Abstract
Purpose :
Laser-induced hyperthermia on retinal pigment epithelial (RPE) cells is discussed as a new therapeutic strategy for different chorioretinal disorders. In this study we investigated the roles of a Ca2+ permeable transient potential receptor vanilloid (TRPV)-1 channel and heat shock protein (HSP) 70 on RPE cell survival and subsequent responses after thermal laser irradiation.
Methods :
Cultured primary porcine RPE cells were heated with a Thulium laser (λ= 1940 nm, spot diameter=30 mm, irradiation time=10s), through which a maximal temperature distribution exhibits a Gaussian decay from central position (peak temperature (Tmax)) to the periphery of the dish. The laser power levels were chosen to reach Tmax of 40, 43, 46, 50 and 58 °C. Cell viability was examined with flow cytometry using FITC-Annexin V and Ethidium Homodimer III, secretion of vascular endothelial growth factor (VEGF) with ELISA, and protein expressions with western blotting against HSP70, VEGF and Thioredoxin, at 3 and 24 h after irradiation performed with or without SB-366791 and VER-155008, a TRPV-1 inhibitor and a HSP70 inhibitor, respectively. As a control situation Tmax-matched non-inhibited RPE-cultures were used.
Results :
Inhibition of TRPV-1 with SB-366791 led to a significant decrease of living cells (15%, p≤ 0.01) 3 h after irradiation with Tmax 58 °C in comparison to the control. Three hours after irradiation the secretion of VEGF declined significantly in cultures irradiated with Tmax 50 °C under TRPV1 inhibition (15%, p≤ 0.05). Inhibition of HSP70 function with VER-155008 led to a significant decline of living cells after 3 h of the irradiation with Tmax 58 °C (5%, P≤ 0.05) and after 24 h of the irradiation with Tmax 50 °C (10%, P≤ 0.05). Western blotting revealed that functional inhibition of HSP70 leads to an enhancement of the laser-induced increase of intracellular HSP70 up to 12 times, intracellular VEGF and Thioredoxin up to 4 times temperature-dependendly, in comparison to the control conditions at 24 h after irradiation.
Conclusions :
From the results,TRPV-1 is suggested to be important for the prevention of heat-induced cell death at an early phase after irradiation, whereas HSP70 to be crucial for longer time (up to days) cell survival and the stabilization of the antioxidant potential of RPE cells after thermal irradiation.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.