Distribution of hMSC to arteries/arterioles, veins and capillaries/end arterioles in the CAM.

<p><b>A.</b> Distribution of hMSC compared to lymphocytes and effects of pre-treatment with anti-SLeX and/or anti-α4 integrin (n = 5). <b>B</b>. Distribution in arteries of hMSC from 5 preparations from 5 different donors of marrow repeated 5 times.</p

Clearance from the circulation of hMSC, melanoma cells and 10 µm inert beads.

<p>Inflexible inert 10 µM beads and B16F1 are cleared from circulation faster than hMSC. <b>A.</b> Values for cellular flux calculated as the average number of cells or 10 µm beads counted within vessels each minute in the CAM at 100× magnification. B. Values expressed as percentage flux were calculated as cells or beads in one minute as % of total observed in 10 minutes (n≥6).</p

3-Dimensional images of cells in rhodamine-labeled vesicles of chick embryo CAM.

<p>Orthologous projections of z-stacked photomicrographs of the CAM at 200× magnification. Crosshairs indicate cell of interest. <b>A.</b> B16F1 melanoma cells primarily embolized in the overlying capillary plexus (arrowhead) and at the ends of tapering arterioles. <b>B</b>. An hMSC, retaining its shape, adhered in a large vessel (dashed lines) lying beneath the capillary plexus.</p

Low passage hMSC express α4 integrin and SLeX.

<p>hMSC derived from four preparations from four different donors were assayed for expression of α4 integrin and SLeX by flow cytometry. Passage 1 cells were plated overnight to recover adherent viable cells and then re-plated at 100 cells/cm². The cells were harvested when 70 to 80% confluent.</p

Real time assay of cells in vessels of the chick embryo CAM.

<p><b>A.</b> Schematic for injecting cells or beads into a large vein of the CAM and capturing images for 3 to 10 minutes at either 40× or 100× magnification. <b>B. (upper panel).</b> Green B16F1 melanoma cells were primarily embolized in the capillary bed and had distorted morphology (∧). <b>(lower panel).</b> Green hMSC retained a regular morphology and were found both within arteries (†) and within the capillary beds (#). Images taken 10 minutes after injection of the cells. Arrows indicate direction of blood flow. Magnification 100×.</p

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 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 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 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 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