Retinal Imaging of Prognostic Indicators of Atherosclerosis

描述（由申请人提供）：动脉粥样硬化涉及多种生物分子和细胞介质的参与。目前的成像技术不能提供这些参与者的同时成像，因为他们在血管疾病期间协同工作。我们试图利用视网膜荧光成像系统同时成像多达四个细胞和/或生物分子在动脉粥样硬化模型视网膜血管的表达。我们的显像剂利用与抗体相连的量子点（QD）的光学特性，在同一成像领域内对不同的生物标志物进行颜色编码。我们假设使用我们的系统可视化视网膜血管异常分子表达在动脉粥样硬化的生物学和医学评估中具有诊断和预后效用。我们建议将我们的视网膜成像策略与QD相结合，在体内检测视网膜血管中的炎症生物标志物，并在小鼠动脉粥样硬化模型中将表达水平与近端主动脉斑块严重程度相关联。在具体目标1中，我们建议确定视网膜血管粥样硬化成像的候选生物标志物。我们将从3个不同年龄组（6周、24周和44周）的ApoE -/-动脉粥样硬化小鼠模型和年龄匹配的对照组中切除视网膜，并使用体内注射的qd抗体偶联物检测斑块表面的炎症标志物VCAM-1、MCP-1、MSR和二聚体纤维蛋白（d -二聚体），这些标志物可作为动脉粥样硬化疾病的相对早期和晚期指标。此外，分离的单核细胞和T细胞将在体外使用光谱不同的QD进行标记，并重新注入小鼠模型和年龄匹配的对照组，并在视网膜组织中进行量化，以将细胞募集（病变进展的标志）与分子表达联系起来。在相同的动物中，将采集主动脉并探测qd标记的物种，并定量评估免疫细胞浸润和脂质含量。在专项目标2中，我们将在动脉粥样硬化过程中对视网膜血管系统进行体内成像。我们将把qd标记的抗体和/或在Aim 1中形成的炎症细胞注射到ApoE -/-小鼠模型和年龄匹配的对照组中，并在体内对视网膜血管进行成像，以监测分子表达和细胞向炎症内皮的募集。我们的实验设计将允许我们在一段时间内跟踪单个动物的疾病进展。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.