CD36 mediates H2O2-induced calcium influx in lung microvascular endothelial cells

Abstract

Elevated levels of reactive oxygen species and intracellular Ca2+play a key role in endothelial barrier dysfunction in acute lung injury. We previously showed that H2O2- induced increases in intracellular calcium concentrations ([Ca2+]i) in lung microvascular endothelial cells (LMVECs) involve the membrane Ca2+channel, transient receptor potential vanilloid-4 (TRPV4) and that inhibiting this channel attenuated H2O2-induced barrier disruption in vitro. We also showed that phosphorylation of TRPV4 by the Src family kinase, Fyn, contributes to H2O2-induced Ca2+influx in LMVEC. In endothelial cells, Fyn is tethered to the cell membrane by CD36, a fatty acid transporter. In this study, we assessed the effect of genetic loss or pharmacological inhibition of CD36 on Ca2+responses to H2O2. H2O2-induced Ca2+influx was attenuated in LMVEC isolated from mice lacking CD36 (CD36_/_). TRPV4 expression and function was unchanged in LMVEC isolated from wild-type (WT) and CD36_/_mice, as well as mice with deficiency for Fyn (Fyn_/_). TRPV4 immunoprecipitated with Fyn, but this interaction was decreased in CD36_/_LMVEC. The amount of phosphorylated TRPV4 was decreased in LMVEC from CD36_/_mice compared with WT controls. Loss of CD36 altered subcellular localization of Fyn, while inhibition of CD36 fatty acid transport with succinimidyl oleate did not attenuate H2O2-induced Ca2+influx. Lastly, we found that CD36_/_mice were protected from ischemiareperfusion injury in vivo. In conclusion, our data suggest that CD36 plays an important role in H2O2-mediated lung injury and that the mechanism may involve CD36-dependent scaffolding of Fyn to the cell membrane to facilitate TRPV4 phosphorylation.