Chemical structures of purified <i>FK1</i> and <i>FK2</i>.

<p>Chemical structures of <b><i>FK1</i></b> (<i>e</i>GGCE) (<b>A</b>) and <b><i>FK2</i></b> (<i>t</i>GGCE) (<b>B</b>), which are coniferyl neolignans, based on the NMR, MS and UV spectroscopic data described in this paper, with <i>e</i>GGCE and <i>t</i>GGCE containing two chiral carbons (C-7 and C-8). The NMR, MS, UV spectroscopic and pro-angiogenic properties of the isolated <b><i>FK1</i></b> and <b><i>FK2</i></b> were also comparable to commercial <i>e</i>GGCE purchased from BOC Sciences, NY, USA, and to naturally-derived <i>t</i>GGCE provided by the Second Military Medical University, Shanghai, China.</p

Negative ion mode ESI-MS/MS spectra analysis of <i>FK1</i> and <i>FK2</i>.

<p><b><i>FK1</i></b>, <i>erythro</i>-guaiacylglycerol-8-<i>O</i>-4´-(coniferyl alcohol) ether (<i>e</i>GGCE) (A) and <b><i>FK2</i></b>, <i>threo</i>-guaiacylglycerol-8-<i>O</i>-4´-(coniferyl alcohol) ether (<i>t</i>GGCE) (B) spectra, selecting <i>m/z</i> 375 ([M-H]^-) for CID at 10 eV.</p

HUVEC proliferation assay in the presence of eGGCE <i>(FK1)</i> and tGGCE <i>(FK2)</i>.

<p>Effect of <i>e</i>GGCE and <i>t</i>GGCE, at concentrations from 5 × 10⁻⁶ M to 5 × 10⁻¹² M on confluent serum-starved HUVEC proliferation in serum free media (SFM) with and without the mitogen bFGF (12.5 ng/mL). All the treatments were undertaken in the same experiment and control cultures contained the same diluent dilution as the test compounds. Cell proliferation was measured as ³H thymidine incorporation after 24 hr incubation. Data analysis was performed by Student–Newman–Keuls test after one-way ANOVA comparing each group to control in each treatment. Error bars represent SEM (n = 6). *, P ≤ 0.05, **, ≤ 0.01, ***, ≤ 0.001. Also, a two-way ANOVA data analysis showed a significant difference for all tested concentrations comparing their effect in SFM to SFM+bFGF (12.5 ng/mL) treatments, P ≤0.0001.</p

Pro-angiogenic activity of <i>e</i>GGCE <i>(FK1)</i> and <i>t</i>GGCE (<i>FK2</i>) in the <i>in vitro</i> rat aorta assay.

<p>The independently sourced <i>e</i>GGCE and <i>t</i>GGCE preparations were tested at concentrations ranging from 5 × 10⁻⁶ M to 5 × 10⁻⁹ M. Control cultures contained the same diluent dilution as the test compounds. Data are expressed as a percentage of aorta vessel outgrowths observed on days 5, 6 and 7. Data analysis was performed by Student–Newman–Keuls test after one-way ANOVA comparing each group to control in each treatment. Error bars represent SEM (n = 6). *, P ≤ 0.05, **, ≤ 0.01.</p

Biological activity of HPLC fractionated soybean xylem sap.

<p>A, UV absorbance at 254 nm of HPLC separated xylem sap material, the injection volume of 50 μL containing a sap concentrate equivalent to 20 mL of sap. Fractions were collected at 30 s intervals and tested by the rat aorta ring assay and fractions with pro-angiogenic activity are highlighted in red. B, Separation of <b><i>FK1</i></b> and <b><i>FK2</i></b> using a shallow gradient for a large scale purification (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196843#sec002" target="_blank">methods</a>). C, D The pro-angiogenic activity of the test compounds is expressed as a percentage of aorta vessel outgrowths observed on days 5, 6 and 7 based on the field of view, with (C) depicting 0% outgrowth and (D) 60% outgrowth. E, Biological activity of fractions containing purified <b><i>FK1</i></b>, <b><i>FK2</i></b> or <b><i>P6</i></b> using the rat aorta ring assay. Day 5–7 results are shown. Control cultures received medium with the diluent only or diluent with the anti-angiogenic compound, PI-88, at 100 μg/ml.</p

Summary of the effects of the soybean-derived neolignans <i>e</i>GGCE and <i>t</i>GGCE on various aspects of angiogenesis.

<p>Summary of the effects of the soybean-derived neolignans <i>e</i>GGCE and <i>t</i>GGCE on various aspects of angiogenesis.</p

Time course of the effect of <i>e</i>GGCE (<i>FK1</i>) on fibronectin-mediated HMEC cell adhesion.

<p>Effect of <i>e</i>GGCE on cell adhesion, at concentrations ranging from 5 × 10⁻⁶ M to 5 × 10⁻¹² M, was measured using wells coated with 10 μg/mL of fibronection and after 5–30 min incubation as optical density of Rose Bengal staining adherent cells. Data analysis was performed by one-way and two-way ANOVA, comparing each group to control and other groups. Error bars represent SEM (n = 6). No significant difference in cell adhesion was observed.</p

Effect of eGGCE <i>(FK1)</i> on various parameters of a HUVEC tube formation assay on Matrigel.

<p>(A) Microscopic view of isolated endothelial cells ranging from 4 × 10⁴ to 1 × 10⁴ cells/well cultured for 6 hr on Matrigel in the absence of compound, or following addition of 5 × 10⁻⁶ M <i>e</i>GGCE which enhanced tube formation. (B-E) Effect of <i>e</i>GGCE, at concentrations from 5 × 10⁻⁶ M to 5 × 10⁻¹² M, on HUVEC tube formation (4 × 10⁴ cells/well) as measured by (*, B) percentage denuded area, (→, C) number of sprouting cells, (→, D) total tube length and (E) number of tubes. Parameters shown in (B) and (C) were measured after 4 hr culture and in (D) and (E) after 6 hr culture. Control cultures contained the same diluent dilution as the test compounds. Data analysis was performed by Student–Newman–Keuls test after one-way ANOVA comparing each group to control in each treatment. Error bars represent SEM (n = 6). **, ≤ 0.01, ***, ≤ 0.001.</p

Lipo-Chitin Oligosaccharides, Plant Symbiosis Signalling Molecules That Modulate Mammalian Angiogenesis <i>In Vitro</i>

<div><p>Lipochitin oligosaccharides (LCOs) are signaling molecules required by ecologically and agronomically important bacteria and fungi to establish symbioses with diverse land plants. In plants, oligo-chitins and LCOs can differentially interact with different lysin motif (LysM) receptors and affect innate immunity responses or symbiosis-related pathways. In animals, oligo-chitins also induce innate immunity and other physiological responses but LCO recognition has not been demonstrated. Here LCO and LCO-like compounds are shown to be biologically active in mammals in a structure dependent way through the modulation of angiogenesis, a tightly-regulated process involving the induction and growth of new blood vessels from existing vessels. The testing of 24 LCO, LCO-like or oligo-chitin compounds resulted in structure-dependent effects on angiogenesis <i>in vitro</i> leading to promotion, or inhibition or nil effects. Like plants, the mammalian LCO biological activity depended upon the presence and type of terminal substitutions. Un-substituted oligo-chitins of similar chain lengths were unable to modulate angiogenesis indicating that mammalian cells, like plant cells, can distinguish between LCOs and un-substituted oligo-chitins. The cellular mode-of-action of the biologically active LCOs in mammals was determined. The stimulation or inhibition of endothelial cell adhesion to vitronectin or fibronectin correlated with their pro- or anti-angiogenic activity. Importantly, novel and more easily synthesised LCO-like disaccharide molecules were also biologically active and de-acetylated chitobiose was shown to be the primary structural basis of recognition. Given this, simpler chitin disaccharides derivatives based on the structure of biologically active LCOs were synthesised and purified and these showed biological activity in mammalian cells. Since important chronic disease states are linked to either insufficient or excessive angiogenesis, LCO and LCO-like molecules may have the potential to be a new, carbohydrate-based class of therapeutics for modulating angiogenesis.</p></div

LCO enhancement or inhibition of integrin-mediated attachment of endothelial cells to extracellular matrix components <i>in vitro.</i>

<p>(A–B) A 2-way ANOVA analyses showed the anti-angiogenic compound 14 inhibits HMEC (human microvascular endothelial cell) attachment to immobilised fibronectin and vitronectin whereas compound 15 affects HMEC attachment to fibronectin only (compounds added at 25 µg/ml, adhesion 60 min). The vitronectin and fibronectin concentrations refer to the concentrations used to coat the plates. (C) One-way ANOVA analyses showed pro-angiogenic compounds 7 and 8 enhances HMEC attachment to vitronectin after incubation for 40 min. “C” designates control in A–C. *(p<0.05); **(p<0.01), ***(p<0.001), ****(p<0.0001). Vertical bars represent SEM (n = 6).</p