Temsirolimus inhibits proliferation and migration in retinal pigment epithelial and endothelial cells via mTOR inhibition and decreases VEGF and PDGF expression

Abstract

Due to their high prevalence, retinal vascular diseases including age related macular degeneration (AMD), retinal vein occlusions (RVO), diabetic retinopathy (DR) and diabetic macular edema have been major therapeutic targets over the last years. The pathogenesis of these diseases is complex and yet not fully understood. However, increased proliferation, migration and angiogenesis are characteristic cellular features in almost every retinal vascular disease. The introduction of vascular endothelial growth factor (VEGF) binding intravitreal treatment strategies has led to great advances in the therapy of these diseases. While the predominant part of affected patients benefits from the specific binding of VEGF by administering an anti-VEGF antibody into the vitreous cavity, a small number of non-responders exist and alternative or additional therapeutic strategies should therefore be evaluated. The mammalian target of rapamycin (mTOR) is a central signaling pathway that eventually triggers up-regulation of cellular proliferation, migration and survival and has been identified to play a key role in angiogenesis. In the present study we were able to show that both retinal pigment epithelial (RPE) cells as wells as human umbilical vein endothelial cells (HUVEC) are inhibited in proliferating and migrating after treatment with temsirolimus in non-toxic concentrations. Previous studies suggest that the production of VEGF, platelet derived growth factor (PDGF) and other important cytokines is not only triggered by hypoxia but also by mTOR itself. Our results indicate that temsirolimus decreases VEGF and PDGF expression on RNA and protein levels significantly. We therefore believe that the mTOR inhibitor temsirolimus might be a promising drug in the future and it seems worthwhile to evaluate complementary therapeutic effects with anti-VEGF drugs for patients not profiting from mono anti-VEGF therapy alone.

Figure 1. Viability of HUVEC and pRPE cells.

Viability of HUVECs and primary human RPE cells after treatment with various concentrations of temsirolimus for 24 h, as measured by a colorimetric test (MTT). Untreated HUVECs or RPE cells of the same passage served as controls. Results are the mean percentages of control cell survival from three experiments, each carried out in triplicate, with error bars indicating SEM. Temsirolimus concentrations up to 12,5 µg/ml for RPE cells and 7,5 µg/mL for HUVEC showed no significant reduction in viability of either cell type (Wilcoxon test with a correction for multiple testing) compared to controls. Data are expressed as means ± SD. (*p<0.05 for HUVECs, #p<0.05 for RPE) Controls are set to 100% to simplify result reading.

Figure 2. Proliferation of HUVEC and pRPE cells.

Inhibition of HUVEC (2A) and RPE cell (2B) proliferation after incubation with three different concentrations of temsirolimus over 24 h measured by a colorimetric assay (MTT), (0 µg/mL  =  control). Data are expressed as means ± SD and results are displayed as % control, the control being set to 100%. (*p<0.05).

Figure 3. Migration of HUVEC and pRPE cells.

HUVEC and RPE cell migration was significantly inhibited after exposure to temsirolimus in a dose-dependent manner (µg/mL) assessed by a modified Boyden chamber method. A) Example of cells attached to a permeable membrane at different concentrations compared to an untreated control. B) Evaluation of migrated HUVEC in percent control after treatment with three different temsirolimus concentrations. C) Migrated RPE cells compared to untreated cells in percent control. Data are expressed as means ± SD with *p<0.05.

Figure 4. VEGF mRNA expression in HUVEC and pRPE cells.

VEGF (VEGF-A) expression of untreated HUVECs and primary RPE cells and after exposure to hypoxia for 24 h, as measured by quantitative RT-PCR. When cells were exposed to hypoxia, a significant increase in VEGF mRNA could be detected in both tested cell lines. When cells were additionally treated with 0.05 µg/ml temsirolimus, in HUVEC (A) and RPE cells (B) the hypoxia-induced increase in VEGF mRNA expression was significantly less. Y-axis: RR of VEGF mRNA normalized to 18S rRNA, expressed in decimal format. (*p<0.05).

Figure 5. PDGF mRNA expression in HUVEC and pRPE cells.

PDGF (PDGF-BB) expression of untreated HUVECs (A) and primary RPE cells (B) and after exposure to hypoxia for 24 h, as measured by quantitative RT-PCR. Experiments were carried out in a similar manner as described in Figure 4. After hypoxia, additional treatment with 0.05 µg/mL temsirolimus led to a significantly less increase of PDGF expression. Y-axis: RR of PDGF mRNA normalized to 18S rRNA, expressed in decimal format. (*p<0.05).

Figure 6. Protein levels of VEGF and PDGF in HUVEC and pRPE cells by Western Blot analysis.

Representative Western blots showing the expression of VEGF (VEGF-A) and PDGF (PDGF-BB) in untreated HUVEC and RPE cells (Co) and treated with 0.05 µg/ml of temsirolimus (Tem) after 24 h exposure to normoxic conditions or hypoxia. Ten micrograms of protein were loaded per lane. An even protein load in each lane was confirmed by staining of the polyvinyl difluoride membranes with Coomassie Brilliant Blue after the blotting procedure.

Figure 7. Protein levels of VEGF in HUVEC and pRPE cell supernatants (ELISA).

Inhibitory effect of temsirolimus on VEGF expression, as measured by ELISA from supernatants. After exposure to 0.05 µg/ml temsirolimus for 24 h, a significant decrease in VEGF protein expression of both HUVEC (A) and RPE cells (B) was detected. Each value was normalized to a standard curve of VEGF, and normalized to the untreated control of each cell line. Data are expressed as means ± SD. (*p<0.05).

Figure 8. Protein levels of PDGF in HUVEC and pRPE cell supernatants (ELISA).

Protein levels of PDGF were measured in a similar manner as VEGF expression from supernatants with an ELISA assay. Cells were exposed to 0.05 µg/ml temsirolimus for 24 h. A significant decrease in PDGF protein expression for both HUVEC (A) and RPE cells (B) was detected. Each value was normalized to a standard curve of PDGF, and normalized to the untreated control of each cell line. Data are expressed as means ± SD. (*p<0.05).