Project Summary Vascular dysfunction plays a key role in hypertension and cardiovascular disease associated with inactivation of mitochondrial deacetylase Sirt3, but mitochondria-targeted treatments are not available. Sirt3 inactivation induces inhibition of mitochondrial superoxide dismutase (SOD2) and impairs fatty acid metabolism leading to mitochondrial oxidative stress and formation of harmful lipid peroxidation products, isolevuglandins (isoLG). We suggest that a feed- forward cycle between Sirt3 inactivation and mitochondrial isoLG promotes vascular dysfunction and hypertension. We developed new mitochondria-targeted isoLG scavenger, mito2HOBA, which protects Sirt3 activity and attenuates hypertension. In this proposal, we will advance this research by defining the novel role of mitochondrial isoLG in Sirt3 inactivation and vascular dysfunction, and we will establish the therapeutic potential of targeting mitochondrial isoLG. Our overall objective is to define the specific mechanism of isoLG-mediated Sirt3 inactivation and directly test the therapeutic potential of targeting mitochondrial isoLG using new transgenic mice, new mitochondria-targeted drugs, and vascular tissue from patients with essential hypertension. We will pursue the following aims: AIM 1.Test the hypothesis that inactivation of endothelial Sirt3 induces endothelial dysfunction which is prevented by targeting mitochondrial isoLG. In this aim we will examine the pathophysiological role of endothelial Sirt3 impairment and mitochondrial isoLG in endothelium specific Sirt3 depleted (EcSirt3KO) and wild-type male and female mice. We will define the role of Sirt3 inactivation and mitochondrial isoLG in endothelial inflammation, cell senescence, endothelial barrier disruption, and impaired relaxation. AIM 2.Test the hypothesis that inactivation of smooth muscle Sirt3 induces vascular dysfunction, and blocking mitochondrial isoLG improves vascular function. We will study the role of smooth muscle Sirt3 impairment in smooth muscle Sirt3 depleted (SmcSirt3KO) mice. The specific roles of SOD2-K68 acetylation and metabolic dysfunction will be tested in available SOD2-deacetylation mimetic SOD2K68R and Sirt3-/--SODK68R mice. The role of mitochondrial isoLG in smooth muscle hypertrophy, inflammation and aortic remodeling will be defined. AIM 3. Determine the therapeutic potential of targeting Sirt3 inactivation and mitochondrial isoLG in mouse models and human vascular tissue from patients with essential hypertension. We will test (A) if treatment with novel mitochondria-targeted isoLG scavengers, such as mito2HOBA, after onset of hypertension increases Sirt3 activity and improves vascular function in mouse models, and (B) if targeting mitochondrial isoLG in vascular tissues from hypertensive human subjects ex vivo reduces inflammation, improves Sirt3 activity, and relaxation. We are in an ideal position to perform these interdisciplinary studies. We developed new Sirt3 transge...