Gabryś Dorota, Greco Olga, Patel Gaurang, Prise Kevin M, Tozer Gillian M, Kanthou Chryso
Department of Radiation Oncology, Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Gliwice, Poland.
Int J Radiat Oncol Biol Phys. 2007 Dec 1;69(5):1553-62. doi: 10.1016/j.ijrobp.2007.08.039. Epub 2007 Oct 24.
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.
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.
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.
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.
研究辐射对内皮细胞骨架及内皮单层通透性的影响，并评估相关信号通路，以揭示辐射对血管已知效应的潜在机制。
对培养的内皮细胞进行X射线照射，通过免疫荧光检测肌动蛋白丝、微管、中间丝和血管内皮（VE）-钙黏蛋白连接。通过荧光葡聚糖透过细胞单层来测定通透性。使用RhoA、Rho激酶和应激激活蛋白激酶-p38（SAPK2/p38）抑制剂，通过三磷酸鸟苷-RhoA激活测定和用RhoAT19N转染来分析信号转导通路。通过蛋白质印迹法评估连接蛋白表达水平以及肌球蛋白轻链和SAPK2/p38的磷酸化情况。通过克隆形成试验验证辐射对细胞死亡的影响。
辐射诱导微血管内皮细胞而非脐静脉内皮细胞中肌动蛋白应激纤维快速且持续形成以及VE-钙黏蛋白连接重新分布，微管和中间丝未受影响。辐射还导致微血管通透性快速且持续增加。RhoA-鸟苷三磷酸酶和Rho激酶被辐射激活，导致下游肌球蛋白轻链磷酸化以及观察到的细胞骨架和通透性变化。SAPK2/p38被辐射激活，但不影响细胞骨架或通透性。
本研究首次表明辐射可在内皮细胞中快速激活RhoA/Rho激酶，并证明了该通路与细胞骨架重塑和通透性之间的联系。结果还表明，RhoA通路可能是调节辐射通透性及其他效应以实现治疗获益的有用靶点。
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