Radiation effects on the cytoskeleton of endothelial cells and endothelial monolayer permeability.
OBJECTIVE
To investigate the effects of radiation on the endothelial cytoskeleton and endothelial monolayer permeability and to evaluate associated signaling pathways, which could reveal potential mechanisms of known vascular effects of radiation.


METHODS
Cultured endothelial cells were X-ray irradiated, and actin filaments, microtubules, intermediate filaments, and vascular endothelial (VE)-cadherin junctions were examined by immunofluorescence. Permeability was determined by the passage of fluorescent dextran through cell monolayers. Signal transduction pathways were analyzed using RhoA, Rho kinase, and stress-activated protein kinase-p38 (SAPK2/p38) inhibitors by guanosine triphosphate-RhoA activation assay and transfection with RhoAT19N. The levels of junction protein expression and phosphorylation of myosin light chain and SAPK2/p38 were assessed by Western blotting. The radiation effects on cell death were verified by clonogenic assays.


RESULTS
Radiation induced rapid and persistent actin stress fiber formation and redistribution of VE-cadherin junctions in microvascular, but not umbilical vein endothelial cells, and microtubules and intermediate filaments remained unaffected. Radiation also caused a rapid and persistent increase in microvascular permeability. RhoA-guanosine triphosphatase and Rho kinase were activated by radiation and caused phosphorylation of downstream myosin light chain and the observed cytoskeletal and permeability changes. SAPK2/p38 was activated by radiation but did not influence either the cytoskeleton or permeability.


CONCLUSIONS
This study is the first to show rapid activation of the RhoA/Rho kinase by radiation in endothelial cells and has demonstrated a link between this pathway and cytoskeletal remodeling and permeability. The results also suggest that the RhoA pathway might be a useful target for modulating the permeability and other effects of radiation for therapeutic gain.
Gabryś
Dorota
D
Department of Radiation Oncology, Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Gliwice, Poland.
Greco
Olga
O
Patel
Gaurang
G
Prise
Kevin M
KM
Tozer
Gillian M
GM
Kanthou
Chryso
C
D000199:Actins / Q000528:radiation effects; D015820:Cadherins / Q000528:radiation effects; D016923:Cell Death / Q000528:radiation effects; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000528:radiation effects*; D003911:Dextrans / Q000493:pharmacokinetics; D042783:Endothelial Cells / Q000528:radiation effects* / Q000648:ultrastructure; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism / Q000528:radiation effects; D006801:Humans; D007382:Intermediate Filaments / Q000528:radiation effects; D008870:Microtubules / Q000528:radiation effects; D048290:Mitogen-Activated Protein Kinase 11 / Q000502:physiology; D010539:Permeability; D011836:Radiation Tolerance; D015398:Signal Transduction / Q000502:physiology / Q000528:radiation effects; D020742:rhoA GTP-Binding Protein / Q000502:physiology
D000199:Actins; D015820:Cadherins; D003911:Dextrans; D048290:Mitogen-Activated Protein Kinase 11; D020742:rhoA GTP-Binding Protein