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Scott Cousins, Priyatham Mettu, Tiffany Pridgen, Sara Crowell, M. Grazia Spiga; Circulating Mesenchymal Progenitor Cells in Patients with Neovascular Age-Related Macular Degeneration. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3663.
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To characterize circulating vascular progenitor cells (VPCs) from the blood of subjects with neovascular age-related macular degeneration (NVAMD). Previously our group has shown that bone marrow-derived mesenchymal progenitor cells (MPCs) regulate the severity and vascular maturation of laser-induced choroidal neovascularization (CNV) in mice.
Peripheral blood mononuclear cells were isolated from subjects with NVAMD (n=40) and from young, healthy subjects (n=20) and were cultured on a fibronectin/Matrigel substrate supplemented with specific mesenchymal growth factors, using EGM2-MV growth medium. Late-outgrowth colonies appearing after day 7 were characterized until day 30 (passage zero or P0) by number of colonies, morphologic appearance, and population doubling time. Endothelial progenitor cell (EPC) and MPC markers were analyzed in P0, P3, and P5 samples via quantitative RT-PCR and immunofluorescence. In vitro angiogenesis tube assay was used with VPCs co-cultured with HUVEC on Matrigel to assess the functional contribution of VPCs in tube formation and stability for up to 7 days. In NVAMD subjects, VPC culture markers were correlated with CNV morphologic subtypes determined by indocyanine green angiography.
Late-outgrowth colonies were successfully isolated from 35 of 40 NVAMD subjects and from 16 of 20 young, healthy subjects. P0 cultures demonstrated a mixed population of cells in a 90:10 MPC:EPC distribution. P0 MPCs were characterized as CD34+, PDGFR-β+, NG2+, and SMA+ (low), while EPCs were characterized as CD34+ and vWF+. Multiple passages of VPCs beyond P0 demonstrated changing patterns of gene and marker expression with each subsequent passage, especially change in MPC and EPC marker distribution, suggesting culture-induced artifact. Co-culture of VPCs with HUVECs in the tube assay extended the duration of tube stability as compared to HUVECs alone (7 vs. 2 days). P0 VPCs isolated from NVAMD patients with CNV manifesting mature branching arterioles appeared to demonstrate greater proportion of NG2+ (pericyte-like) MPCs as compared to patients with immature vessels or young, healthy controls.
Circulating MPCs can be isolated from the peripheral blood of patients with NVAMD. Isolation of P0 cells with high proportion of NG2 positivity may identify a subset of subjects prone to develop mature CNV with feeder vessels and branching arterioles.
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