PROJECT SUMMARY/ABSTRACT Angiogenesis is the process of blood vessel formation in which proliferating endothelial cells (ECs) sprout from preexisting vessels to extend a vascular network. Either impaired or excessive angiogenesis is associated with the pathogenesis of human retina diseases such as retinal hypovascular familial exudative vitreoretinopathy and hypervascular diabetic retinopathy. EC primarily uses the glycolysis pathway which is further enhanced during angiogenesis. Recent studies suggest that mitochondrial activity is critical for angiogenesis, but its mechanism is not entirely clear. We have investigated the angiogenic functions of three mitochondrial proteins with distinct activities - mitochondrial transcriptional factor (TFAM), respiratory complex IV component (COX10) and a mitochondrial redox protein thioredoxin 2 (TRX2). Our data show that: 1) silencing of Tfam, Cox10, or Trx2 in an in vitro 3D sprouting assay attenuates EC sprouting, which correlated with reduced EC proliferation, but not with ROS generation or EC apoptosis; 2) mice with an inducible deletion of Tfam, Cox10, or Trx2 exhibit retarded retinal vessel growth without penetration into the deep plexi at early ages (P5–P12), and this attenuated retinal sprouting was not correlated with mtROS production; 3) the three mutant mice develop arteriovenous malformations (AVM) with enhanced arterialization and microaneurysm formation in the microvessels at advanced ages (P12–P30). The hypovasculature and microaneurysm phenotypes resemble that of aged human retinas and human retinal vascular abnormalities such as Coats' disease, Leber's military aneurysms and familial exudative vitreoretinopathy (FEVR); 4) Furthermore, single-cell RNA-seq analyses of retinal ECs suggest that the three mutant mice have common EC clusters with reduced gene expression in angiogenic and metabolic pathways but increased TGFR signaling. Based on these data, we hypothesize that mitochondrial activity regulates common angiogenic and metabolic pathways (rather than ROS/apoptosis) in vascular growth and maturation. We propose the following specific aims: 1) To identify critical angiogenic and metabolic pathways in mitochondrial dysfunction-induced defects in retinal sprouting angiogenesis, AVM and microaneurysm formation as well as in retinal function; 2) To define how mitochondrial dysfunction regulates TGFR-Smad2/3 signaling; 3) To determine the role of the TGFR-Smad2/3 signaling in mitochondrial dysfunction-induced vascular retardation and malformation by pharmacological blockade and genetic deficiency. Our proposed study will define the mechanism by which mitochondrial activities regulate normal retinal vascular growth and maturation, and will provide a novel model and therapeutic intervention for human retinal vascular diseases associated with many pathological complications such as diabetes, hypertension and aging that can result in vision loss. 1