VEGF-A
VEGF-A mRNA expression was measured by real-time PCR. In samples collected after 24 h, the mRNA expression measured at lower temperatures was decreased compared to expression in cells cultured at 37°C, with an observed decrease of 14.8% at 35°C, 39.3% at 33°C, and 43.3% at 31°C. In samples collected after 48 h, compared with cells cultured at 37°C, the decreases measured at each reduced temperature were 35°C, 37.3%; 33°C, 52.0%; and 31°C, 59.3%. This temperature-dependent decrease was statistically significant (Fig. 1a, left).
VEGF-A protein concentration in the conditioned medium was measured by enzyme-linked immunosorbent assay (ELISA). In samples collected after 24 h, the decreases measured at each reduced temperature compared to 37°C were as follows: 35°C, 9.55%; 33°C, 25.3%; and 31°C, 46.6%. In samples collected after 48 h, the decreases measured at each reduced temperature were 35°C, 7.52%; 33°C, 27.3%; and 31°C, 52.6%. Significant temperature-dependent decreases in protein abundance were also observed (Fig. 1a, right). Overall, expression of both VEGF-A mRNA and protein decreased as the culture temperature decreased in samples collected after either 24 or 48 h.
PEDF
PEDF mRNA expression was measured by real-time PCR. In samples collected after 24 h, no significant change in PEDF mRNA expression was found over the range from 37–33°C. However, PEDF mRNA expression at 31°C was significantly lower than at 37°C. Similarly, in samples collected after 48 h, no significant expression change was found over the range from 37–33°C, but at 31°C, expression significantly decreased by 45.0% compared with expression in cells cultured at 37°C (Fig. 1b, left). The concentration of PEDF protein in the conditioned medium was measured by ELISA. PEDF protein abundance showed no significant change over the range of 37–31°C (Fig. 1b, right).
VEGF165
VEGF165 expression in conditioned medium was measured by western blot analysis, and the results are shown in Fig. 2. In samples collected after 24 h, decreased expression was observed at all temperatures compared to expression at 37°C, with a percent decrease of 10.7% at 35°C; 12.7% at 33°C; and 39.9% at 31°C. In samples collected after 48 h, the corresponding decreases were 35°C, 6.41%; 33°C, 15.7%; and 31°C, 42.3%. Expression at 31°C was significantly decreased in samples collected after 24 and 48 h. Generally, expression was observed to decrease in a temperature-dependent fashion.
VEGF121
VEGF121 expression was not detected by western blot analysis in any of the samples examined (Fig. 2a).
PlGF
PlGF expression was not detected by real-time PCR or ELISA in any of the samples examined (data not shown).
Tube formation assay
To investigate possible changes in the angiogenic environment produced by RPE, we performed endothelial tube formation (in vitro angiogenesis) assays using conditioned medium from ARPE-19 cells cultured at various temperatures, from 37 to 31°C, and for either 24 or 48 h. Representative photomicrographs and image quantitative analysis are shown in Fig. 3. Typical tube formation was not found in any of the samples. However, significant differences in the number of branching points and total skeleton length were observed among the conditions. With conditioned medium samples collected after 24 h, the number of branching points was reduced compared to the values observed at 37°C by the following percentages: at 35°C, 26.6%; at 33°C, 56.5%; and at 31°C, 60.9%. In samples collected after 48 h, the percent decrease in expression was as follows: at 35°C, 47.1%; at 33°C, 61.7%; and at 31°C, 73.8%. With respect to total skeleton length, in samples collected after 24 h and compared with conditioned medium from cells cultured at 37°C as the baseline, the decreases observed at each temperature were as follows: at 35°C, 19.3%; at 33°C, 44.6%; and at 31°C, 80.7%. In samples collected after 48 h, the decreases observed at each temperature were as follows: at 35°C, 14.4%; at 33°C, 47.1%; and at 31°C, 83.6%. Overall, the number of branching points and total skeleton length in samples collected after either 24 h or 48 h showed significant decreases as the culture temperature decreased.
Measurement of cellular metabolism
The metabolism of cells cultured at each adjusted temperature was evaluated by dehydrogenase activity. In samples collected after incubation for 24 h, the decreases measured at each reduced temperature compared to 37°C were as follows: 35°C, 12.3%; 33°C, 37.8%; and 31°C, 36.0%. In samples collected after incubation for 48 h, the decreases measured at each reduced temperature were 35°C, 25.6%; 33°C, 25.7%; and 31°C, 52.8%. Significant temperature-dependent decreases in dehydrogenase activity were observed in samples collected after either 24 or 48 h (Fig. 4).
The balance between VEGF-A and PEDF plays an important role in choroidal neovascularization [6, 16–18]. In the present study, VEGF-A expression significantly decreased in RPE with temperature dependence, as analyzed using real-time PCR and ELISA. Western blot analysis showed a temperature-dependent decrease in VEGF165 expression. In contrast, PEDF protein abundance did not show significant change. Thus, RPE cultured at temperatures lower than 37°C may exhibit an anti-angiogenic environment because of the decreased VEGF-A expression in conjunction with sustained PEDF expression. The results of the tube formation angiogenesis assay also supported this hypothesis, although typical tube formation was not observed, probably because of insufficient VEGF expression.
The sustained PEDF that is secreted from RPE predominantly to the apical side [19–22] may play a role in preventing wet AMD development by the following two mechanisms. First, because the secreted PEDF plays an autocrine role in maintaining RPE function [20], healthy RPE maintained by sufficient PEDF should prevent an invasion of choroidal neovascularization to the retinal side. Second, PEDF in the basal side, even if it is not a large amount, may act as an antagonist for VEGF-A. Becerra et al. investigated PEDF localization in monkey eye and detected PEDF not only in the apical side but also in the Bruch membrane [21].
The measurement of dehydrogenase activity at each adjusted temperature showed a cellular metabolism decrease in association with the culture temperature decrease. We confirmed no significant difference in the number of cells in the dish at the end of incubation between each sample (data not shown). This result implies that a decrease in VEGF-A expression is correlated with a decrease in metabolism in cells cultured under hypothermia, although it is still unclear why PEDF expression is not so closely correlated.
The retinal temperature in a vitrectomized eye is lower than that in a non-vitrectomized eye, but this measurement of real-time retinal temperature in humans may be possible only during surgery. The anterior chamber temperature is remarkably cooled [13], and ophthalmologists often observe a warm current of aqueous humor convective flow caused by cooling of the anterior chamber by outside air. Because of thermal diffusion from the posterior to the anterior chamber through the lens, the anterior vitreous cavity temperature may be lower than the posterior vitreous cavity temperature. In the vitreous cavity not filled with viscous vitreous in a vitrectomized eye, a more convective flow may occur because of the difference in temperature between the anterior and posterior vitreous cavities; however, because of the viscous vitreous in a non-vitrectomized eye, such a convective flow may not easily occur. Our hypothesis regarding this intraocular thermal diffusion is shown in Fig. 5. The strict measurements taken of retinal temperature immediately adjacent to the fovea by Landers et al. during surgery were as follows: before vitrectomy, 34.9°C; at the end of vitrectomy after plugging the sclerotomies and closing the infusion line for 5 min, 32.6°C [14]. This difference of 2.3°C may have been enough to decrease VEGF-A expression in the eye, as our present results revealed that a 2°C decrease caused a significant decrease in VEGF-A expression in vitro.