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Matthew W. Wilson, Qiuhua Zhang, Jena J. Steinle; Exposure of Human Retinal Endothelial Cells to High Dose Chemotherapy Upregulates Inflammation and Chemotaxis. Invest. Ophthalmol. Vis. Sci. 2011;52(14):6653.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate apoptotic, migratory, and proliferative capabilities of a number of key chemotherapy agents, including melphalan, carboplatin, nutlin, topotecan, etopostide, and vincristine in retinal endothelial cells.
Human retinal vascular endothelial cells were grown in high glucose medium (25mM). Once cells reached confluence, they were tested in 3 assays of cellular survival, apoptotic cell death (Roche Cell Death ELISA), cell migration (BD Science Angiogenic Assay) and cell proliferation (Millipore MTT assay). Cells were treated with varying doses of melphalan, carboplatin, nutlin, topotecan, etoposide, and vincristine in each assay according to manufacturer’s instructions. In additional experiments, retinal endothelial cells were grown in high glucose and then treated with melphalan for 24 hours and collected for RNA analyses via microarray or protein analyses via ELISA.
Melphalan induced a 6-fold increase in cell death over a 24-hour period at the 4ug/ml dose. Interestingly, surviving cells were highly migratory (increased migration of 41%) and proliferative. Carboplatin also increased cell death by 4-fold at 1mM but did little to increase cell migration at that dose. Nutlin increased cell death at a stimulation of greater than 1uM; however, nutlin had lesser proliferative and migratory actions than melphalan. Etoposide, vincristine, and topotecan had modest actions in all assays in the retinal endothelial cells. In microarray data, melphalan significantly increased levels of 55 genes, including IL-8, ICAM1, and a number of interferon gamma-related proteins. ELISA analyses for IL-8 and ICAM1 supported the microarray findings.
At the dose currently used for retinoblastoma therapies, melphalan caused a 6-fold increase in cell death. Surviving cells were more angiogenic, suggesting that they were trying to repair damage. Microarray and ELISA analyses showed increased expression of key chemotactic and inflammatory genes and proteins. Both carboplatin and nutlin were effective in cell death as chemotherapy agents; however, lesser angiogenic actions were observed. Our results suggest that in vivo doses below 4ug/ml of melphalan should be used to preserve retinal vasculature in children with retinoblastoma treated with super-selecetive intra-ophthalmic artery chemotherapy. Further pharmacokinetic studies are needed.
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