Inhibition by apoEdp of VEGF-induced Flk-1.

<p>HUVECs were pre-treated with apoEdp at the indicated concentrations for 1 hr; VEGF (10 ng/mL) added and incubated at 37°C for 10 min, to measure the phosphorylation of Flk-1(7-A), c-Src(7-B), Akt(7-C), eNOS(7-D), FAK(7-E), and Erk1/2(7-F). Equal loading of total proteins was also measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015905#s4" target="_blank">Materials and Methods</a>. Values were normalized by arbitrarily setting the densitometry of control cell signals 1.0 (mean ± SEM, n = 5). The asterisks indicate significant differences compared with VEGF stimulation (*p<0.05, **p<0.01 and ***p<0.001).</p

ApoEdp inhibits angiogenesis in rabbit corneal micropocket assay.

<p>Slow-releasing micro pellets (500 µm×500 µm) containing 160 ng of VEGF were corneally implanted in New Zealand white rabbits (1.5–2.5 kg). The pellets were positioned about 2.0 mm from the corneal limbus. Treatment began one day post-implantation (PI) and continued for five consecutive days. The right eye of all 5 rabbits was treated using 1% peptide apoEdp, and left eye of the same rabbit received saline drops as mock treatment. Each eye drop (50 µL) was applied topically 5 times per day every 2 hr starting at 8 AM and ending at 4 PM. Data and photos were obtained on days 3 through 10 after pellet implantation. (A)The area of neovascular response, vessel length, and clock hours of new blood vessel of rabbits in each group were calculated according to the formula Area (mm²)  = C/12×3.1416 [r²−(r−L)²] where C  =  the number of clock hours at the limbus involved in the neovascular response, L  =  length of the longest neovascular pedicle from the limbus onto anterior cornea, and r  =  radius of the cornea. (B). ApoEdp significan.tly inhibited VEGF-induced angiogenesis in rabbit corneal micro pocket assay. **p<0.01 and ***p<0.001.</p

ApoEdp affects HUVEC survival but not MDA-MB-231.

<p><b>A. HUVEC</b>: ApoEdp has a significant effect on VEGF-induced HUVEC viability. Cells were treated with apoEdp at the indicated concentrations and incubated with constant 50 ng/mL VEGF for 48 hr. All values were normalized to cell control of no VEGF and no apoEdp. Cell viability values (mean ± SEM) are expressed as % of the value of the VEGF control. <b>B. MDA-MB-231</b>: ApoEdp has no effect on MDA-MB-231viability. ApoEdp has no significant effect on MDA-MB-231 survival. Cells were treated with apoEdp at the indicated concentrations and incubated at 37°C for 2 days. Cell viability values (mean ± SEM) are expressed as % of the value of the control (untreated cells). The asterisks indicate significant differences compared with VEGF stimulation. ** <i>p</i><0. 01; and *** <i>p</i><0.001.</p

ApoEdp inhibited capillary tubule formation (<i>in vitro</i> angiogenesis).

<p>About 4×10⁴ (per well) HUVECs in medium containing 50 ng/mL of VEGF were plated in 24-well plates previously coated with growth factor-reduced matrigel, and incubated for 12–16 hr at 37°C in the absence or presence of apoEdp. Tubular structures were quantitated by manual counting under low power fields. Representative photomicrographs of tubule formation in the (A) VEGF control and (B) apoEdp-treated wells are shown. (C) “Percentage (%) of inhibition” is the mean number (± SEM) of tubules expressed as a proportion of that in the VEGF control group. ** p<0.01 and *** p<0.001.</p

A working model to explain anti-angiogenic activity of apoEdp driven by VEGF.

<p>ApoEdp peptide suppresses tumor growth through inhibition of VEGF-induced EC proliferation, migration, invasion, and capillary tube formation.</p

ApoEdp inhibits wound-healing migration <i>in vitro</i>.

<p>Monolayers of HUVECs were scraped and incubated in medium containing 50 ng/mL VEGF in the presence or absence of various concentrations of apoEdp. VEGF stimulated the migration of HUVECs in the scraped area. Representative photomicrographs of cells treated with (A) VEGF alone or with (B) VEGF and apoEdp together are shown. Solid lines indicate the initial scraping. (C) ApoEdp significantly inhibited the migration of HUVEC to the wounded area. The bar diagram is the quantitative measurement of cell migration inside the scraped area. The data shown are representative of three independent experiments. ** <i>p</i><0. 01; and *** <i>p</i><0.001.</p

ApoEdp inhibits endothelial cell invasion.

<p>About 4×10⁴ HUVECs were placed into each insert of Boyden chamber (0.8-µm pore size) (BD Biosciences) of 24-well transwell plate. The bottom chambers were filled with 600 µL medium supplemented with 50 ng/mL VEGF. The top chamber was seeded with approximately 4×10⁴ HUVEC cells/well in 100 µL containing different concentrations of apoEdp and incubated at 37°C for 16 hs. HUVEC cells were then fixed and stained with H&E and counted under the microscope. Representative photographs of (A) VEGF-treated and (B) apoEdp-treated membranes are shown. The results show that the (C) HUVECs migrating across the transwell membrane are significantly suppressed by the peptide apoEdp in a dose-dependent manner. **p<0.01 and ***p<0.001.</p

ApoEdp inhibits tumor growth <i>in vivo.</i>

<p>MDA-MB-231 breast cancer cells were used for anti-tumor studies. A suspension of 3×10⁶ cells in 0.1 mL of PBS was injected in the right dorsal flank. Tumors were monitored weekly in two diameters with digital calipers. Tumor volumes were determined using A×B²×0.52 (where A is the longest and B is the shortest diameter). Tumors were allowed to grow to ∼100 mm³ and mice were randomized. Single intralesional administration daily of PBS or 40 mg/kg/day of apoEdp for 3 consecutive days was performed and then stopped. Representative photographs of mouse xenograft treated with (A) apoEdp or PBS are shown. (B) ApoEdp treatment significantly inhibited tumor growth throughout the examination period. *p<0.05, and ***p<0.001.</p