HDAC inhibitors have a biphasic effect on antiangiogenesis by PEDF. (A) HMVECs grown on coverslips in low serum were stimulated with bFGF and treated with PEDF (10nM) and/or HDAC inhibitor vorinostat (SAHA, 0-100μM as indicated) overnight for 16 hours; the cells were counterstained with propidium iodide (red) and apoptosis detected by TUNEL (green). Representative overlay images are shown. Apoptotic ECs appear yellow. 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. (B) The experiment in panel A was quantified using MetaMorph software. Note the apoptotic effect of vorinostat alone at 100μM (P < .001), a dose-dependent enhancement of PEDF apoptotic activity by vorinostat at concentrations up to 30μM, and the inhibition of PEDF-dependent apoptosis by 100μM vorinostat. (C) DIVAA was performed with fixed concentrations of PEDF antiangiogenic peptide (the 34-mer, 100nM) and increasing doses of SAHA. Note similar cooperation between PEDF and 20μM SAHA (P < .005) and the reversal of PEDF angioinhibitory activity by SAHA at high dose (200μM, P = .03). (D) The acetylation of p65 was measured by Western blot with phospho-specific antibodies. Three independent experiments were performed with similar results. The ratio between phospho-p65 and total p65 is shown below.