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Large-Scale Purification of Porcine or Bovine Photoreceptor Outer Segments for Phagocytosis Assays on Retinal Pigment Epithelial Cells
Published on: December 12, 2014
Retinal pigment epithelium exhibits gene expression and phagocytic activity alterations when exposed to
Rodrigo Cerna-Chavez1, Agata Rozanska1, Giulia Lodovica Poretti1
1Biosciences Institute, Newcastle University, UK.
Insights
Chemotherapy for retinoblastoma (Rb) can harm retinal pigment epithelium (RPE) cells. While not directly cytotoxic, these drugs impair RPE barrier function and phagocytosis, potentially impacting vision.
Area of Science:
- Ophthalmology
- Cell Biology
- Developmental Biology
Background:
- Retinoblastoma (Rb) is a pediatric eye cancer.
- Chemotherapeutic agents for Rb can cause retinal pigment epithelium (RPE) damage.
- Developing effective and safe treatments requires understanding drug effects on RPE.
Purpose of the Study:
- To develop and utilize pluripotent stem cell-derived RPE (PSC-RPE) models.
- To assess the cytotoxicity and functional impact of common Rb chemotherapeutics on RPE cells.
- To investigate the molecular mechanisms underlying drug-induced RPE alterations.
Main Methods:
- Generation of two distinct PSC-RPE models.
- Treatment of PSC-RPE models with Melphalan, Topotecan, and TW-37 at clinical concentrations.
- Measurement of trans-epithelial electrical resistance (TEER) to assess barrier integrity.
- Assessment of RPE phagocytic activity.
- Transcriptional analysis (gene expression profiling).
Main Results:
- Chemotherapeutic agents altered RPE barrier function (decreased TEER) and phagocytic activity.
- Transcriptional analysis revealed altered gene expression in melanin/retinol processing, tight junction, and polarity pathways.
- No significant cytotoxic effects, changes in apical-basal polarity, tight junction network, or cell cycle were observed at clinical concentrations.
- In vitro drug application compromised phagocytosis and barrier function, with gene expression changes potentially affecting the visual cycle.
Conclusions:
- Common Rb chemotherapeutics do not induce significant cytotoxicity in RPE cells at clinical doses.
- These drugs can compromise RPE barrier function and phagocytosis, impacting visual health.
- Careful drug delivery is crucial to minimize damage to adjacent healthy RPE during retinoblastoma treatment.
Abstract:
Retinoblastoma (Rb) is a rare malignant disorder affecting the developing retina of children under the age of five. Chemotherapeutic agents used for treating Rb have been associated with defects of the retinal pigment epithelium (RPE), such as hyperplasia, gliosis, and mottling. Herein, we have developed two pluripotent stem cell (PSC)-RPE models to assess the cytotoxicity of known Rb chemotherapeutics such as Melphalan, Topotecan and TW-37. Our findings demonstrate that these drugs alter the RPE by decreasing the monolayer barrier's trans-epithelial resistance and affecting the cells' phagocytic activity. Transcriptional analyses demonstrate an altered expression of genes involved in melanin and retinol processing, tight junction and apical-basal polarity pathways in both models. When applied within the clinical range, none of the drug treatments caused significant cytotoxic effects, changes to the apical-basal polarity, tight junction network or cell cycle. Together, our results demonstrate that although the most commonly used Rb chemotherapeutic drugs do not cause cytotoxicity in RPE, their application in vitro leads to compromised phagocytosis and strength of the barrier function, in addition to changes in gene expression that could alter the visual cycle in vivo. Our data demonstrate that widely used Rb chemotherapeutic drugs can have a deleterious impact on RPE cells and thus great care has to be exercised with regard to their delivery so the adjacent healthy RPE is not damaged during the course of tumor eradication.
