Lipo-Chitin Oligosaccharides, Plant Symbiosis Signalling Molecules That Modulate Mammalian Angiogenesis In Vitro

Abstract

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 in vitro 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.

LysMD3 is a type II membrane protein without an role in the response to a range of pathogens

“…The intracellular receptors NOD1 and NOD2 have been shown to bind the peptidoglycan fragments Tri-DAP and muramyl-dipeptide, respectively (7), but it is unknown if the mammalian immune system is capable of recognizing the ubiquitous polymeric glycan backbone of peptidoglycan. Furthermore, a recent study suggested that chitin oligomers may modulate mammalian angiogenesis through interactions with a yet unknown receptor (8). We hypothesized that the LysMD family may represent a novel family of pattern recognition receptors, potentially capable of recognizing important microbial ligands such as peptidoglycan or chitin.…”

Promotion of mammalian angiogenesis by neolignans derived from soybean extracellular fluids

“…An activity-guided rat aorta ring bioassay with a proven ability to identify anti-angiogenic compounds [ 33 ] was modified by reducing the concentration of HIFCS from 20% to 5% [ 36 ]. This slowed the rate of tube formation but enabled the screening of pro- and anti-angiogenic activities over three consecutive days.…”

“…The rat aorta angiogenesis bioassay [ 33 ] was modified to enhance the ability to screen for pro-angiogenic activity in the HPLC-derived fractions by reducing the concentration of heat inactivated foetal calf serum (HIFCS) from 20% to 5% and incubating for 7 days in 48 well culture plates (Costar, Corning, Lowell, MA) at 37°C in 5% CO 2 [ 36 ]. Six technical and three biological replicates were used and the medium was changed on day 4 of culture.…”

“…In this paper we used a modification of the rat aorta assay [ 36 ] that enabled us to screen for, and discriminate between, pro- and anti-angiogenic activities in fractionated extracts of soybean xylem sap. Using this approach we identified three fractions of soybean xylem sap that enhanced angiogenesis and, subsequently, characterised the proangiogenic molecules in two of these fractions and determined their mode of action.…”

Detection of fungal and bacterial carbohydrates: Do the similar structures of chitin and peptidoglycan play a role in immune dysfunction?

“…Recently, such a mechanism has been shown to operate in the legume Lotus japonicus , in which a LysM domain-containing kinase that mediates the response to pathogens exhibits much higher sensitivity to chitin as compared to Nod factors [ 19 ]. In mammalian cells, the ability of Nod factors (lipochitin oligosaccharides) to stimulate mammalian angiogenesis [ 20 ], taken with the previously mentioned observation that a Nod factor is able to suppress a plant innate immune reaction in species that lack the Nod receptor [ 14 ], suggests that physiologically relevant cross-activation is possible. For example, a chitin derived molecule could bind a LysM domain that normally binds Nod factor ( Fig 2 , left).…”