Assessment of the sprouting of tubes from rat aorta rings induced by pro-and anti-angiogenic LCO and LCO-like compounds.

<p>(A B) Tube sprouting induced by pro-angiogenic compounds 7–9 (A) compound 11 (B) compared to mock-treated controls (“C”). (C). Tube sprouting induced by the anti-angiogenic disaccharide compounds, 14 and 15, relative to a mock-treated control (“C”) and compound 16. (* = p<0.05; ** = p<0.01; *** = p<0.001; one-way ANOVA).</p

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

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

Angiogenesis modulating activity of compounds determined by the rat aorta ring angiogenesis assay.

<p>The generic structure indicates the positions of the substitutions, R¹–R⁵, for the 24 compounds tested. Compounds activities were measured as a % of untreated control: positive values represent enhanced angiogenesis; negative values represent inhibited angiogenesis. PI-88 (Muparfostat; 100 µg/ml) was included in all experiments as an anti-angiogenesis control. This compound is in phase III cancer trials and used therapeutically at these concentrations <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112635#pone.0112635-Parish1" target="_blank">[34]</a>. In multiple assays PI-88 typically inhibited angiogenesis by 45–65%. The Table includes results for the 24 LCO and LCO-like compounds only which were accumulated from the results of multiple experiments each with mock- and Muparfostat-treated controls. Chitin oligomers were purchased from Sapphire Chemicals and Compound 12 from Ciba Geigy. Compounds 11, 13–16 were synthesised to purity and confirmed by high resolution mass spectrometry and NMR (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112635#pone.0112635.s001" target="_blank">Fig. S2 and Methods S1 in S1 File</a>). All other compounds were synthesised as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112635#pone.0112635-Grenouillat1" target="_blank">[40]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112635#pone.0112635-DemontCaulet2" target="_blank">[58]</a>. Compound 11 was also derived independently from the supernatant of transgenic <i>E. coli</i>. The benzamide of compound 6 is described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112635#pone.0112635.s001" target="_blank">Fig. S1 in S1 File</a>; the benzamide of compound 15 has an <i>O-</i>linked aliphatic C₁₃H₂₇. All compounds were tested at 100 µg/ml unless otherwise stated. NSE = no significant effect. *p<0.05; **p<0.01, ***p<0.001. Compound 24 was provided by Prof. William Broughton (University Genéve, Switzerland).</p