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Hisashi Iwami, Lars Ptaszynski, Veit Danicke, Ralf Brinkmann, Yoko Miura; Sublethal Hyperthermia-induced Vascular Endothelial Growth Factor Secretion And Its Contribution To Adoptive Response Of Retinal Pigment Epithelial Cell. Invest. Ophthalmol. Vis. Sci. 2012;53(14):4782.
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To investigate temperature increase-induced secretion of vascular endothelial growth factor (VEGF) from retinal pigment epithelial (RPE) cells and its contribution to adoptive response relating to cell defence system against oxidative stress.
Porcine RPE cells on 35 mm culture dish were used in the study. Thulium laser (=1940 nm, spot size 33 mm was utilized as a heat source. Temperature increase during irradiation for different power and time setting at cell level was measured with thermocouple, and power and time setting of the experiment was determined based on this calibration. Culture medium was replaced by 1.2 ml phosphate buffer saline and then laser was irradiated with different power settings for 10 seconds, so that the peak temperature reaches from 40°C to 65°C. Cellular viability after laser irradiation was examined with MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay immediately after irradiation. VEGF secretion was investigated with enzyme-linked immunosorbent assay (ELISA) at 2 and 24 hrs after irradiation. Contribution of a temperature-dependent calcium channel, TRPV (transient receptor potential vanilloid) channels in laser-induced VEGF secretion was investigated using TRPV channel blocker, ruthenium red (20 µM). TRPV channel blocker-containing medium was replaced by the normal medium soon after laser irradiation. Hydrogen peroxide (H2O2) or advanced glycation endproduct (AGE)-was exposed after 6 hrs of laser irradiation and cell viability was examined with MTT assay.
Peak temperature threshold for immediate RPE cell death was found around 55 °C with our irradiation setting. VEGF secretion was increased after sub-lethal irradiation in power-dependent manner, which was partially suppressed by TRPV channel blocker. Sublethal laser irradiation reduced H2O2 and AGE-induced cell death and this effect was smaller in the cells treated with TRPV channel inhibitor during laser irradiation.
Sublethal temperature increase-induced VEGF production might contribute to the enhancement of RPE cell defence system against oxidative stress.
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