Integration of advanced imaging and multiOMICs to elucidate pro-atherogenic effects of endothelial-to-Immune cell-like transition (EndICLT)

项目摘要/摘要 动脉粥样硬化是一种多因素疾病，是导致发病率和死亡率的主要原因。这个 内皮，血管壁的内层，传递恒定和节律性的管壁剪应力(WSS)。 血液粘度和流量。动脉分叉内侧壁有稳定的单向层流(S流) 发展减弱全身炎症反应，而双向和轴向错位的血流 侧壁决定慢性低度炎症反应的局灶性但偏心性，内皮细胞 动脉粥样硬化病变的重编程和优先形成。我们不偏不倚的scRNA序列和基因本体论 分析表明，D-Flow诱导内皮细胞向免疫细胞样转化(EndICLT)和促动脉粥样硬化 小路。然而，EndICLT在体内的病理生理意义以及它是否可以作为一种 抗动脉粥样硬化的治疗目标仍然难以捉摸。长期存在的技术挑战之一是 询问细胞信号机制并同时发挥作用，以揭开关键的作用机制和 病理生理里程碑的动态变化。对动脉粥样硬化和内皮细胞的全面了解 动态可以通过可视化斑块中的多组图谱来实现。尖端荧光 光学清除斑块的显微镜可提供动脉粥样硬化的定性和定量评估。 然而，目前的荧光成像方式和传统组织的耗时程序 清除技术限制了高空间分辨率的高通量成像。多尺度亚尺度技术的出现 体素光片荧光显微镜结合斑块的快速清除可以解决未满足的问题 挑战。在这笔赠款中，我们将使用组织清除的新方法，先进的图像获取来阐明 流动敏感机制，由此EndICLT促进动脉粥样硬化。在Aim1中，我们将检查是否 新的二氯甲烷-组氨酸梯度介质净化技术具有快速提取的潜力 光学透明斑块中的多组体信息。传统的荧光显微镜技术包括 广视场和衍射受限的共焦显微镜会产生离焦照明的干涉和 降低了穿过试件的轴向穿透深度。在目标2中，我们将重点建立一个先进的 基于机器学习的亚体素重建LSFM成像平台 可伸缩提取高空间分辨率多组特征的分割。最后，在目标3中，我们将 整合目标1和目标2，以探索EndICLT依赖的动脉粥样硬化形成的关键潜在机制。一起， 这些目标将允许范式的转变，以确定动脉粥样硬化的新治疗靶点。这项提议将 请允许我深化我的研究经验，并提供关键支持，为 从事心血管研究。

Project Summary/Abstract Atherosclerosis is a multifactorial disease accounting for a leading cause of morbidity and mortality. The endothelium, the inner lining of vessel walls, transduces constant and rhythmic wall shear stress (WSS) from blood viscosity and flow. At the medial wall of arterial bifurcation, stable unidirectional laminar flow (S-flow) develops attenuates systemic inflammatory responses, whereas bidirectional and axially misaligned flow in the lateral wall determine focal but eccentric nature of chronic low-grade inflammatory responses, endothelial cell reprogramming and preferential formation of atherosclerotic lesion. Our unbiased scRNA seq and gene ontology analysis suggested that D-flow induces endothelial-to-immune cell like-transition (EndICLT) and pro-atherogenic pathways. However, the pathophysiological significance of EndICLT in vivo and whether it could serve as an anti-atherogenic therapeutic target is yet remains elusive. One of the longstanding technical challenges is interrogating cell signaling machinery and function simultaneously to unravel key mechanisms in action and dynamic changes in pathophysiological milestones. Comprehensive insights into atherosclerosis and endothelial dynamics can be achieved by visualizing multiomic atlas throughout the plaque. Cutting-edge fluorescence microscopy in optically cleared plaque provides qualitative and quantitative evaluations of atherosclerosis. However, current modalities of fluorescence imaging and time demanding procedures of conventional tissue clearing techniques limits high throughput imaging with high spatial resolution. The advent of multi-scale sub- voxel light-sheet fluorescence microscopy combined with a rapid clearing of plaques may address unmet challenges. In this grant, we will use novel approaches of tissue clearing, advanced image acquisition to elucidate flow-sensitive mechanisms, whereby EndICLT promotes atherosclerosis. In Aim1, we will examine whether the novel dichloromethane - histodenz gradient medium clearing technique has potential for rapid extraction of multiomic information in optically cleared plaque. Conventional fluorescence microscopy techniques including wide-field and diffraction-limited confocal microscopy creates interference from out-of-focus illumination and reduced axial penetration depth across the specimen. In Aim 2, we will focus on establishing an advanced imaging platform of LSFM followed by sub-voxel reconstruction (SV-LSFM) and machine-learning based image segmentation for scalable extraction of multiomic features in high spatial resolution. Finally, in Aim 3, we will integrate Aim 1 and 2 to explore key underlying mechanisms in the EndICLT-dependent atherogenesis. Together, these aims will allow a paradigm shift to identify novel therapeutic targets of atherosclerosis. This proposal will allow me to deepen my research experiences and provide a critical support to build a strong foundation for a career in cardiovascular research.