Mechanosensitive Ca ²⁺ signaling potentiates EndMT in lung vascular endothelial cells

Pulmonary vascular remodeling is the main cause of elevated pulmonary vascular resistance in patients with pulmonary arterial hypertension (PAH). Endothelial-to-mesenchymal transition (EndMT), a process in which endothelial cells (ECs) are phenotypically converted to highly proliferative mesenchymal-like cells, has been implicated in vascular remodeling in PAH. Recent studies show that single-stranded RNA (ssRNA) derived from bacteria activates Piezo1, a mechanosensitive Ca 2+ channel highly expressed in lung vascular ECs. We have shown that Piezo1 is upregulated in the lung peripheral tissues in animals with experimental pulmonary hypertension (PH) in comparison to controls. We thus aim to examine whether ssRNA-mediated activation of Piezo1 contributes to EndMT in lung ECs. We measured the changes of cytosolic free Ca 2+ concentration ([Ca 2+ ] cyt ) with digital imaging fluorescence microscopy and recorded macroscopic Ca 2+ currents with patch clamp techniques in human pulmonary arterial endothelial cells (PAECs). We examined the levels of phosphorylated AKT, SNAI1/2, and EC markers in human PAECs with immunoblotting. Activation of Piezo1 channels by Yoda1, a selective activator of Piezo1, caused a rapid increase in [Ca 2+ ] cyt in human PAECs, while removal of extracellular Ca 2+ significantly inhibited Yoda1-mediated increase in [Ca 2+ ] cyt . Yoda1-mediated Ca 2+ influx in human PAECs was associated with a significant increase in phosphorylated AKT1 (at Ser473). Extracellular application of ssRNA40, an HIV-1LTR-derived ssRNA, significantly increased Ca 2+ currents and caused rapid increases in [Ca 2+ ] cyt and phosphorylated AKT (at Ser473) in PAECs. Downregulation of AKT1 with siRNA significantly inhibited membrane stretch-mediated increase in Jag-1, a Notch ligand, and Notch signaling is involved in EndMT. Our in vivo animal experiments showed that knockout of Akt1 ameliorated experimental PH in mice. Our data indicate that ssRNA, synthesized and released from bacteria and viruses and damaged organ cells, may serve as an important pathogenic trigger for enhancing the mechanosensitive Ca 2+ signaling via Piezo1 channels in PAECs. Increases in the endothelial Ca 2+ and phosphorylated AKT1, subsequently contribute to the induction and/or enhancement of EndMT via the pAKT1-mediated upregulation of Jag-1 and activation of Notch signaling, and ultimately the development and progression of pulmonary vascular remodeling in PAH. This study is supported in part by a grant from the NHLBI/NIH (HL171538) This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.