Flow-Activated Calcium Signaling Microdomains in Small Pulmonary Arteries

ABSTRACT Endothelium-dependent vasodilation of small pulmonary arteries (PAs) lowers PA pressure (PAP) under normal conditions. The goal of this application is to identify novel calcium signaling mechanisms that regulate endothelium-dependent dilation of small PAs and their impairments in pulmonary hypertension (PH). Our studies during the previous funding cycle showed that TRPV4 channels, a crucial calcium entry pathway in endothelial cells, lower PAP under normal conditions. Moreover, we showed that endothelial pannexin 1 (Panx1), an ATP efflux pathway, activates TRPV4 channels. PAs are a high-flow vascular bed, but flow-induced signaling mechanisms in the pulmonary endothelium are unknown. Our preliminary data show that intraluminal flow activates endothelial Panx1–TRPV4 channel signaling to dilate small PAs. Moreover, mechanosensitive Piezo1 channels lie upstream of Panx1 in the flow-induced pathway for dilation of small PAs. Additionally, scaffolding protein caveolin-1 (Cav1) ensures nanometer proximity between Piezo1, Panx1, and TRPV4 channels in the pulmonary endothelium. Therefore, we will first determine the role of Cav1-scaffolded Piezo1–Panx1–TRPV4 signaling in flow-induced dilation of small PAs and regulation of PAP. We will then determine whether a mis- localization of Piezo1:Panx1:TRPV4 channels in the pulmonary endothelium impairs flow-induced dilation of small PAs in PH. Our prior studies indicated a crucial role for peroxynitrite, a reactive nitrogen species, in impairing endothelium-dependent dilation of small PAs in PH. Therefore, we will test the hypothesis that peroxynitrite disrupts Cav1-scaffoled Piezo1–Panx1–TRPV4 signaling in small PAs in PH. Tumor necrosis factor (TNF), an inflammatory cytokine, is a well-known promoter of cellular peroxynitrite formation. Proinflammatory effects of TNF are mediated mainly by TNF receptor I (TNFRI). Our preliminary data show that endothelial TNFRI deletion improves endothelial function and lowers RVSP in a mouse model of PH. Therefore, we will test the hypothesis that endothelial TNFRI signaling elevates endothelial peroxynitrite levels in PH, impairing flow- induced dilation and elevating PAP. Collectively, these studies will fill major gaps in our understanding of endothelial calcium signaling mechanisms that regulate PAP and their abnormalities in PH.