Figure 1.
Cathepsin S expression and activity is up-regulated by pro-angiogenic stimuli.
(A) Total RNA was isolated from endothelial cell lines (HUVEC and HMEC-1) and used to assess Cathepsin S mRNA by RT-PCR. GAPDH serves as a control. (B,C) Human recombinant VEGF (10 ng/ml) up-regulates cathepsin S mRNA and protein in HUVECs when assessed by RT-PCR and western blot using GAPDH and tubulin as respective controls (D) Human recombinant VEGF (10 ng/ml) stimulates Cathepsin S-like activity, as assessed by the cleavage of fluorigenic substrate, Cbz-Val-Val-Arg-AMC, by 37% in HUVEC cell lysates. (E,F) To demonstrate the effect of hypoxia on Cathepsin S up-regulation total RNA and protein was isolated from HUVEC endothelial cells (grown in normal or hypoxic conditions) and examined by RT-PCR and western blot. GAPDH and tubulin serves as a control.
Figure 2.
Fsn0503 demonstrates anti-angiogenic effects in vitro.
(A) Fsn0503 attenuates HUVEC invasion using a modified Boyden chamber assay at 500 nM. (B) Fsn0503 (500 nM) inhibits HUVEC degradation of quenched fluorescent substrate DQ gelatin. Cells treated with Fsn0503 or controls were analysed for the presence of fluorescent degradation products by confocal microscopy (representative images shown). (C) A reduction of 70% of DQ gelatin degradation was observed when quantified by mean energy per cell (p = 0.03) (5 fields per assay). (D) Fsn0503 (400 nM) inhibits HUVEC tube formation compared to isotype control (representative images shown). (E) Tube formation was assessed by counting nodes with 1, 2, 3, or more branches and the average calculated per field of view. The number of nodes with 3 or more branches, which are indicative of normal tube formation, were significantly reduced in Fsn0503 treated samples (p<0.01).
Figure 3.
Fsn0503 inhibits angiogenesis in vivo; CD34 Immunohistochemical analysis of Fsn0503 treated tumors.
(A) Pattern of small vessel (white arrows) and large vessel (black arrows) distribution in isotype control and Fsn0503 treated tumors (10 mg/kg 5 times a week for 4 weeks) (20×). (B) Analysis of total vessel number as characterized by CD34 staining shows that Fsn0503 caused an increase in small vessel number and a significant decrease in the number of large vessels (p<0.001). (C) A significant reduction is observed in the mean vessel area of tumours treated with Fsn0503 (p<0.001).
Figure 4.
Fsn0503 in combination with an anti-VEGF antibody synergistically inhibit angiogenesis in vitro.
(A) Representatives tube assay images of VEGF control, anti-VEGF antibody treated, Fsn0503 treated and the combination of anti-VEGF antibody and Fsn0503 which shows the greatest disruption of tube formation. (B, C) Tube formation was assessed by counting nodes with 1, 2, 3, or more branches and the average calculated per field of view. The number of nodes with 3 or more branches, which are indicative of normal tube formation were assessed to demonstrate efficacy. Quantification of tube formation shows the single agents reducing the number of nodes with 3 or more branches and the combination synergistically reducing the number of nodes with 3 or more branches and increasing the number of single branches.