PEDF increases FasL expression by activating chromatin at the FasL promoter. (A) Quantitative PCR showing a 6-fold increase in FasL mRNA in ECs treated with bFGF plus PEDF (P < .001) reversed by BMS-345541 (P < .031). (B) Mice were injected with Matrigel plugs containing bFGF, 34-mer peptide, and BMS-345541 where indicated. Sectioned plugs were stained for CD31 (red) and FasL (green). Colocalization of red and green fluorescence appears yellow. (C) Digital images (2 or 3 sections per treatment condition, 3 or 4 fields per section) were quantified using MetaMorph software. The percentages of the FasL-positive ECs (yellow) of the total ECs (red and yellow) were calculated. *Significantly different value from bFGF alone (P < .001). (D) Mice were injected with Matrigel containing control, noninfected HMVECs (NI), or HMVECS infected with adenoviral IκB super-repressor (SR) or β-Gal (AdC). Angiogenesis was induced with VEGF (200 ng/mL) and blocked with 100nM PEDF 34-mer peptide; 5-μm sections were stained for CD31 (red) and FasL (green) as in panel B. Images were viewed with a Nikon Eclipse TE 2000-U microscope with a 10×/0.45 objective and Fluoromount G imaging medium (Southern Biotech), and captured with a Nikon LH-M100 C-1 camera. Metamorph, Adobe Photoshop CS3 Extended Version 10.01, and Corel Photo-Paint 10 software were used. (E) TSP1 and PEDF increased NF-κB recruitment to the FasL promoter: ChIP was performed with p65 antibody, followed by PCR with primers for the FasL promoter fragment encompassing the κB consensus sequence. (F-G) PEDF increased HAT recruitment and histone acetylation at the FasL promoter: ChIP with the antibodies for AcH3 (F) and p300 (G) followed by quantitative PCR for the same fragment of FasL promoter. Note significant increases in AcH3 (P < .01) and p300 (P = .04) in the presence of PEDF. Both are reversed by the IKK inhibitor, BMS-345541 (P < .025, in both cases).