Project Summary Peripheral artery disease (PAD) results in the progressive reduction of blood flow to peripheral body parts including the lower limbs. The resulting ischemic muscle triggers an increased arterial pressure during lower limb movements that is due to an exaggerated exercise pressor reflex (EPR). The EPR is a neural feedback mechanism that leads to a physiological increase in blood pressure upon muscle contraction. When the EPR is exaggerated in PAD, such that even low levels of muscle contraction (e.g., elicited by walking) cause abnormally high blood pressure responses. This significantly contributes to the high risk of cardiovascular morbidity and mortality in patients with PAD. Therefore, exercise, which has been shown to be beneficial for improving muscle function and repair in patients with PAD cannot be safely prescribed to the patients. This emphasizes the need for therapies that can normalize the EPR. The mechanically sensitive arm of the EPR mediates its exaggeration in PAD, as has been shown by rodent femoral artery ligation (FAL), a well-established model of PAD. FAL chronically sensitizes the mechanically sensitive receptors e.g., Piezo2, but the mechanism is unknown. Preliminary studies from our lab show that Piezo2, in part, mediates the exaggerated EPR. The ischemic muscles of patients and rodent models with PAD exhibit increased expression of pro-inflammatory genes and immune cell infiltration. The effect that the resulting inflammatory cytokines from the ischemic muscle has on the muscle mechanosensitive afferent neurons and their resident ion channels is unknown. Previous findings have reported a signal transduction pathway that begins with an inflammatory mediator and culminates in the heightened activity of a mechanosensitive channel in somatosensory neurons. Therefore, the hypothesis of this proposal is that the pro-inflammatory signals from the ischemic muscle increase the expression and activity of Piezo2 channels in mechanosensitive afferent neurons. To test this hypothesis, the proposed aims (Aim 1) will determine the effect of proinflammatory mediators on the activity and expression of Piezo2 channel and other mechanosensitive channels in mouse dorsal root ganglia (DRG) sensory neurons and hiPSC-derived sensory neurons (hiPSC-SN). The effects of proinflammatory mediators in the absence of ischemia on Piezo2 and other mechanosensitive channels’ expression and activity will also be investigated in vivo. To establish a definitive cause-and-effect relationship between muscle inflammation and Piezo2 sensitization, the proposed experiments involve macrophage depletion under ischemic conditions. The effects of this depletion on the activity and expression of Piezo2 and other mechanosensitive channels in the DRG will be investigated. The resulting findings will offer insights into the interplay between neural and immune responses in PAD, potentially leading to novel therapeutic strategies to better managing the card...