Ultrasound (US) is used for a variety of therapeutic applications and has been proposed for increasing tissue perfusion in ischemic cardiovascular disease. The most important non- thermal bioeffect by which US can increase perfusion is convective motion which increases shear mechanotransduction. Microbubble (MB) contrast agents used in patients to enhance the blood pool can potentially augment shear through microstreaming during acoustic cavitation. We have recently demonstrated that US-mediated MB cavitation can augment limb tissue perfusion by up to 10-fold and that this effect can persist for greater than 24 hours. The overall aim of this proposal is to assemble a multidisciplinary team to optimize the technology of US- mediated MB cavitation and transition it from pre-clinical models to the treatment of patients with severe symptomatic peripheral artery disease (PAD). In Aim 1 preclinical small animal models will be used to define optimal conditions for augmenting limb perfusion with regards to acoustic pressure, frequency, MB concentration, and pulsing interval which governs the vascular level at which cavitation occurs. Contrast ultrasound perfusion imaging will be used to assess effect and the type of cavitation (stable versus inertial) will be determined from frequency-power spectra in order to understand the physical determinants for flow augmentation. In Aim 2 we will use large animal models of PAD to spatially characterize and quantify changes in limb perfusion in relation to the volume of tissue in which cavitation is produced. In both of the first two Aims, data will also be generated to define the safety profile for microbubble cavitation. Understanding the biologic determinants for acute and long-term (>24 hour) flow augmentation will be the subject of Aim 3 where we will examine the role of shear- and pressure-dependent mediators of vascular tone. Potential candidate mediators will include NO, adenosine, eicosanoids, and ATP. In Aim 4, an existing 3-D with optimized acoustic conditions for MB cavitation will be applied to evaluate limb tissue perfusion in (a) healthy human subjects, and (b) patients with PAD and critical limb ischemia. These studies represent the translational steps for development of a non-invasive therapy for severe PAD that can be used to achieve meaningful and immediate increases in perfusion, and that can maintain limb tissue viability in situations where immediate revascularization is either not immediately possible or available.