Enhancing of GM3 synthase expression during differentiation of human blood monocytes into macrophages as in vitro model of GM3 accumulation in atherosclerotic lesion

Abstract

In previous studies, we showed that ganglioside levels (GM3 being the main ganglioside) in human aortic intima isolated from atherosclerotic lesions were 5 times greater compared to intima from non-diseased vascular areas. Recently, we found that GM3 and GM3 synthase levels in differentiated in vitro macrophages were five and ten times higher, respectively, compared to freshly isolated human monocytes. In this article, we report that GM3 synthase mRNA levels were significantly higher in differentiated human monocyte-derived macrophages compared to monocytes and in atherosclerotic aorta compared to normal aorta. The depletion of GM3 synthesis in cultured monocyte-derived macrophages with DL-threo-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol, an inhibitor of ganglioside synthesis, delayed the acquisition of CD206 antigen, prevented the loss of CD163 antigen and enhanced anti-inflammatory cytokine (CCL18) secretion. In the current study, we performed purification of CMP-N-acetylneuraminic acid:lactosylceramide alpha2,3-sialyltransferase (GM3 synthase) from Triton X-100 extract of human blood mononuclear cells by immunoaffinity chromatography on Sepharose coupled with anti-GM3 synthase antibody. Comparison with several glycolipid substrates showed high specificity of the purified enzyme for lactosylceramide. The apparent K(M) for lactosylceramide and CMP-NeuAc were 101 and 180 muM, respectively. Analysis of the purified enzyme by SDS-PAGE followed by the anti-GM3 synthase antibody probing detected two bands with apparent molecular masses of 60 and 64 kDa. There were no other protein bands as revealed by Coomassie Blue staining. Thus, ganglioside GM3 may be considered as a physiological modulator of macrophage differentiation in human atherosclerotic aorta. The presented data suggest that up-regulation of GM3 levels is an element of monocyte/macrophage differentiation that provides a tool for control of macrophage accumulation in inflammatory loci.

A Cytofluorometric Study of Membrane Rafts in Human Monocyte Subsets in Atherosclerosis

“…These findings, in conjunction with our earlier results [ 13 , 14 , 30 ], indicate a probable relationship between biosynthesis activation and, as a consequence, an increase in the level of gangliosides and a higher expression of lipid rafts in cultured macrophages compared to freshly isolated monocytes. An increasing amount of data confirms that monocytes are already pre-activated, circulating before their migration to inflammation areas during inflammatory pathologies accompanied by an increased production of cytokines, such as TNF-α, M-CSF, or IL-6 [ 10 - 12 ].…”

“…Previously, we observed a significant increase in the synthesis of gangliosides during the in vitro differentiation of human monocytes into macrophages [ 14 ]. We also found that the mRNA level of GM3 synthase (a key enzyme of the ganglioside synthesis) was significantly higher in cultured monocytes/macrophages than in freshly isolated monocytes and in the intima of an atherosclerotic plaque compared to the intima of unaffected regions of the human aorta [ 30 ]. It should be noted that macrophages are the main cells in the intima of the atherosclerotic arteries, whereas they are actually absent in the normal intima.…”

Activation of ganglioside GM3 biosynthesis in human blood mononuclear cells in atherosclerosis

“…Полученные данные позволяют сделать вывод о том, что повышение содержания GM3 -один из признаков активации моноцитов и лимфоцитов в крови пациентов с атеросклерозом. Так как этот признак характерен для дифференцированных макрофагов в культуре [11], можно предположить, что моноциты периферической крови пациентов с атеросклерозом приобретают некоторые свойства макрофагов.…”

“…Подавление биосинтеза ганглиозидов специфическим ингибитором глюкозилцерамидсинтазы D-1-трео-1-фенил-2-пальмитоиламино-3-пирролидино-1-пропанолом приводит к нарушению процесса макрофагальной дифференцировки [11].…”

Vascular Diseases and Gangliosides

“…In the human pre-myeloid leukemia cell line HL-60 and histiocytic lymphoma cell line U937, exogenous GM3 induces monocytic cell differentiation and notably, GM3 increase during macrophage-like cell differentiation [47]. GM3 synthase levels are significantly higher in human monocyte-derived macrophages than in monocytes and GM3 has been considered as a physiological modulator of macrophage differentiation in human atherosclerotic aorta [78]. In bone marrow-derived macrophages, peritoneal macrophages and the Raw264.7 macrophage cell line, exogenous GM1 contributes to the induction of arginase-1, a major M2 macrophage marker, and to the secretion of monocyte chemoattractant protein-1 (MCP-1) through CD206-mediated activation of signal transducer and activator of transcription (STAT) 6 [48].…”