Piezo1 activation in endothelial cells aggravates microvascular ischemia-reperfusion injury in limbs by enhancing ferroptosis

Acute limb ischemia-reperfusion injury (ALIRI) prominently involves microvascular dysfunction, with notable contributions from damage to microvascular endothelial cells (MECs). Previous research suggests that the mechanosensitive ion channel Piezo1 becomes active in response to mechanical stress conditions, including ischemia and trauma. However, its precise function within the ALIRI context remains elusive. Notably, the expression of Piezo1 was markedly elevated postreperfusion in mouse hind limb ischemia/reperfusion (I/R) models, implicating its crucial involvement in limb survival. Employing specific inhibitors of cell death pathways, the study delineated key molecular drivers of Ferroptosis during limb damage. Here evaluations of limb vitality, western blot, quantitative PCR and immunofluorescence implicated that activation of Piezo1 by its agonist exacerbates I/R-induced microvascular perfusion deficits, tissue swelling, skeletal muscle damage and increased tissue infarction and MECs damage. Conversely, these detrimental impacts were mitigated through pharmacological blockade of Piezo1 or specific deletion of Piezo1 in MECs. Comprehensive untargeted metabolomic analysis revealed significant changes primarily in glycerophospholipid and arachidonic acid metabolism pathways. Further experiments demonstrated that RNA interference-mediated inhibition of cytosolic Phospholipase A2 (cPLA2) and acyl-CoA synthetase long-chain family member 4 (ACSL4) negated the protective effects against Ferroptosis and limb damage that were observed with Piezo1 deletion. Moreover, this study confirmed that protein kinase C phosphorylates ACSL4, which mediates Piezo1-induced Ferroptosis and exacerbates limb damage, as shown through immunoprecipitation studies. In summary, Piezo1 contributes to the exacerbation of microvascular and skeletal muscle damage in ALIRI by facilitating the cPLA2-dependent release of arachidonic acid and promoting ACSL4-driven lipid peroxidation, thereby intensifying Ferroptosis in MECs.