Molecular ultrasound and MRI of Vascular Development. - Renewal - 1

描述（由申请人提供）：基因工程小鼠已被广泛用于血管发育的体内研究，并用于研究中风、癌症和缺血性心脏病等多种人类疾病的血管变化及其发病机制。因此，迫切需要体内方法来分析小鼠模型在发育和疾病过程中三维血管形态和基因表达模式的动态变化。为了在这一领域取得进展，报告小鼠需要进行比传统光学显微镜更具穿透性的血管成像。在之前的资助期间，我们建立了两个新的报告系统，用于超声和磁共振成像（MRI），可用于发育和成年小鼠的高分辨率3D血管成像的体内方法。具体来说，我们开发了一种用于细胞表面生物素化的“Biotag”转基因基因，并产生了Tie2-Biotag转基因小鼠，用于血管内皮细胞（VECs）的超声和MRI靶向成像。我们还发现，二价金属转运蛋白DMT1可以作为mn增强MRI （MEMRI）的高效报告基因，MEMRI是一种体内成像方法，具有标记两种主要血管细胞类型VECs和平滑肌细胞（SMCs）的潜力。我们现在建议充分利用这些分子成像技术的突破，并产生和验证下一代通用报告小鼠，用于成像从胚胎到成年阶段的血管形态和基因表达模式。这些报告小鼠将用于建立野生型（WT）小鼠胚胎和Gli2-/-突变体中发育中的血管的体内分子成像方法，我们发现这些突变体在脑动脉中存在模式缺陷。我们还将利用成人血管生成的体内模型来测试和验证通用报告小鼠。该项目的具体目标是：1)优化从胚胎到成人阶段血管成像的超声和MRI方案；2)建立通用的Biotag报告小鼠，用于多种VEC基因的体内多模态表达成像；3)建立通用DMT1报告小鼠，用于vec和SMCs的体内MEMRI成像。该研究将生成并验证通用报告小鼠，用于MRI和超声在体内血管成像，从而实现从胚胎到成年阶段血管形态和基因表达模式动态变化的前所未有的研究。

DESCRIPTION (provided by applicant): Genetically-engineered mice have been utilized widely for in vivo studies of vascular development and for studying vascular changes underlying pathogenesis in a wide range of human diseases such as stroke, cancer and ischemic heart disease. As a result, there is a critical need for in vivo methods to analyze dynamic changes in three-dimensional (3D) vascular morphology and gene expression patterns during development and disease in mouse models. To progress in this area, reporter mice are required for vascular imaging with more penetration than conventional optical microscopy. During the previous funding period, we established two novel reporter systems for ultrasound and magnetic resonance imaging (MRI), in vivo methods that can be used for high-resolution, 3D vascular imaging in developing and adult mice. Specifically, we developed a "Biotag" transgene for cell surface biotinylation, and generated Tie2-Biotag transgenic mice for targeted imaging of vascular endothelial cells (VECs) using avidinated contrast agents for both ultrasound and MRI. We also discovered that the Divalent Metal Transporter, DMT1 can be utilized as a highly effective reporter gene for Mn-enhanced MRI (MEMRI), an in vivo imaging method that has the potential for labeling both VECs and smooth muscle cells (SMCs), the two major vascular cell types. We now propose to take full advantage of these breakthroughs in molecular imaging technology, and to generate and validate the next generation universal reporter mice for imaging vascular morphologies and gene expression patterns from embryonic to adult stages. These reporter mice will be used to establish in vivo approaches for molecular imaging of the developing vasculature in wild type (WT) mouse embryos, and in Gli2-/- mutants, which we showed have patterning defects in the cerebral arteries. We will also utilize in vivo models of adult angiogenesis to test and validate the universal reporter mice. The specific aims of the project are: 1) Optimize ultrasound and MRI protocols for vascular imaging from embryonic to adult stages; 2) Establish a universal Biotag reporter mouse for in vivo, multi-modality expression imaging of a variety of VEC genes; and 3) Establish a universal DMT1 reporter mouse for in vivo MEMRI imaging of both VECs and SMCs. This research will generate and validate universal reporter mice for in vivo vascular imaging with MRI and ultrasound, enabling unprecedented studies of dynamic changes in vascular morphologies and gene expression patterns, from embryonic to adult stages.