Bubbles Induce Endothelial Microparticle Formation via a Calcium-Dependent Pathway Involving Flippase Inactivation and Rho Kinase Activation

Abstract

Background/Aims: Intravascular bubbles can exert pleiotropic detrimental effects, partly by inducing endothelial microparticles (EMPs) production, which play critical roles in cell communication and vascular inflammation cascades. However, the underlying mechanisms remain unclear. This study aimed to delineate the possible mechanisms involving bubble-induced EMPs formation. Methods: Human umbilical vein endothelial cells (HUVECs) were contacted by bubbles and EMPs level in supernatant were quantified by flow cytometry. Cytoplasmic calcium (Ca²⁺) was measured by the Ca²⁺ binding dyes Fluo-3 AM and flippase activity was assessed by translocation rate of fluorescent phosphatidylserine (PS) analogue NBD-PS. Protein levels of phospho-myosin light chain (MLC, a Rho kinase substrate) and phospho-extracellular signal-regulated kinase 1 or 2 (ERK1/2) were determined by western blotting. The score of actin colocalization was assessed by phalloidin-FITC using an immunofluorescent microscopy. Results: EMPs level markedly increased after bubble stimulus. Cytoplasmic calcium (Ca²⁺) significantly elevated (P< 0.05), flippase activity decreased (P< 0.05), protein levels of phospho- MLC and phospho- ERK1/2 significantly increased (P< 0.05, P < 0.05), and the score of actin colocalization markedly reduced (P< 0.05) in bubble-stimulated HUVECs. All the above changes except the increase in phospho-ERK1/2 can be reversed by Ca²⁺ channel blocker LaCl₃ (P< 0.05). Additionally, MLC phosphorylation was significantly inhibited and actin colocalization markedly increased by Rho kinase inhibitor pretreatment and more importantly, bubble-induced EMPs markedly decreased. Conclusions: These results demonstrate that bubble stimulates EMPs formation by cytoplasmic Ca²⁺ elevation and subsequently activating Rho kinase pathway and cytoskeleton reorganization. Simultaneously, cytoplasmic Ca²⁺ inhibits the flippase activity and subsequently increases phosphatidylserine exposure, which also contributes to EMPs formation.