDLc and/or total cholesterol levels that are otherwise observed 38,39 . In addition, evidence suggests that treatment with atorvastatin and ezetimibe may reduce vascular lipid deposition 39,73 .…”

“…To examine the anti-atherogenic potential of combined treatment with atorvastatin and ezetimibe, we overfed larvae of three backgrounds on a cholesterol-supplemented diet – as described earlier – in the presence or absence of 6μg atorvastatin and 80μg ezetimibe per 1g of dry food 73 from 5 to 9dpf. At 10dpf, larvae were optically screened for atherogenic traits (see ‘ Imaging ’) and used for enzymatic assessment of whole-body lipid and glucose levels (see ‘ Lipid, glucose and protein quantification ’).…”

Comparing Raman and fluorescence lifetime spectroscopy from human atherosclerotic lesions using a bimodal probe

“…In particular, FLIm is useful for quickly measuring lipid to collagen ratios in the fibrous cap , which can provide information about plaque stability. Raman spectroscopy can provide further chemical specificity, and is able to, for instance, distinguish between calcifications, cholesterol or carotenoids . FLIm can create maps of the surface composition at comparable speed with IVUS or OCT whereas Raman spectroscopy is slower and more suitable for point measurements rather than mapping.…”