PROJECT SUMMARY/ABSTRACT Aging is the primary risk factor for cardiovascular diseases (CVD) and Alzheimer's disease and its related dementias (ADRD),1, 2 due in part to adverse changes in peripheral vascular endothelial3 and cerebral vascular function,4 respectively (i.e., vascular aging). Regular aerobic exercise (AE) protects against vascular aging by favorably targeting several pillars of aging (i.e. inflammation, mitochondrial dysfunction, cell senescence, impaired nutrient sensing and proteostasis) and reducing oxidative stress. However, there is considerable heterogeneity in the endothelial and cerebral vascular benefits to AE in middle-age and older (MA/O) adults, including sex differences. The reason(s) for AE response variation are poorly understood but could be related to age/sex differences in the biological changes underlying vascular aging and/or the molecular transducers (i.e., circulating exerkines including endothelial microvesicles [EMVs]) that communicate and coordinate the effects of AE on the vasculature in the periphery and brain. Here, we propose a randomized controlled 12- week intervention of AE (3 d/week, 60-80% heart rate reserve, ~1 h duration) or non-exercise control in groups of young (18-39 years), middle-aged (40-59 years) and older (≥60 years) adults, balanced by sex. Outcomes to be assessed before and after the intervention include: 1) peripheral endothelial function (brachial artery flow- mediated dilation -- primary outcome), 2) cerebrovascular function (cerebrovascular reactivity to a hypercapnic stimulus -- primary outcome), and 3) biospecimens: blood and/or vascular endothelial cells assayed for transcriptomic, proteomic, metabolomic and EMVs and their cargo (e.g., micro RNA) obtained (a) before, (b) during (blood only) and (c) after acute treadmill AE (40 minutes; 60-80% VO2max) or control period. Aim 1 will determine if pre- and post-intervention molecular signatures associated with the pillars of aging are associated with age and sex differences in vascular adaptations to chronic AE training. Aim 2 will determine the influence of age and sex on the dynamic circulating molecular responses to acute and chronic AE, and their association with changes in vascular function with chronic AE. Aim 3 will determine if circulating exerkines (e.g., EMVs) are linked to changes in peripheral and cerebral vascular endothelial cell function with chronic AE, as well as the corresponding influences of age and sex, by assessing peripheral (aortic) and cerebral endothelial cell culture nitric oxide, endothelial nitric oxide synthase, and reactive oxygen species production pre-post AE or non-exercise control after serum or EMV exposure. The proposed research will advance our understanding of the molecular signals and pathways underlying the systemic and local effects of AE on endothelial and cerebral vascular function that may explain the heterogeneity in AE responses with age and sex. This knowledge will allow for the developm...