Women’s health across the lifespan has recently been highlighted as a national research priority. Arterial stiffening is a process that occurs naturally with aging, and it is an independent risk factor for cardiovascular (CV) disease (CVD). Although arterial stiffness develops later in life in women than men, it develops at a faster rate and has a stronger association with CVD morbidity and mortality in women and has direct clinical implications in CV conditions that predominantly affect women, such as isolated systolic hypertension and heart failure with preserved ejection fraction. Post-menopausal women and men have lower levels of circulating estrogens than premenopausal women, suggesting a greater presence of unliganded estrogen receptor alpha (ERα) in aging women and men. Previously thought to be inert, recent in vitro data support a detrimental role for unliganded ERα in vascular health. Preliminary data from our lab support a link between circulating estrogens, ERα, and expression of the vasoprotective angiotensin II type 2 receptor (AT2R). AT2R expression and activity has previously been shown to be estrogen-dependent in many tissues, but this has yet to be investigated in the vasculature or in the context of aging-associated arterial stiffness. We propose to test the novel hypothesis that unliganded smooth muscle cell (SMC)-ERα contributes to aging-associated arterial stiffness via repression of AT2R and subsequent increase in pro-fibrotic pathways downstream of AT2R. To investigate the role of SMC- ERα in aging-associated arterial stiffness in females and males, I will complete the following three aims: 1) Determine the role of SMC-ERα in aging-associated arterial stiffness in vivo in aging female and male SMC-ERα-intact and SMC-ERα-knockout (KO) mice; 2) test the hypothesis in vitro that SMC-ERα regulates pro-fibrotic genes in an AT2R-dependent manner in human aortic SMC from young and aged women and men; and 3) test the in vivo therapeutic potential of chronic AT2R activation to prevent aging- associated arterial stiffness in middle-aged (12 mo.) female and male wildtype mice treated with either vehicle or NP-6A4, a novel AT2R agonist, for 4 weeks. In vivo arterial stiffness will be measured by aortic pulse wave velocity, intrinsic SMC stiffness will be measured by atomic force microscopy, and aortic fibrosis will be assessed with Masson’s trichrome staining. Aortic protein and gene expression of AT2R and downstream pro-fibrotic targets will be quantified and circulating sex hormones will be measured via mass spectrometry. Results from the proposed studies will have substantial clinical significance, enhancing the basic understanding of sexually dimorphic molecular mechanisms of aging-associated arterial stiffness and providing support for innovative therapeutic treatments to improve CVD outcomes in postmenopausal women and men, thus clearly supporting the National Institutes of Health core mission to enhance health, lengthen life, an...