Anti-Proliferative Actions of the mTOR Inhibitor Temsirolimus in Primary Human Retinal Pigmentepithelium (RPE) and Vascular Endothelial Cells

Abstract

Purpose: : The mammalian target of rapamycin (mTOR) plays a central role in cell growth and proliferation. Dysregulation of mTOR has been linked to proliferative retinal disease such as age-related macular degeneration (AMD) and diabetic retinopathy (DR). This study investigates potential anti-proliferative effects of the mTOR inhibitor temsirolimus on primary human retinal pigmentepithelium (RPE) and vascular endothelial cells

Methods: : Human umbilical vein endothelial cells (HUVEC) and RPE cells were incubated for 24 hours with different concentrations of temsirolimus (0.1 to 25µg/mL). Propidium iodide (PI) and Hoechst 33342 staining and tetrazolium dye-reduction assay (MTT) were performed to exclude toxic concentrations. To determine cell proliferation, cells were incubated with temsirolimus at the maximum slope of the growth curve for 24 hours and MTT test was performed. In addition, cell attachment was assessed by the MTT test and migration was determined by a modified Boyden chamber method. 3D-Angiogenesis assay was performed.

Results: : Temsirolimus concentrations from 0.1 to 10µg/mL for RPE, and 0.1 to 5µg/mL for HUVEC showed no effects on cellular viability. At a concentration of 0.5µg/mL, temsirolimus effectively inhibited angiogenesis, proliferation, attachment, and migration of HUVEC and RPE cells. 0,5µg/mL temisirolimus accounted for the following: inhibition of cell proliferation to 41% for HUVEC and 82% for RPE cells, reduction in cell attachment to 63% (HUVEC) and 81% (RPE), and inhibition of cell migration to 41%(HUVEC) and 28% (RPE), compared to the untreated control. All effects were dose dependent. No toxic effects were detected compared with controls.

Conclusions: : In our experimental set-up, temsirolimus showed significant inhibitory action on RPE and HUVEC angiogenesis, proliferation, migration and attachment. Anti-proliferative effects were significant stronger in HUVEC, compared to RPE cells. Our in vitro results provide early evidence that temsirolimus provides promising properties for treatment of proliferative retinal disease.

Keywords: retinal neovascularization • age-related macular degeneration • diabetic retinopathy