Intra-ophthalmic artery chemotherapy triggers vascular toxicity through endothelial cell inflammation and leukostasis

Abstract

Purpose. Super-selective intra-ophthalmic artery chemotherapy (SSIOAC) is an eye-targeted drug-delivery strategy to treat retinoblastoma, the most prevalent primary ocular malignancy in children. Unfortunately, recent clinical reports associate adverse vascular toxicities with SSIOAC using melphalan, the most commonly used chemotherapeutic. Methods. To explore reasons for the unexpected vascular toxicities, we examined the effects of melphalan, as well as carboplatin (another chemotherapeutic used with retinoblastoma), in vitro using primary human retinal endothelial cells, and in vivo using a non-human primate model, which allowed us to monitor the retina in real time during SSIOAC. Results. Both melphalan and carboplatin triggered human retinal endothelial cell migration, proliferation, apoptosis, and increased expression of adhesion proteins intracellullar adhesion molecule-1 [ICAM-1] and soluble chemotactic factors (IL-8). Melphalan increased monocytic adhesion to human retinal endothelial cells. Consistent with these in vitro findings, histopathology showed vessel wall endothelial cell changes, leukostasis, and vessel occlusion. Conclusions. These results reflect a direct interaction of chemotherapeutic drugs with both the vascular endothelium and monocytes. The vascular toxicity may be related to the pH, the pulsatile delivery, or the chemotherapeutic drugs used. Our long-term goal is to determine if changes in the drug of choice and/or delivery procedures will decrease vascular toxicity and lead to better eye-targeted treatment strategies.

Figure 1.

The mean (percentage of control) and SEM of retinal endothelial cells either left untreated (control) or treated with escalating doses (n = 3) of melphalan (A–C) or carboplatin (D–F). Cell death increased with increasing dose (A, D). For melphalan, migration increased with increasing dose (B), and proliferation was greatest at 0.4 μg/mL (C). Migration and proliferation decreased with increasing dose for carboplatin (E, F). *P < 0.05 versus control.

Figure 2.

Increased production of adhesion genes IL-8 (in pg/mL) and ICAM-1 (in ng/mL) after treatment with 4 μg/mL melphalan for 24 hours (A, B) or 1 mM carboplatin for 24 hours (C, D). *P < 0.05 versus control (NT). NT, not treated.

Figure 3.

Flow chamber adhesion analysis. Monocytes perfused over retinal endothelial cell monolayer were analyzed over 120 minutes in the absence (light red) or presence (dark red) of melphalan (4 μg/mL). Data in 30-minute increments are expressed as the mean number of adhering cells/field ± SD for at least four different fields of view. Adhesion was significantly increased after melphalan exposure compared with control. *P < 0.05; **P < 0.005.

Figure 4.

Representative images of control retinal endothelial cells with no treatments: (A) DAPI nuclear stain, (B) P-selectin, (C) ICAM-1, (D) overlay. Representative images of retinal endothelial cells treated with melphalan (4 μg/mL) for 2 hours: (E) DAPI nuclear stain, (F) P-selectin, (G) ICAM-1, and (H) overlay.

Figure 5.

Serial sections of the same macular blood vessels in the ganglion cell layer (nasal to temporal) showing a progression of smudging of the endothelial cells and their basement membrane (A), with associated leukostasis (B), and terminal vessel occlusion (C).