Abstract
Purpose: :
Dendritic cells (DCs) are innate immune cells that have recently been shown to support angiogenesis in tumors, endometriosis and lymph nodes. A major cause of legal blindness is Age-Related Macular Degeneration (ARMD) wherein abnormal blood vessels grow under the retina, known as choroidal neovascularization (CNV). The purpose of this study was to investigate the role of DCs in the development of CNV.
Methods: :
Laser photocoagulation was used to induce CNV in C57BL/6J mice. We analyzed CNV lesions for the presence of DCs using flow cytometry and immunostaining at designated time points. Furthermore, we analyzed the effects of intravenous bone marrow derived dendritic cell (BMDC) transplantation on CNV development by measuring the lesion area 1 week after laser injury. BMDCs were grown by subjecting mouse BM to GM-CSF (10 ng/ml) for 8 days in culture, after which DCs were collected for transplantation.
Results: :
Analysis of DCs infiltrating the CNV legions by flow cytometry, revealed that without laser injury, ocular-infiltrating CD11c+ MHCII+ DCs were rarely detected (day 0, 0.43%). However, following laser injury, they were clearly detected (day 1-7). The number of infiltrating DCs increased and peaked at 2-4 days (14-16%) and gradually decreased thereafter (day 7, 6.52%). Immunostaining of the CNV lesions confirmed that DCs were located at the sites of newly formed blood vessels. We next tested the effect of DC transplantation on the growth of the CNV. We injected 1 x 106 chloromethylbenzamido (CM-DiI) labeled DC intravenously every 48 hours (0, 2, 4 days) immediately after laser. On day 7, we stained the CNVs and observed that labeled BMDCs incorporated into the CNVs and that CNVs were larger following the incorporation of BMDCs when compared with CNVs following injection of vehicle. To demonstrate a direct role for DCs in angiogenesis, we monitored CNV growth after transplantation. Starting immediately after laser injury, 1 x 106 BMDCs, PBS or splenocytes were injected intravenously every 48 hours (0, 2, 4 days). On day 7 mice were euthanized and choroids were removed and analyzed. BMDC injection led to a ~2 fold increase in CNV size (P<0.001) as compared to PBS injection, while splenocyte injection did not increase CNV size.
Conclusions: :
The pathogenesis of ARMD remains unresolved, however, our results indicate a role for DCs in promoting angiogenesis and lesion growth in laser-induced CNV. Since the infiltration of DCs results in increased choroidal neovascularization, therapies designed to inhibit DC infiltration or function should be studied as possible treatments for ARMD.
Keywords: choroid: neovascularization • age-related macular degeneration • laser