Multiple A2E treatments lead to melanization of rod outer segment-challenged ARPE-19 cells.

PURPOSE: Daily phagocytosis of outer segments (OSs) and retinoid recycling by the RPE lead to the accumulation of storage bodies in the RPE containing autofluorescent lipofuscin, which consists of lipids and bisretinoids such as A2E and its oxidation products. Accumulation of A2E and its oxidation products is implicated in the pathogenesis of several retinal degenerative diseases. However, A2E accumulates in the RPE during normal aging. In this study, we used a cell model to determine the homeostatic mechanisms of RPE cells in response to A2E accumulation. METHODS: To distinguish between pathologic and normal responses of the RPE to A2E accumulation, we treated established ARPE-19 cells (cultured for 3 weeks after reaching confluence) with low micromolar amounts of A2E for several weeks. We compared the lysosomal function, lysosomal pH, degree of OS digestion, and melanization of the treated cells to untreated control cells in response to a challenge of purified rod OSs (ROSs). A2E was analyzed with high-performance liquid chromatography (HPLC); and A2E and melanin were identified with mass spectrometry. RESULTS: We found that post-confluent ARPE-19 cells took up and accumulated A2E under dim light conditions. Spectral analysis of the HPLC separations and mass spectrometry showed that A2E-fed cells contained A2E and oxidized A2E (furan-A2E). A2E accumulation led to a modest increase (up to 0.25 unit) in lysosomal pH in these cells. The specific activity of cathepsin D and lysosomal acid phosphatase was reduced in the A2E-treated cells, but ROS degradation was not impaired. We found that, upon challenge with ROSs, melanin pigment was induced in the lysosomal fraction of the A2E-treated ARPE-19 cells. Thus, the ARPE-19 cells responded to the A2E treatment and ROS challenge by producing a melanin-containing lysosome fraction. We speculate that this prevents them from becoming impaired in OS processing. CONCLUSIONS: We used a modified ARPE-19 cell model in which melanization was elicited as a response to chronic accumulation of A2E. We found that although A2E treatment led, as has been previously reported, to modest lysosomal alkalinization and lysosomal impairment of ARPE-19 cells, a potential homeostatic mechanism may involve production of a special type of lysosomes containing melanin.

SU5416 induces premature senescence in endothelial progenitor cells from patients with age-related macular degeneration.

PURPOSE: We recently demonstrated increased frequency and growth potential of late outgrowth endothelial progenitor cells (OECs) in patients with neovascular age-related macular degeneration (nvAMD). This study investigated the effects of short- and long-term in vitro inhibition of vascular endothelial growth factor (VEGF) Receptor-2 (VEGFR-2) signaling by SU5416 and other inhibitors of the VEGF signaling pathway in OECs. METHODS: OECs, from the peripheral blood of patients with nvAMD, and human umbilical vein endothelial cells were grown in the presence of SU5416, other VEGFR-2 tyrosine kinase inhibitors (TKIs), and inhibitors of phosphatidylinositol 3'-Kinase (PI3K)/protein kinase B (Akt) and protein kinase C (PKC) in complete angiogenic medium. Apotosis was assessed after 48 h using the fluorescein isothiocyanate Annexin V method. Cell counts were performed for 10 days, and features of senescence were analyzed using senescence-associated ß-galactosidase staining, the telomeric repeat amplification protocol for telomerase activity, Southern blot analysis for mean telomere length, flow cytometric analysis for cell-cycle arrest, and western blot for p53 and p21. Control OECs, cells treated for 7 days with inhibitors, as well as naturally senescent OECs were analyzed for expression of different endothelial antigens, including VEGFR-2 and the receptor for stromal cell-derived factor 1, chemokine receptor 4 (CXCR-4). Migration in vitro to VEGF and stromal cell-derived factor 1 of OECs was assessed. RESULTS: SU5416, other VEGFR-2 TKIs, and inhibitors of PI3K, Akt, and PKC induced apoptosis, inhibited long-term proliferation, reduced telomerase activity, and induced premature senescence and cell-cycle arrest in OECs as well as in human umbilical vein endothelial cells. Naturally senescent cells and cells rendered senescent by VEGFR-2 TKIs had reduced VEGFR-2 and CXCR-4 expression and demonstrated reduced migratory ability to VEGF. CONCLUSIONS: This study demonstrates apoptosis upon short-term inhibition and inhibition of long-term survival of OECs from patients with nvAMD by SU5416, presumably via PI3K/Akt and/or PKC-mediated reduction in telomerase activity and subsequent induction of premature senescence, which is accompanied by impaired endothelial activity. Therefore, induction of premature senescence in endothelial cells may represent a potential therapeutic target in nvAMD.

Late outgrowth endothelial progenitor cells in patients with age-related macular degeneration.

PURPOSE: To evaluate the feasibility of isolating and expanding endothelial progenitor cells (EPCs), in the form of late outgrowth endothelial progenitor cells (OECs), from the peripheral blood of an aged population, particularly patients affected by different forms of AMD. METHODS: Peripheral blood mononuclear cells were collected from young control subjects (n = 18) and from elderly subjects with non-AMD/low-risk dry AMD (n = 15), high-risk dry AMD (n = 6), or neovascular AMD (nvAMD; n = 32); cultured in established conditions; and observed for appearance of OEC clusters and growth characteristics on expansion. Expression of VEGF receptor-2 (KDR) in OECs after expansion was determined by Western blot. Plasma samples of study subjects were analyzed for CRP and VEGF levels. RESULTS: OEC cultures were successfully generated from a similar number of subjects in each group. After adjustment for all other variables, subjects with high-risk dry AMD had a 5.6-fold higher number of OEC clusters per 20 mL blood, and subjects with nvAMD had a 5.1-fold high number than did subjects with non-AMD/low-risk dry AMD (P < 0.05). High-risk dry AMD generated 63 times more (NS) and nvAMD 32-times more (P < 0.05) OECs on expansion of clusters than did non-AMD/low-risk dry AMD. Population doubling occurred significantly faster in cultures from nvAMD eyes compared to non-AMD/low-risk dry AMD eyes. In addition, a significant correlation between the number of OEC clusters, expanded OECs and levels of KDR was demonstrated. CONCLUSIONS: An OEC population was isolated and expanded from the blood of elderly control and AMD-affected patients and demonstrated significantly higher number of initial OEC clusters and expansion potential of OECs in patients at risk for or already affected by nvAMD. OECs may be used for further phenotypic, genetic, and functional analyses in patients with nvAMD.