Expression of heat shock protein 70 and cell death kinetics after different thermal impacts on cultured retinal pigment epithelial cells

Abstract

Recent technologies are broadening the possibility to treat the retinal pigment epithelium (RPE) with different thermal impacts, from sublethal to lethal ranges. Thus temperature-dependent subcellular molecular responses need to be elucidated in more detail. In this study, RPE cell viability and expression of heat shock protein 70 (Hsp70) were investigated after thermal irradiation with different temperature increase using an in-vitro model. Primary porcine RPE cell cultures were irradiated with different laser power of a thulium laser (λ = 1940 nm, beam-diameter 30 mm) for 10 s, such that the maximal temperatures at the center of the culture dish (T_max) reach 40, 44, 47, 51 or 59 °C after 10-s irradiation. The temperature distribution across the culture dish shows a Gaussian decay from central position to the periphery of the dish. At 3, 24 and 48 h after irradiation cell viability was assessed with fluorescence microscopy using cell viability-indicating fluorescence dyes, followed by the determination of the threshold temperature for apoptotic change and death of RPE cells. Intracellular localization and amount of Hsp70 were investigated with immunofluorescence and western blotting, respectively. The threshold temperature (at the 10th second of irradiation: T_10s) for cellular apoptosis and complete cell death showed a decrease over time after irradiation, suggesting a long-term process of thermally induced cell death. For complete cell death the threshold T_10s was 52.1 ± 0.6 °C, 50.1 ± 1.4 °C, and 50.1 ± 0.8 °C, for 3, 24 and 48 h, respectively, whereas for the apoptotic changes 48.6 ± 1.8 °C, 47.2 ± 1.3 °C, and 46.7 ± 0.9 °C, respectively. Quantitative analysis of Hsp70 with western blotting showed a significant increase in intracellular Hsp70 at lethal irradiation with T_max ≥ 51 °C, up to 19.6 ± 2.3 fold after 48 h at 59 °C, whereas sub-lethal irradiations with T_max ≤ 44 °C led to a slight tendency of time-dependent increases (up to 1.8 ± 1.1 fold) over 48 h. Immunostainings for Hsp70 showed a circle- or ring-pattern of the Hsp70 staining during 3-48 h after irradiation, and the range of the 1st and 3rd quartiles of T_10s for heat-induced Hsp70 expression over this time period was between 44.8 °C and 48.2 °C. A very strong staining of Hsp70 was observed at the border to the damaged zone, where many cells show the strong staining in the whole cytoplasmic space, while some cells in the nucleus, or some cells show the signs of cell migration and proliferation. Moreover, among the cells showing high intensity of Hsp70 staining, there are small round cells like apoptotic cells. Results suggest that RPE cell death after thermal irradiation may take time, and mostly undergoes through apoptosis, unless cells are immediately killed. Thermal irradiation-induced Hsp70 expression is not only temperature-dependent, but also depends largely on the existence of neighboring cell death, suggesting the crucial role of Hsp70 in apoptosis and wound healing processes of RPE cells. The increase of Hsp70 over 24-48 h indicates its long-term roles in cell responses both after sublethal and lethal thermal laser irradiations.