Non-thermal plasma enhances endothelial cell proliferation through fibroblast growth factor-2 release

Abstract

Summary form only given. Non-thermal atmospheric pressure dielectric barrier discharge plasma is currently being developed for a wide variety of clinical applications, including skin sterilization, blood coagulation, malignant cell apoptosis, and wound healing. Angiogenesis, the growth of new blood vessels from existing vessels, is critical in wound healing. Endothelial cells, which line the inner surface of blood vessels, control the angiogenic process through production, release, and response to growth factors. We hypothesized that low level plasma treatment can enhance endothelial cell proliferation through growth factor release. Porcine aortic endothelial cells were seeded in vitro on 18 mm diameter cover glasses in 12 well plates near confluence (4 x 10 <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">5</sup> cells per well). Cells were cultured for at least 24 hours prior to plasma treatment to allow full attachment and spreading. We then treated the endothelial cells using non-thermal dielectric barrier discharge plasma at a power level of 1 W/cm <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sup> for varying exposure times (short exposures of 5 s, 15 s and 30 s and long exposures of 60 s and 120 s). Viable endothelial cell number was determined by Coulter counting trypsinized cells. Endothelial cell apoptosis was measured using an Annexin V-Propidium Iodide assay. Cell proliferation was quantified via a BrDU assay. FGF-Release was quantified by ELISA. Our previous results demonstrated that non-thermal plasma is non-toxic to endothelial cells at short exposure times up to 30 s, while longer exposure times are cytotoxic. Cells treated with low levels of non-thermal plasma demonstrated enhanced proliferation as compared to untreated cells five days after treatment. Proliferation was related to cellular soluble factor release, since conditioned media from plasma treated cells induced similar proliferation when applied to untreated cells. Fibroblast growth factor-2 (FGF2), a potent angiogenic factor, is a prime candidate for plasma release, since it is only known to be released with cell membrane damage. Endothelial cell FGF2 release increased up to 3 h after plasma treatment, and the cell proliferative response to plasma treatment was negated by an FGF2 blocking antibody. Reactive oxygen species generated by non-thermal plasma in liquid may mediate FGF2 release from endothelial cells after plasma treatment, since effects were blocked with a reactive oxygen species scavenger. These data suggest that low level non-thermal plasma treatment enhances endothelial cell proliferation through FGF2 release. Future work includes three-dimensional models of angiogenesis.