Structural statistics for the 15 final NMR structures of VpepB.

a)<p>Ordered residues (residues with sum of phi and psi order parameters <1.8) are considered for r.m.s.d. calculations and Ramachandran statistics.</p

The pY747 peptide has no effect on β3^−/− or DiYF mice.

<p><b>a</b>) pY747 peptide does not inhibit VEGF-induced aortic ring growth from β3^−/− mice. Mouse aortic rings were embedded in matrigel in the presence of 40 ng/mL of VEGF and 40 µM of peptides as indicated. <b>b</b>) Quantification of aortic ring assay as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031071#pone-0031071-g004" target="_blank">Fig. 4a</a>. <b>c</b>) pY747 could not inhibit bFGF-induced aortic ring growth, Mouse aortic rings were isolated from wild type (WT), β3^−/−, and DiYF mice and embedded in matrigel in the presence of 40 ng/mL of VEGF, 20 ng/mL of bFGF or pY747 peptides as indicated. Aortic rings were incubated for 3 days for wild type and β3^−/− aortic rings and 4 days for DiYF aortic rings (longer incubation was used to obtain visible aortic sprouting which is diminished in these mice). <b>d</b>) pY747 peptide does not inhibit angiogenesis in DiYF mice. Peptides' effect on <i>in vivo</i> angiogenesis in wild type mice and DiYF mice was tested as described. <b>e</b>) Quantification of blood vessels in matrigel plus assay as indicated.</p

Summary of the <i>in vitro</i> evidence for a direct interaction between VEGFR2 and β₃CTs and structure of the VEGFR2 ⁸⁰¹YLSI motif in bound conformation.

<p>Chemical shift titrations were performed in water at 25°C at pH 6.1 with β₃ concentrations of in a range of 30–100 µM. Expanded region of ¹⁵N HSQC spectra show chemical shift perturbations for <b>a</b>) β₃NP. <b>b</b>) β₃MP in presence of VpepA at the ratio 1∶1. <b>c</b>) Chemical shift changes in ¹⁵N labeled β₃NP upon addition of VpepA at the ratios of 1∶1 (red) and 1∶2 (green). <b>d</b>) Chemical shift changes in ¹⁵N labeled β₃MP upon addition of VpepA at the ratios of 1∶1 (red) and 1∶2 (green). Delta [ppm] refers to the combined HN and N chemical shift changes according to the equation: Δδ(HN,N) = ((Δδ_HN²+0.2(Δδ_N)²)^1/2, where Δδ = δ_bound-δ_free. Transferred NOEs: all the NOESY experiments were performed in 50 mM NaCl and 25 mM Na-phosphate buffer at pH 6.1 and 25°C with peptide to protein ratio of 50 to 1 and peptide concentrations of 1 mM; <b>e</b>) VpepB alone is shown in black and VpepB in combination with GST-β₃ is shown in green; <b>f</b>) VpepC alone (black) and VpepC in combination with GST-β₃ (green). <b>g</b>) Ribbon representation of VpepB structure. Hydrophobic residues of ⁸⁰¹YLSI region are shown in dark gray. <b>h</b>) Backbone superimposition of the 15 lowest energy conformers of VpepB. Residues used for superimposition are ⁸⁰¹YLSI. Molecular graphics images were produced using the UCSF Chimera package (Pettersen et al., 2004).</p

The pY747 peptide inhibits VEGFR2-induced angiogenesis in <i>ex vivo</i> and <i>in vivo</i>.

<p><b>a</b>) Inhibition of <i>ex vivo</i> endothelial sprouting by the pY747 peptide. Mouse aortic rings were embedded in matrigel in the presence or absence of 40 ng/mL of VEGF and peptides as indicated. Photographs were taken at three days and the number of endothelial sprouts originating from each ring was determined. <b>b</b>) Quantification of aortic ring assay as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031071#pone-0031071-g003" target="_blank">Fig. 3a</a>. <b>c</b>) Inhibition of <i>in vivo</i> angiogenesis by pY747 peptide. Results of matrigel plug angiogenesis assay are shown. The indicated peptides at 200 µM concentration were mixed with growth factor-reduced matrigel containing VEGF (500 ng/mL) and injected subcutaneously into wild type mice. Seven days later, the matrigel implants were removed, sectioned, and blood vessels were stained with CD31 Ab (red) and nuclei with DAPI (blue). Vessel area was determined using ImagePro. <b>d</b>) Quantified results of matrigel plug assay as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031071#pone-0031071-g003" target="_blank">Fig. 3c</a>.</p

pY747 peptide inhibits VEGF-induced VEGFR2 phosphorylation and ERK activation.

<p>Serum starved HUVEC were incubated with the indicated concentrations of pY747 or F747 peptides for 3 h, then stimulated with 20 ng/mL VEGF for five min at 37°C or left unstimulated. The cells were lysed and equal amounts of protein from total cell lysates were subjected to Western blot analysis with <b>a</b>) anti-p-VEGFR2 (Y1775) Ab or <b>b</b>) anti-p-ERK1/2 antibodies. The blots were reprobed with <b>a</b>) anti-total VEGFR2 or <b>b</b>) anti-total ERK1/2 antibodies as loading control. Bands were quantified by densitometric analysis and fold increase over unstimulated cells are displayed (right panels).</p

The pY747 peptide inhibits VEGFR2-induced angiogenesis <i>in vitro</i>.

<p><b>a</b>) The amino acid sequences of VpepA, human integrin β₃ cytoplasmic tail (CT), and derived peptides. The conserved YLSI (VpepA), HDRKE (Integrin β₃CT) along with the two tyrosine phosphorylation motifs are shown in orange. HIV-TAT leader sequence (shown in blue) was added to allow delivery of the peptides across the cell membrane. <b>b</b>) Representative images of HUVEC tube formation in the presence with or without VEGF (20 ng/mL). <b>c</b>) Example images of inhibition of <i>in vitro</i> endothelial tube formation by the pY747 peptide. HUVEC were plated on matrigel-coated 48 well plates in the presence of 20 ng/mL VEGF and peptides at indicated concentrations. The cells were allowed to form tubes for 16 hours, bright field images at 2.5× magnification were taken and analyzed (using computer algorithms) for number, length, and thickness of branches. <b>d</b>) Quantitative result of branch thickness under different treatments. <b>e</b>) Representative images of tube formation in the presence of pY747 and pY759 peptide. <b>f</b>) Quantitative result of tube length as indicated.</p

Detection and Biological Activities of Carboxyethylpyrrole Ethanolamine Phospholipids (CEP-EPs)

Oxidation of docosahexaenoate phospholipids produces 4-hydroxy-7-oxo-hept-5-eonyl phospholipids (HOHA-PLs) that react with protein lysyl ε-amino residues to generate 2-ω-carboxyethylpyrrole (CEP) derivatives, endogenous factors that induce angiogenesis in the retina and tumors. It seemed likely, but remained unproven, that HOHA-PLs react with ethanolamine phospholipids (EPs) <i>in vivo</i> to generate CEP-EPs. We now show that CEP-EPs are present in human blood at 4.6-fold higher levels in age-related macular degeneration plasma than in normal plasma. We also show that CEP-EPs are pro-angiogenic, inducing tube formation by human umbilical vein endothelial cells by activating Toll-like receptor 2. CEP-EP levels may be a useful biomarker for clinical assessment of AMD risk and CEP-associated tumor progression and a tool for monitoring the efficacy of therapeutic interventions