Regulation of vimentin intermediate filaments in endothelial cells by hypoxia

Abstract

Hypoxia triggers responses in endothelial cells that play roles in many conditions including high-altitude pulmonary edema and tumor angiogenesis. Signaling pathways activated by hypoxia modify cytoskeletal and contractile proteins and alter the biomechanical properties of endothelial cells. Intermediate filaments are major components of the cytoskeleton whose contribution to endothelial physiology is not well understood. We have previously shown that hypoxia-activated signaling in endothelial cells alters their contractility and adhesiveness. We have also linked p38-MAP kinase signaling pathway leading to HSP27 phosphorylation and increased actin stress fiber formation to endothelial barrier augmentation. We now show that vimentin, a major intermediate filament protein in endothelial cells, is regulated by hypoxia. Our results indicate that exposure of endothelial cells to hypoxia causes vimentin filament networks to initially redistribute perinuclearly. However, by 1 hour hypoxia these networks reform and appear more continuous across cells than under normoxia. Hypoxia also causes transient changes in vimentin phosphorylation, and activation of PAK1, a kinase that regulates vimentin filament assembly. In addition, exposure to 1 hour hypoxia increases the ratio of insoluble/soluble vimentin. Overexpression of phosphomimicking mutant HSP27 (pm-HSP27) causes changes in vimentin distribution that are similar to those observed in hypoxic cells. Knocking-down HSP27 destroys the vimentin filamentous network, and disrupting vimentin filaments with acrylamide increases endothelial permeability. Both hypoxia-and pmHSP27 overexpression-induced changes are reversed by inhibition of phosphatase activity. In conclusion hypoxia causes redistribution of vimentin to a more insoluble and extensive filamentous network that could play a role in endothelial barrier stabilization. Vimentin redistribution appears to be mediated through altering the phosphorylation of the protein and its interaction with HSP27. permeability; edema; pulmonary; vascular; cytoskeleton HYPOXIA has been shown to alter the structure and function of endothelial cells in a manner that contributes to a variety of diseases. Signaling pathways that are activated by hypoxia such as p38 mitogen-activate protein (MAP) kinase and Rho kinase pathways alter the actin cytoskeleton and contractile proteins leading to changes in biomechanical forces in these cells (3). These forces likely play an important role in endothelial cell migration and interaction with leukocytes as well as in regulation of endothelial barrier permeability which is altered in hypoxia (10,14,19). Intermediate filaments constitute one of the three major components of the cytoskeleton which also include actin microfilaments and tubulin microtubules.These structures act in concert to control cell morphology and biomechanics. The role of intermediate filaments in regulation of endothelial function has not been sufficiently studied.We have focused on the regulation of vimentin filaments in rat pulmon...

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