Increases in reactive oxygen species enhance vascular endothelial cell migration through a mechanism dependent on the transient receptor potential melastatin 4 ion channel. SFKs is posttranslationally regulated by several mechanisms (1, 31). Importantly, association with membrane anchor proteins retains Fyn at the cell membrane, allowing Fyn to exert its kinase activity on nearby membrane proteins (8). In the endothelium, Fyn associates with CD36, a fatty acid transporter (18, 32, 33). The role of CD36 in lung disease is under active investigation, with evidence showing that endothelial CD36 contributes to LPS-induced barrier dysfunction (4). Though principally involved in fatty acid transport across the cell membrane, CD36 has been recently implicated in many other cellular processes including angiogenesis and apoptosis (32). While the mechanism by Rabbit Polyclonal to RAD21 which CD36 mediated these effects is still Nimodipine under investigation, there is growing evidence to suggest that CD36 participates in cell signaling events independent of fatty acid transport. As mentioned above, CD36 can act as a membrane anchor for the Src kinase Fyn. In addition, following activation by ligands such as thrombospondin, CD36 has been shown to serve as Nimodipine a nidus for recruitment and activation of various intracellular kinases (10, 23, 35). Moreover, the presence of CD36 on the membrane has been shown to be important for basal Ca2+ dynamics, with loss of CD36 attenuating Ca2+ influx in CHO cells following thapsigargin-induced store depletion (20). Collectively, these data suggest a critical role for CD36 in Ca2+ signaling and Nimodipine kinase activation, although the specifics of these two functions in LMVECs are not known. CD36 has also been implicated in IR pathobiology. Loss of CD36 attenuates IR injury in various vascular beds including the brain and the heart (9, 26) and, in the lung, we recently showed that loss of CD36 is protective against H2O2-induced barrier dysfunction in LMVEC in vitro and malaria-induced ARDS in vivo (2). However, to our knowledge, a role for CD36 in lung IR injury has not been previously described in vivo. Given the involvement of CD36 in IR injury in other tissues, our data showing that endothelial dysfunction due to increased H2O2 is dependent on elevations in intracellular Ca2+ and prior data linking CD36 to intracellular Ca2+ flux, we hypothesized that CD36 participates in IR injury by modulating H2O2-induced Ca2+ influx. Specifically, we hypothesized that CD36-mediated tethering of Fyn to the cell membrane facilitates phosphorylation of TRPV4, a step we previously showed was necessary for subsequent Nimodipine activation of TRPV4 by H2O2 (36). Thus in this study we determined whether loss of CD36 alters H2O2-induced Ca2+ influx in vitro and whether loss of CD36 is protective against IR injury in vivo. METHODS All procedures were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Animal Care and Use Committee of The Johns Hopkins University School of Medicine. Isolation and culture of mouse lung microvascular endothelial cells. Adult (8C10 wk), wild-type (WT; C57/B6) and male mice were generously provided by Dr. Alan Scott (Johns Hopkins University). All mice were subjected to genotyping before use. Mice were euthanized by cervical dislocation, and the lungs were quickly removed by dissection and immersed in DMEM (GIBCO). As described by us previously (36), peripheral mouse tissue was obtained by dissection, minced, and digested in complete medium using collagenase (Type 1A; 1 mg/ml). Following centrifugation and resuspension of the cell pellet, the cell suspension was incubated with CD31-conjugated beads.