Fig. 3

Analysis of antiangiogenic effect of Thalidomide, Lenalidomide, and Pomalidomide at potent, anti-inflammatory concentrations. Effects upon angiogenesis was analyzed, using 24 hpf Fli1:EGFP zebrafish, following incubation for 3 and 24 h with each compound, or DMSO. (A–A2) DMSO control embryos exhibit normal angiogenesis. (B–B2) CPS49 10-μg/mL treatment abolishes blood vessels. (C–C22) Lenalidomide 200 μg/mL (772 μM) treatment consistently showed areas of angiogenic inhibition. (D–D22) Pomalidomide 60 µg/mL (219 μM) treated embryos showed normal angiogenesis and no significant changes in mean stalk length (E) or Mean stalk number (F). To confirm angiogenic sensitivity, HUVEC were plated on ECMatrix and incubated for 18 h. (G–J) Angiogenic growth following compound or DMSO incubation indicates Pomalidomide has no effect as quantified by measuring area of filopodia outgrowth (K). (L–O2) HUVEC were treated with drug or DMSO, then analyzed for α-tubulin (green) and F-actin (red). DNA was visualized with Hoechst dye (blue). HUVEC were treated with 0.1% DMSO, CPS49 10 μg/mL, Lenalidomide 200 μg/mL, or Pomalidomide 60 μg/mL for 4 h, then fixed and stained. (P and Q) Fluorescence intensity (FI) for F-actin and α-tubulin was measured. F-actin was up-regulated following CPS49 treatment and α-tubulin was down-regulated with antiangiogenic drugs. (R) Here, qRT-PCR showed a significant decrease in COX2 expression following Lenalidomide, but not Pomalidomide treatment. (E and F) Statistical significance was analyzed using two-way ANOVA with Bonferroni posttest or (K, P–R) one-way ANOVA with Tukey’s post hoc test; *P < 0.05; **P < 0.005, ***P < 0.0005; ****P < 0.0001; NS, P > 0.05, FI represent fluorescent intensity. (Scale bars: 100 μm.)