Project Summary This grant proposes to address a fundamental question, “How does the pulmonary artery change as we grow older?” There are two specific aims intended to determine age-related changes in the pulmonary artery’s structure, function, and genetic expression. We will find out how extracellular collagen changes in the pulmonary artery as it ages to explain how the artery stiffens over time. Specifically, our first aim will calculate material stiffness based on multiple measurements of deformation of pulmonary arteries of mice (ages 9, 15, 20, 24, and 27 months), including diameter, pressure, and force, while the artery is submerged in physiologic solution. We will calculate mean fiber orientation of collagen fibers using 2-photon imaging and fast fourier transform analysis. We will also perform single cell RNA sequencing to investigate cell populations in tissue with near complete genomic profile of individual cells. We hypothesize that pulmonary artery material stiffness correlates with collagen fiber orientation and that age-related changes in cell phenotypes of fibroblasts, smooth muscle cells, and endothelial cells are observable by measuring genetic expression. We will identify differential gene expressions in these pulmonary arterial cells and associate them with changes in collagen orientation and vaso- activity as a function of age. These findings will provide sufficient information to model pulmonary arterial aging in mice and be the first study of pulmonary arterial aging using an ex-vivo model of the pulmonary artery. The overall objective of this proposal is to identify physical and functional parameters of pulmonary arterial aging that we will validate in human tissue, a study currently underway. These results will enable us to perform human clinical trials in the future to evaluate potential biomarkers or therapeutic targets to promote healthy aging.