Figure 7
Increased induction of endothelial cell tube formation by CM of RPE cells expressing S179C-TIMP3 is mediated by bFGF. (a) Parental porcine aortic endothelial cells (Parental PAE, top panel) and PAE expressing FGF receptor-1 (PAE/FGFR-1, 2nd panel) were embedded in three-dimension (3D) collagen gels and incubated with conditioned medium (CM) of RPE cells expressing empty vector (V), wild-type TIMP3 (W1) or S179C-TIMP3 (M1, M5) in the presence of 10 μg/ml anti-bFGF neutralizing antibody (third panel) or a MMP2/9 inhibitor (SB-3CT, 5 μg/ml) for 48 hours. Differentiated cells were photographed using an inverted microscope (original magnification 200x) and (b) quantitated using customized macros generated in Image-Pro Plus 5.1. Data are presented as means ± SD n = 3. *p ≤ 0.05 vs vector control **p ≤ 0.01 vs vector control. (c) RF6A monkey choroid endothelial cells were subjected to tube formation assay in the presence of conditioned medium (CM) of RPE cells expressing empty vector (V), wild-type TIMP3 (W1) or S179C-TIMP3 (M1, M5) in the presence of anti-bFGF or SB-3CT (lower panel) and compared to the absence of inhibitors (upper panel). (d) Migration of RF6A choroidal endothelial cells towards conditioned medium of RPE cells expressing empty vector (V), wild-type TIMP3 (W1) or S179C-TIMP3 (M1, M5). Migrating cell number is expressed as mean +/− SD of quadruplicate samples.
