Article Figures & Data
Figures
- Fig. 1.
Treatment with VEGF increases the number of cells and the level of rhodopsin protein in the retinal cultures. Dissociated retinal cells were prepared from P1 animals, plated at a density of 425 cells/mm2, and treated with VEGF-2 (A, B) or VEGF-1 (C, D). After 2 d (open squares), 5 d (filledsquares), 7 d (open circles), or 9 d (filled circles) the total number of cells in the cultures was estimated by measuring the calcein emission. Then the cultures were fixed, and the levels of rhodopsin protein were quantitated by ELISA and expressed in terms of optical density. The experiment that is shown is representative of three to four independent experiments. The data points are the mean of five to six determinations ± SEM.
- Fig. 2.
Photomicrographs of immunohistochemically stained retinal cultures. The cultures were derived from P1 animals and maintained in vitro for a period of 8 d in the presence of 100 ng/ml of VEGF-2. Then the cells were fixed and immunohistochemically stained for rhodopsin (A–C) or syntaxin (D–F). Scale bars: A, D, 25 μm; B, C, E, F, 50 μm.
- Fig. 3.
The number of rhodopsin-immunopositive cells is increased after treatment with VEGF. The retinal cells were maintainedin vitro for 8 d in the presence of either VEGF-1 or VEGF-2. Then the cultures were fixed and immunohistochemically stained for rhodopsin. The experiment that is shown is representative of the three experiments that were conducted, and the data points are the mean of five to six determinations ± SEM.
- Fig. 4.
Treatment with VEGF increases the number of BrdU-immunopositive cells and [3H]thymidine incorporation in retinal cultures. The cells were isolated from P1 animals and plated at a density of 425 cells/mm2. The cultures were treated initially at 4 hr after plating with either VEGF-2 (A) or VEGF-1 (B). After 1, 2, or 3 d the cultures were labeled for 4 hr with BrdU. Then the cells were fixed and processed for BrdU immunohistochemistry.C, The cultures were treated with the factors as described above for the BrdU labeling. After the appropriate time interval the cells were labeled with [3H]thymidine for 6 hr. The experiment shown is representative of the three experiments that were conducted, and the data points are the mean of five to six determinations ± SEM.
- Fig. 5.
Delaying the addition of VEGF-2 to retinal cultures results in a time-dependent decrease of the VEGF-induced response. The retinal cultures were prepared as previously described in Figure 1. One set of cultures was treated initially with factors at 4 hr after plating (9/0); subsequently, additional sets were treated after 24 or 48 hr (8/1 or7/2, respectively). After 9 d in culture the cells were fixed, and the level of rhodopsin protein was quantitated by ELISA assay.
- Fig. 6.
VEGF treatment increases the number of amacrine cells and the level of high-affinity GABA uptake, but not the level of GFAP protein. Retinal cells were treated for 8 d with the indicated concentrations of VEGF-2. Then the cells were fixed, immunohistochemically stained for syntaxin, and counted (A), analyzed for the level of high-affinity GABA uptake (B), or analyzed for GFAP protein (C). The data that are shown are representative of three to four experiments and are the mean of five to six determinations ± SEM.
- Fig. 7.
Effect of developmental age on the response of retinal cells to VEGF. Retinal cells derived from E15 (A), E20 (B), or P1 (C) animals were plated at a density of 212 (open squares), 318 (filled squares), or 425 cells/mm2 (open circles). At 4 hr after plating the cultures were treated with the indicated concentrations of VEGF-2. After 24 hr the cultures were switched to serum-free medium, and the factors were added again. Then the cultures were labeled with [3H]thymidine after 24 hr. The data points are the mean of five to six determinations ± SEM. At the density of 212, 318, or 425 cells/mm2 the basal values (dpm/well) for [3H]thymidine incorporation at E15 were 1599.7 ± 95.3, 4133.9 ± 409.2, and 6315.6 ± 154.7; at E20 they had risen to 3361 ± 192.8, 4936 ± 271.9, and 6377.5 ± 313.9, respectively. At P1 the basal values were 479.7 ± 33.3, 754.7 ± 70, and 744.7 ± 11.7, respectively.
- Fig. 8.
The response profile of P1 retinal cells to VEGF-2 as compared with EGF, FGF-2, or TGFβ-1. The cultures were seeded at a density of 425 cells/mm2 and treated for 9 d.A, The total number of cells in the cultures was estimated by using calcein. B, The level of rhodopsin protein was determined by ELISA assay. The reagent blanks in this experiment had an optical density of 0.1. The experiment that is shown is representative of the four independent experiments that were conducted; the data points are the mean of five to six determinations.
- Fig. 9.
CNTF inhibits the VEGF-induced increase in rhodopsin, but not the early proliferative response. Retinal cultures were treated 24 hr after plating with the indicated concentrations of CNTF in the presence or absence of 150 ng/ml of VEGF-2. After 8 din vitro the amount of rhodopsin was quantitated (A), and the total number of cells in the cultures was determined (B). C, To determine the effect of CNTF treatment on the early proliferative response induced by VEGF, we treated the cultures with the indicated concentrations of VEGF-2 in the presence or absence of 100 ng/ml of CNTF. After 48 hr the cultures were labeled for 6 hr with [3H]thymidine.
Tables
- Table 1.
Retinal cell survival in the presence or absence of VEGF-2 during the first 48 hr in culture
Time (hr) Treatment Vehicle VEGF-2 0 1272.0 ± 32.6 1195.0 ± 43.4 6 1207.3 ± 55.8 1294.8 ± 37.1 18 1678.0 ± 61.1 1789.3 ± 64.2 24 1728.2 ± 53.1 1831.4 ± 44.2 48 2061.2 ± 58.7 2395.2 ± 74.7 The retinal cells were plated at a density of 425 cells/mm2 and treated with VEGF-2 at the time of plating. The level of calcein emission was determined at time zero by plating the cells in Ham's F-12 in the presence of calcein. For the later time points, the emission levels were determined as described in Materials and Methods. The data points are the mean of five or six determinations ± SEM.
