Atherosclerosis involves the participation of multiple biomolecular and cellular mediators. Current imaging techniques do not provide for the simultaneous imaging of these participants as they work in concert during vascular disease. We seek to utilize a fluorescence retinal imaging system to simultaneously image the expression of up to four cells and/or biomolecules in retinal vasculature in a model of atherosclerosis. Our imaging agents harness the optical properties of quantum dots (QD) linked to antibodies to color-code different biomarkers within the same imaging field. We hypothesize that visualization of abnormal molecular expression in retinal vasculature using our system has diagnostic and prognostic utility in the evaluation of atherosclerosis in biology and medicine. We propose to apply our retinal imaging strategy in conjunction with QD to detect inflammatory biomarkers in retinal vasculature in vivo, and to correlate expression levels with plaque severity in proximal aorta ex vivo in a mouse model of atherosclerosis.
In Specific Aim 1 we propose to identify candidate biomarkers for atherosclerotic imaging in retinal vasculature. We will excise retinas from ApoE -/- mouse models of atherosclerosis and age-matched controls at 3 different age groups (6, 24, and 44 weeks) and use QD-antibody conjugates injected in vivo to detect the inflammatory markers VCAM-1, MCP-1, MSR, and dimerized fibrin (D-dimer) on plaque surfaces, which serve as relatively early and late indicators of atherosclerotic disease. In addition, isolated monocytes and T cells will be labeled ex vivo using spectrally-distinct QD and reinfused into mouse models and age-matched controls, and quantified in retinal tissue, to correlate cellular recruitment (a marker of lesion progression) with molecular expression. In the same animals, aortas will be harvested and probed for QD-labeled species and will be quantitatively assessed for immune cell infiltration and lipid content.
In Specific Aim 2 we will perform in vivo imaging of retinal vasculature throughout atherosclerotic progression. We will inject QD-labeled antibodies and/or inflammatory cells developed in Aim 1 into ApoE -/- mouse models and age-matched controls and image the retinal vasculature in vivo to monitor molecular expression and cellular recruitment to inflammatory endothelium. Our experimental design will allow us to follow disease progression within a single animal over time. If our hypothesis is correct, this approach has clinical potential for non-invasively staging atherosclerosis based on molecular signatures.
Atherosclerosis is a complex disease involving multiple cell types and proteins in various stages of initiation, progression, and eventually plaque rupture which is responsible for mortality and morbidity. Many imaging strategies ranging from ultrasound to MRI have been developed with the intention of detecting atherosclerotic disease early, such that therapeutic interventions can slow progression before vulnerable plaques rupture. However, these approaches do not possess the resolution necessary to detect early lesions, and imaging of arteries located deep in the body, such as the aorta, can be difficult due to the presence of connective tissue and fat. Furthermore, current imaging strategies are not capable of detecting the spectrum of cell types and proteins present in plaques on vessel linings. The """"""""molecular signature"""""""" of plaques, if detectable, would be particularly useful in diagnosis and treatment of atherosclerotic disease. For example, the presence of macrophages in lesions signals a relatively """"""""late stage"""""""" plaque which is more likely to rupture, whereas certain proteins on the vessel wall, the cell adhesion molecules, may signal """"""""early warning signs"""""""" to start treatment to curb risks. In this proposal, we seek to non-invasively image atherosclerosis in blood vessels using a retinal imaging system. Furthermore, we will use optical probes to color-code different biomarkers, such as cells and cell adhesion molecules, with distinct fluorescent emission spectra using semi-conducting nanocrystals or quantum dots. The retina offers a continuously-accessible, noninvasive window into the circulation, and can be used to rapidly and safely acquire the """"""""molecular signature"""""""" of the blood vessels throughout the body in the clinic. We will measure fluorescence due to atherosclerosis-associated mediators in the retinal vessels, and correlate their expression with expression in other inaccessible but major arteries in the body, which are commonly prone to lesion formation. Proven biomarker correlates will then be imaged in vivo in a mouse model of atherosclerosis to validate the utility of our imaging approach.
| Russ, Patricia K; Kupperman, Asher I; Presley, Sai-Han et al. (2010) Inhibition of RhoA signaling with increased Bves in trabecular meshwork cells. Invest Ophthalmol Vis Sci 51:223-30 |
| Jayagopal, Ashwath; Linton, Macrae F; Fazio, Sergio et al. (2010) Insights into atherosclerosis using nanotechnology. Curr Atheroscler Rep 12:209-15 |
