Melphalan-Induced Injury in Human Primary Retinal Endothelial Cells: A Novel Flow-Based Investigation

Abstract

Purpose: : Super-selective intra-ophthalmic artery chemotherapy (SSIOAC) is an organ-specific drug delivery strategy used to treat retinoblastoma, the most common primary intraocular malignancy in children. In this study we constructed an in vitro model which mimics SSIOAC under physiological shear stress conditions to investigate mechanisms of melphalan-induced leukocyte-endothelial cell adhesion.

Methods: : Human primary retinal endothelial cells (REC) were grown to confluence on glass slides. In a parallel-plate flow chamber with a continuous flow-loop and loaded with REC slides (shear stress of 2.0 dyne/cm2), U937 human monocytes were perfused through the chamber in RPMI 1640 medium containing 10% serum. After two hours, melphalan (4µg/mL) was added to the perfusate. Phase contrast images and digital video was collected over 4 hours. Adhesion (leukocytes remaining stationary for > 3s) was monitored over 8 fields of view. Confocal microscopy of fixed vehicle-control and melphalan-treated REC slides was performed using P-selectin and ICAM-1 cell-surface antibodies.

Results: : Negligible leukocyte adhesion was observed when U937 cells were perfused over untreated RECs. Leukocyte adhesion significantly increased within 30 minutes of melphalan injection (p < 0.05). Adhesion was linked to increased expression of P-selectin and ICAM-1.

Conclusions: : Melphalan-induced damage correlates with previous in vivo studies and clinical reports detailing a devastating insult to the retinal vasculature. We have shown significant up-regulation in leukocyte adhesion, a precursor to the irreversible loss of functional vessels, following melphalan exposure within the retina, correlating with increased ICAM-1 and P-selectin levels. Taken together, this data suggests that melphalan induces flow-based damage to the retina.

Keywords: retinoblastoma • pathology: experimental • drug toxicity/drug effects