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

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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.