Human RPE cells were isolated from fetal donor eyes (gestation time over 22 weeks) which were obtained from the Anatomic Gift foundation (Woodbine, GA) as has been described 25. RPE cells were seeded onto laminin-coated 6 well plates (Fisher Scientific, Tustin, CA) using DMEM with 10 % fetal bovine serum, 1 % penicillin/streptomycin and glutamine. Cells from passage 3 to 5 were used in all experiments.

The cyclic integrin antagonist RGDfV, specific for the inhibition of integrins α_vβ₃ and α_vβ₅ 18192021 and the control peptide RADfV were synthesized as described 6 at the USC/ Norris Cancer Peptide synthesis facility. The peptides were dissolved in Hank's Balanced Salt Solution (HBSS), and stored at -80°C. HPLC analysis revealed a purity > 99% of the synthesized peptide.

RPE cells (30,000 cells/well) were treated for 5 days with the cytokines bFGF and PDGF-BB at a concentration of 10 ng/ml in 6 well plates. Cells were detached, fixed with 4 % paraformaldehyde, blocked with 5 % goat serum for 30 minutes, centrifuged at 4°C, and incubated with a monoclonal mouse antibody against α_vβ₃ or α_vβ₅ (5 μg/ml) (Chemicon, CA) for one hour. After washing, a fluorescein conjugated secondary antibody (Chemicon, CA) directed against the mouse antibodies was added in a dilution of 1:100 for one hour. The expression of the integrin receptors α_vβ₃ or α_vβ₅ was measured using a fluorescent-activated cell sorter (FACStar plus, Becton Dickinson, Mountain View, CA). Forward and side light scatter was used to gate the desired scattered events (RPE cells) from dead cells and debris. The fluorescent index was determined by multiplying the percentage of cells by their mean fluorescence. At least 10,000 cells were evaluated/experiment and all experiments were performed in triplicate.

Subconfluent, bFGF (10 ng/ml) or PDGF-BB (10 ng/ml) pre-stimulated RPE cells were seeded at a density of 8 × 10⁴ cells per well in 6 well plates containing DMEM with 10 % FBS and the cyclic integrin antagonist or the control peptide (0.01 μg/ml, 0.1 μg/ml, 1 μg/ml, 30 μg/ml and 300 μg/ml). The cells were exposed during the proliferation assay to 10% FBS or the cytokines bFGF and PDGF-BB (10 ng/ml). After 3 days of culture, the cell number was determined using a hemocytometer. The experiments were performed six times, each in triplicate. Cell viability was estimated by observation of cell morphology and by exclusion of 0.4 % trypan blue solution 26.

RPE cells (10,000 cells/well) were seeded into DMEM with 10 % FBS in 24 well plates with the cyclic integrin antagonist or the control peptide (0.01 μg/ml, 0.1 μg/ml, 1 μg/ml, 30 μg/ml and 300 μg/ml). To determine a possible stronger effect of the cyclic integrin antagonist on RPE cell proliferation, cells were pre-stimulated for 5 days with bFGF (10 ng/ml) or PDGF-BB (10 ng/ml) (R&D Systems, Minneapolis, MN) to induce an upregulation of the integrin receptors α_vβ₃ or α_v β 5. After the pre-incubation, cells were seeded in DMEM with 10 % FBS, with the cyclic integrin antagonist or control peptide, and bFGF (10 ng/ml) or PDGF-BB (10 ng/ml). Cells were treated for three days with the peptides, then ³H-thymidine uptake was measured as described previously 25. Results are presented as the percentage of control values.

Migration was performed in a modified Boyden chamber (Falcon, Becton Dickinson Labware, Franklin Lakes, NJ). Subconfluent RPE cells, which have been pre-exposed to the cyclic integrin antagonist peptide (0.001 ng/ml, 0.01 ng/ml, 0.1 ng/ml, 1 ng/ml, 3 ng/ml and 10 ng/ml) or the control peptide (10 μg/ml) over night were seeded into DMEM with 1 % FBS into the upper part of a Boyden chamber (50,000 cells/chamber). The cyclic integrin antagonist was added directly to the upper part of the chamber (0.001 ng/ml, 0.01 ng/ml, 0.1 ng/ml, 1 ng/ml, 3 ng/ml, or 10 ng/ml). To evaluate nonspecific activity of the integrin antagonist, a control peptide (10 μg/ml) was used. The lower part of the chamber was filled with DMEM with 1 % FBS containing the chemoattractant PDGF-BB (10 ng/ml) or the ECM molecule fibronectin (30 ng/ml). RPE cells were incubated for 8 hours in a humidified CO₂-incubator. Then the cells in the upper part were scraped away and the membrane was fixed with 1/2 strength Karnovsky's solution and stained with 0.25 % Richardson's solution. RPE cells on the lower side of the membrane were counted with the help of an inverted microscope and a grid (Carl Zeiss, Jena, Germany) in five random high power fields (200 X) in triplicate.

Ninety-six well plates were coated with the ECM molecules laminin, fibronectin or collagen IV (25 μg/ml). RPE cells were pre-incubated with the cyclic integrin antagonist (concentrations as above) or control peptide (10 μg/ml) over night. Then 15 × 10³ cells, suspended in 100 μl DMEM with the cyclic or control peptide were seeded in each well and allowed to attach for 60 minutes. A tetrazolium MTT [3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide] solution (20 μl/well) (Boehringer Mannheim, Indianapolis, IN, USA) was added for 5 hours of incubation. Then the supernatants were decanted, 150 μl of 100% DMSO added, and the cells were placed on a shaker for 10 minutes. Absorbance of the cells was measured at 550 nm with a Dynatech MR 600 spectrophotometric microplate reader.

RPE cell invasion was examined in a modified Boyden chambers. The insert membrane was coated overnight with fibronectin (25 μg/ml). Subconfluent RPE cells were pretreated overnight with the cyclic integrin antagonist (3 μg/ml) or the control peptide (3 μg/ml). An RPE cell suspension (50,000 cells/0.1 ml) in DMEM with 0.4 % FBS and 3 μg/ml cyclic integrin antagonist or control peptide was added into the upper part of the chamber. 600 μl DMEM with 0.4% FBS and PDGF-BB (10 μg/ml) was added into the lower part of the chamber. Invasion was measured with and without PDGF-BB stimulation after 5 hours at 37°C (95 % air/ 5 % Co₂). After PBS washing and methanol fixation (10 minutes at 4°C), counterstaining with hematoxylin was performed. Cells in the upper chamber were wiped away. RPE cells that invaded the lower surface of the membrane were quantified by cell counting (320 × magnification).

All experiments were repeated in triplicate. Standard deviations and averages were calculated. The data obtained were analyzed for significance using one way analysis of variance (ANOVA). Data with a p-value of less then 0.05 were considered to be statistically significant.

The RPE cells were stimulated to incorporate thymidine by either 10% serum or by 10 ng/ml bFGF or 10 ng/ml PDGF-BB (p < 0.01). Neither the integrin antagonist, nor control peptide showed any significant effect on serum, PDGF-BB or bFGF stimulated DNA synthesis. (data not shown). In addition, cell counting showed no effects of the integrin antagonist or the control peptide on serum or cytokine stimulated proliferation. Furthermore, a lack of toxicity for the integrin antagonist and the control peptide in the concentration range tested (0.01 μg/ml, 0.1 μg/ml, 1 μg/ml, 30 μg/ml and 300 μg/ml) was determined by use of the trypan blue exclusion assay (data not shown).

Pre-stimulation of RPE cells for 5 days with the cytokines PDGF-BB and bFGF (10 ng/ml) increased the expression of the integrin receptors α_vβ₃ and α_vβ₅ (Fig. 1; representative flow cytometric study). Incubation with 10 ng/ml bFGF upregulated the expression of α_vβ₃ (1.9 fold) and strongly enhanced the expression of α_vβ₅ (2.9 fold). PDGF-BB (10 ng/ml) strongly enhanced α_vβ₃ (2.3 fold), and less strongly upregulated surface expression of α_vβ₅ (1.5 fold).