PROJECT SUMMARY Ischemic heart disease affects over 150 million people worldwide and remains a leading cause of death for mankind. While overall mortality after myocardial infarction has improved, many patients ultimately succumb to heart failure despite pharmacologic, revascularization, and reconstructive therapies due to microvascular perfusion deficits which remain unaddressed. Targeted therapies such as cytokine, stem cell, and tissue- engineering approaches to myocardial revascularization, repair, and regeneration have had varied success, likely due to limitations in mechanistic understanding and suboptimal delivery systems. Thus, novel approaches to microrevascularization are greatly needed. In the recent funding period, we further elucidated the native signaling mechanisms of the angiogenic cytokine stromal cell-derived factor 1 (SDF) with its target, endothelial progenitor stem cells, to better harness its therapeutic effect during post-infarction myocardial repair. Furthermore, we synthesized a supra-efficient engineered SDF analog (ESA) and delivered it via a novel biomaterial for prolonged therapeutic effect. These strategies revealed robust angiogenesis, improved cardiomyocyte survival, preserved myocardial tissue biomechanics, reduced adverse post-injury remodeling, and enhanced cardiac functional recovery. We also designed and constructed a tissue-engineered bilayer cell sheet and demonstrated its ability to improve angiogenesis and ventricular remodeling. Finally, we have translated elements of our work into pre-clinical large animal models to evaluate the potential of these therapies to reach human clinical trials. This renewal application proposes to further define the mechanisms underlying the therapeutic effects of SDF, and to optimize cytokine delivery platforms for large animal and eventual human clinical translation. Aim 1 seeks to delineate SDF/ESA molecular structure to guide synthesis of novel analogs with enhanced efficacy, genetically manipulate SDF signaling to identify therapeutic targeting opportunities, and study the effects of microrevascularization on cellular perfusion, tissue biomechanics, and ventricular function. Aim 2 proposes the development of two innovative bioengineered cytokine delivery platforms for treating myocardial ischemia. One encompasses a novel shear-thinning hydrogel with dual cytokine release modalities and the capacity for percutaneous transcatheter delivery. Another involves fabricating a tissue-engineered SDF-eluting vascular conduit to enable simultaneous macro- and microrevascularization. Aim 3 strives to scale these innovative strategies to large animal pre-clinical models of percutaneous transcatheter therapeutics and minimally invasive coronary artery bypass grafting with synthesized bioconduits. The proposed experiments will yield important knowledge on potential clinical therapies for coronary artery disease, myocardial infarction, and heart failure.