Superoxide-mediated Actin Response in Post-hypoxic Endothelial Cells

Abstract

The mechanism leading to changes in the superstructure of endothelial cells exposed to ischemia and reperfusion remains uncharacterized. We show that in posthypoxic endothelial cells, the simple re-addition of oxygen induces a profound reorganization of the actin cytoskeleton. The total filamentous actin pool increases by 41% and translocation of actin filaments to the submembranous network is observed. Concurrent with the actin polymerization, increased tyrosine phosphorylation of endothelial cell substrates is detected on Western blots. Overexpression of superoxide dismutase using replication incompetent adenovirus inhibits the actin and tyrosine phosphorylation responses to reoxygenation. Inhibition of tyrosine kinases with the isoflavone genistein also suppressed the actin polymerization response to reoxygenation, but unlike superoxide dismutase, genistein also induced the collapse of the superstructure of endothelial cells upon reoxygenation. These experiments support the concept that reoxygenation following a period of hypoxia can induce the remodeling of the actin cytoskeleton in endothelial cells. Such a response requires the intact coupling of superoxide producing pathway(s) with tyrosine kinase pathway(s).Angiogenesis can either rescue ischemic tissues by re-establishing an appropriate blood supply, or neovascularize proliferating tissues such as developing organs and neoplastic lesions (1, 2). Substantial progress has been made in the characterization of growth factors that promote or antagonize the angiogenic response (1-3). Vascular endothelial growth factor has been shown to be produced by the cells of ischemic tissues (glioblastoma and lung) (4, 5), and the receptors for vascular endothelial growth factor (flt and KDR) are up-regulated on the surface of endothelial cells in these tissues (4 -7). However, little is known about the molecular control of the cellular machinery (the actin cytoskeleton) that mediates the translocation of endothelial cells to ischemic areas, a process that is necessary, although not sufficient, for angiogenesis to take place.The vascular endothelial growth factor receptor, like most growth factor receptors, is a receptor tyrosine kinase (8 -10), whose signaling cascade is mediated by the clustering of effector molecules through the binding of SH2 domains to tyrosinephosphorylated residues. We found out recently that, in addition to tyrosine phosphorylation, the activity of growth factor receptors is also coupled to the production of reactive oxygen species (ROS) 1 such as superoxide or hydrogen peroxide (H 2 O 2 ) (11). Thus, activation of rat vascular smooth muscle cells with platelet-derived growth factor (PDGF) leads to H 2 O 2 production. Catalase or ROS chelators block the tyrosine kinase and mitogenic response to platelet-derived growth factor (11). Moreover, while ROS have been recognized in the past as mediators of intercellular interactions, we and others have recently identified ROS as major intracellular signal transduction messengers used by several small GTP...

Elevated superoxide production by active H-ras enhances human lung WI-38VA-13 cell proliferation, migration and resistance to TNF-α

“…The function of superoxide in cell migration is most likely due to the activation of cytoskeletal organization. Crawford et al (1996) demonstrated actin polymerization was concurrent with increased superoxide production in post-hypoxic endothelial cells. Moreover, endothelial cell migration in the wound was signi®cantly retarded in the presence of DPI or MnSOD mimetic (Moldovan et al, 2000).…”

Effect of homocysteine on cytokine production by human endothelial cells and monocytes

“…19,20 These changes could be attributed to cytoskeletal re-arrangements occurring as a response to homocysteine-induced oxidative stress. 30 The signi¢cance of the morphological changes in HPBMs and HMDMs (see Fig. 5) is uncertain; however, it is known that reorganization of the cytoskeleton plays a role in respiratory burst activity in monocytes.…”

Intracellular Localization and Preassembly of the NADPH Oxidase Complex in Cultured Endothelial Cells

“…In an experimental model of endothelial cell hypoxia, the reorganization of actin microfilament structure was modulated by the redox state of the cells, and the incorporation of actin into filaments could be inhibited by diphenyleneiodonium, suggesting a role for NADPH oxidase in microfilament formation (42). Although these interactions were not the focus of the present study, the localization of functional NADPH complexes that we report here may be relevant to a role of cell redox state and ROS generation in modulating cellular structure and motility.…”