Involvement of endothelial Man and Gal-binding lectins in sensing the flow in coronary arteries

Abstract

The coronary endothelial luminal membrane (CELM) glycocalyx has diverse molecules involved in blood flow signal transduction. Evidence suggests that some of these structures may be lectinic. To test this, we synthesized two monosaccharide polymers (Mon-Pols) made of Mannose (Man-Pol) or Galactose (Gal-Pol) covalently coupled to Dextran (70 kDa) and used them as lectin affinity probes. In situ intracoronary infusion of both polymers resulted in CELM-binding but only Man-Pol caused a reduction in flow-induced positive inotropism and dromotropism. To demonstrate that our lectinic probes could bind to CELM lectins, a representative CELM protein fraction was isolated via intracoronary infusion of a cationic silica colloid and either Mannose- or Galactose-binding lectins were purified from the CELM protein fraction using the corresponding Mon-Pol affinity chromatography resin. Resin-bound CELM proteins were eluted with the corresponding monosaccharide. 2D-SDS-PAGE (pH 4-7) revealed 9 Mannose- and approximately 100 Galactose-selective CELM lectins. In summary, the CELM glycocalyx contains Mannose- and Galactose-binding lectins that may be involved in translating coronary flow into a cardiac parenchymal response.

Flow enhances the agonist-induced responses of the bradykinin receptor (B2) because of its lectinic nature: Role of its oligosaccharide environment

“…In four previous papers we have reliably isolated the whole protein fraction from the coronary luminal endothelial surface membrane and glycocalyx thanks to the method developed and refined by different groups [8,12,14,26,29,30]. This procedure consists of coating of luminal endothelial membrane proteins with cationic colloidal silica particles (20-to 50-nm diameters) due to strong electrical binding to glycocalyx anionic proteins and polymerizing luminal endothelial membrane protein-bound silica with an anionic polymer.…”

“…Oligosaccharide polymer pretreatment: Our previous confocal microscopic and functional studies have shown that oligosaccharide polymers (a combination of mannose, galactose, and N-acetylglucosamine covalently bound to dextran) bind irreversibly with a high affinity to endothelial surface layer structures and, once bound, are not washed away [8,16,12,26]. As described above, control concentration-response curves to bradykinin were obtained at 11 ml/ min (basal pressure 51.1 ± 2.9 mmHg) and at 16 ml/min (basal pressure 81.2 ± 4.1 mmHg).…”

“…As negative control, no sugars were added for binding but replaced by large amounts of ethanolamine. The negative control polymers do not have a physiological effect and do not bind to the endothelial surface layer [8,12,16,26,28].…”

“…Synthesis of the soluble oligosaccharide polymer: Well established procedures in our laboratory were utilized [8,12,14,16,26,27]. Briefly, glucose polymer, 70 kDa dextran, was dissolved in NaCO 3 (0.5M, pH 11), mixed with sufficient divinyl sulfone (DVS), so each of the three -OH groups per glucose moiety reacted with one of the two vinyl moieties of each DVS, leaving one free vinyl moiety per each DVS molecule.…”

“…As described above, control concentration-response curves to bradykinin were obtained at 11 ml/ min (basal pressure 51.1 ± 2.9 mmHg) and at 16 ml/min (basal pressure 81.2 ± 4.1 mmHg). Thereafter, soluble oligosaccharide polymer was infused for 5 min at a concentration of 400 µM followed by a wash period of 10 min so that only oligosaccharide polymer remains bound to the endothelial surface layer and there is no free oligosaccharide polymer in the perfusion solution [816, 26,38]. The concentrationresponse curves were repeated at a flow of 11 ml/min (basal pressure 46.0 ± 2.1 mmHg) and at 16 ml/min (basal pressure 78.9 ± 3.1).…”