Figure 7.

Heparan sulfate chain composition is altered by hypoxia. (A,B) RPE cells were maintained under normoxic or hypoxic (1% pO₂) conditions for 72 h. During the last 2.5 h of culture, the cells were treated with or without 0.25 units/mL Heparinase III at 37 °C (hep’ase) prior to fixing. The cells were washed, fixed, blocked, and incubated with anti heparan sulfate antibodies clone 10E4 (A) or clone 3G10 (B) and HRP linked goat anti mouse IgG was used to detect 10E4 and 3G10. The data is expressed as blank corrected absorbance values corrected for cell number ±SEM where N = 4 for each condition. (C) After the hypoxia conditioning period and 2.5 h heparinase III treatment, RPE cells were washed and fluorescently labeled RECs allowed to attach for 1 h, washed, fixed, and counted. Data (4 fields/well; 3 wells/condition; n = 4; N = 3) is expressed as the average number of cells per field ±SD. Statistical analysis revealed that hypoxia significantly increased 10E4 and 3G10 binding to RPE cells (A,B), and that hypoxia increased REC attachment to RPE cells (C).