Retinal Imaging of Prognostic Indicators of Atherosclerosis

描述(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。目前的成像技术没有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。我们试图利用荧光视网膜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中视网膜血管中的表达。我们的成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记物进行颜色编码。我们推测，使用我们的系统对视网膜血管中异常分子表达的可视化在生物学和医学上对动脉粥样硬化的评估具有诊断和预后价值。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性生物标志物，并在体外将表达水平与动脉粥样硬化小鼠模型中近端主动脉斑块的严重程度相关联。在具体目标1中，我们建议识别视网膜血管系统动脉粥样硬化成像的候选生物标记物。我们将在3个不同的年龄组(6、24和44周)从ApoE/-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抗体结合物来检测斑块表面的炎症标志物VCAM-1、MCP-1、MSR和二聚化纤维蛋白(D-dimer)，它们作为动脉粥样硬化性疾病的相对早期和晚期指标。此外，分离的单核细胞和T细胞将使用光谱不同的QD进行体外标记，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)与分子表达相关联。在相同的动物中，将采集主动脉并探测QD标记的物种，并将对免疫细胞渗透和脂肪含量进行定量评估。在特定的目标2中，我们将对动脉粥样硬化进展过程中的视网膜血管进行活体成像。我们将把在Aim 1中开发的QD标记抗体和/或炎性细胞注射到ApoE/-小鼠模型和年龄匹配的对照组中，并在体内成像视网膜血管系统，以监测分子表达和细胞对炎性内皮的募集。我们的实验设计将允许我们跟踪单个动物内随着时间的推移疾病的发展。 如果我们的假设是正确的，这种方法具有基于分子特征的非侵入性动脉粥样硬化分期的临床潜力。 公共卫生相关性：动脉粥样硬化是一种复杂的疾病，涉及多种细胞类型和蛋白质，在起始、进展和最终斑块破裂的不同阶段，这是导致死亡和发病率的原因。从超声到核磁共振的许多成像技术已经被开发出来，目的是早期发现动脉粥样硬化性疾病，这样治疗干预可以在脆弱的斑块破裂之前减缓进展。然而，这些方法不具备发现早期病变所需的分辨率，而且由于存在结缔组织和脂肪，对位于身体深处的动脉(如主动脉)的成像可能很困难。此外，目前的成像策略不能检测血管衬里斑块中存在的细胞类型和蛋白质的光谱。斑块的“分子特征”，如果可以检测到，将在动脉粥样硬化性疾病的诊断和治疗中特别有用。例如，病变中巨噬细胞的存在标志着相对“晚期”的斑块更有可能破裂，而血管壁上的某些蛋白质--细胞黏附分子--可能发出“早期预警信号”，开始治疗以遏制风险。 在这项建议中，我们寻求使用视网膜成像系统对血管中的动脉粥样硬化进行非侵入性成像。此外，我们将使用光学探针对不同的生物标记物进行颜色编码，如细胞和细胞黏附分子，使用半导体纳米晶体或量子点进行清晰的荧光发射光谱。视网膜提供了一个连续可及的、非侵入性的循环窗口，可用于在临床上快速、安全地获取全身血管的“分子特征”。我们将测量视网膜血管中动脉粥样硬化相关介质的荧光，并将它们的表达与身体其他难以接近但通常容易形成病变的主要动脉的表达联系起来。已证实的生物标记物的相关性将在动脉粥样硬化的小鼠模型中进行体内成像，以验证我们成像方法的实用性。

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.