Temporospatial Single-Cell Characterization of Angiogenesis and Myocardial Regeneration in Small and Large Mammals

项目总结/摘要 缺血性心脏病每年影响全球超过197人。尽管宏观经济取得了进展， 再血管化技术如冠状动脉旁路移植术和经皮冠状动脉介入治疗， 许多患者由于残余的微血管灌注不足而进展为心力衰竭。虽然成年人 心脏似乎不能显着再生心肌损伤后，新生小鼠和猪， 能够在生命的第一周内有效地进行血管生成和心肌再生。内源 血管生成和再生途径与伤口愈合炎性级联反应错综复杂地联系在一起， 细胞外基质重塑、内皮细胞迁移和心肌细胞增殖。因此 梗死边缘区是这些细胞过程的空间交叉点，已被证明是复杂的， 动态微环境研究。在这个建议中，我们描述了一个新的应用程序的多路复用 具有单细胞分辨率的免疫荧光成像平台，称为CODEX（索引共检测）， 是在斯坦福大学发展起来的。在研究培训计划中描述的指导和指导下，我们 已经组建了一个由内科医生和外科医生科学家、心脏病学家、细胞生物学家和 来自斯坦福大学细胞科学成像设施的CODEX专家。如果获得奖学金，这 一个多学科团队处于独特的位置，执行这一首次将CODEX应用于心肌 再生CODEX将用于描述时空关系的纵向变化 在心肌的驻留细胞和控制血管生成的旁分泌途径之间， 心肌再生CODEX是利用抗体的免疫荧光显微镜的延伸 与DNA条形码结合，同时原位定量多达44种抗原。在目标1中，我们将应用CODEX 新生小鼠和猪心肌再生的LAD结扎模型，以确定新的空间表型 新生儿期的血管生成和炎症细胞街区的再生潜力 与非再生的成年人相比。在表征了细胞微环境和细胞信号传导之后， 新生哺乳动物中天然血管生成的活性，在目的2中，我们将研究外源性 调节血管生成和急性炎症反应的成年小鼠和猪。作为动物模型 对于心肌再生，控制自然血管生成和随后的血管生成的调节途径是 小鼠和猪的心肌再生由于其潜在的治疗作用而成为人们非常感兴趣的课题。 对人类有益。通过使用CODEX研究多种细胞类型之间的复杂相互作用，旁分泌 信号通路和细胞间的过程，我们希望能促进我们对自然界的机械理解。 新生儿血管生成和心肌再生，并确定缺血性心脏的新治疗靶点 这种疾病有可能促进全世界数百万患有这种疾病的患者的治疗。 缺血性心脏病

Project Summary/Abstract Ischemic heart disease affects more than 197 people worldwide every year. Despite advances in macro revascularization techniques such as coronary artery bypass grafting and percutaneous coronary intervention, many patients progress to heart failure due to residual microvascular perfusion deficits. Although the adult human heart appears to be unable to significantly regenerate myocardium after injury, neonatal mice and pigs are capable of efficient angiogenesis and myocardial regeneration during the first week of life. Endogenous angiogenic and regenerative pathways are intricately linked to the wound-healing inflammatory cascade, extracellular matrix remodeling, endothelial cell migration, and cardiomyocyte proliferation. Consequently, the infarct border zone, which is the spatial intersection of these cellular processes, has proven to be a complex and dynamic microenvironment to investigate. In this proposal, we describe a novel application of a multiplexed immunofluorescent imaging platform with single cell resolution called CODEX (co-detection by indexing) which was developed here at Stanford. With the guidance and mentorship described in the research training plan, we have compiled an advisory committee of physician and surgeon-scientists, cardiologists, cell biologists, and the experts in CODEX from Stanford's Cell Sciences Imaging Facility. If awarded this Fellowship, this multidisciplinary team is uniquely positioned to execute this first of its kind application of CODEX to myocardial regeneration. CODEX will be used to characterize the longitudinal changes in the spatiotemporal relationship between the resident cells of the myocardium and the paracrine pathways that govern angiogenesis and myocardial regeneration. CODEX is an extension of immunofluorescence microscopy that utilizes antibodies conjugated to DNA barcodes to simultaneously quantify up to 44 antigens in situ. In Aim 1 we will apply CODEX to neonatal mouse and pig LAD ligation models of myocardial regeneration to define novel spatial phenotypes of angiogenic and inflammatory cellular neighborhoods throughout the neonatal period of regenerative potential in comparison to non-regenerating adults. After characterizing the cellular microenvironments and cell-signaling activity of natural angiogenesis in neonatal mammals, in Aim 2 we will investigate the effect of exogenous modulators of angiogenesis and the acute inflammatory response in adult mice and pigs. As an animal model for myocardial regeneration, the regulatory pathways that govern natural angiogenesis and subsequent myocardial regeneration in the mouse and pig are the subject of great interest due to their potential therapeutic benefit in humans. By using CODEX to study the complex interplay between multiple cell types, paracrine signaling pathways, and intercellular processes we hope to advance our mechanistic understanding of natural neonatal angiogenesis and myocardial regeneration and identify new therapeutic targets for ischemic heart disease, which has the potential to advance the treatment of millions of patients worldwide who are suffering from ischemic heart disease.