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

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.

Expression of GM3 synthase in human atherosclerotic lesions.

We have previously demonstrated that amounts of ganglioside GM3 are markedly higher in human atherosclerotic lesions compared to that in non-diseased arterial tissue. Because the fatty acid composition of GM3 in blood plasma low density lipoproteins (LDL) and the fatty acid composition of GM3 in atherosclerotic lesions differed, we hypothesized that, in addition to GM3 originating from LDL infiltrating the arterial wall from the blood, excessive GM3 may be synthesized locally in atherosclerotic lesions. In the present work, using an anti-GM3 antibody developed by us, we showed that the levels of GM3 synthase in membrane fractions isolated from the atherosclerotic intima were higher compared to those in non-diseased arterial tissue. Using an immunohistochemical approach, we examined the expression of GM3 synthase in sections of atherosclerotic plaques and non-diseased arterial wall. GM3 synthase immunopositivity was found to be low in non-diseased arterial intima but large numbers of GM3 synthase-immunopositive cells were observed in atherosclerotic plaques. GM3 synthase was overexpressed by macrophages and dendritic cells and double immunostaining demonstrated cellular co-localization of GM3 synthase and GM3. Further in vitro experiments showed that both monocyte-derived dendritic cells and macrophages expressed high levels of GM3 synthase. The findings of the present study indicate that, at least partially, excessive amounts of GM3 in atherosclerotic lesions can be synthesized by macrophages and dendritic cells directly within the arterial wall.

Sialyltransferase activity of human plasma and aortic intima is enhanced in atherosclerosis.

Sialyltransferase activity has been determined in membrane preparations containing the Golgi apparatus that were isolated from atherosclerotic and normal human aortic intima as well as in plasma of patients with documented atherosclerosis and healthy donors by measuring the transfer of N-acetylneuraminic acid (NeuAc) from CMP-NeuAc to asialofetuin. The asialofetuin sialyltransferase activity was found to be 2 times higher in the atherosclerotic intima as compared to the normal intima and 2-fold higher in patients' plasma than in that from healthy donors. The mean values of the apparent Michaelis constant (K(m)) for the sialylating enzyme for both tissues did not differ and were close for the intima and plasma. In contrast, the maximal velocity (V(max)) was 2 times higher for the atherosclerotic intima than for the normal intima and 3 times higher for patients' plasma than for that of the donors. These results suggest that the activity of asialofetuin sialyltransferases of aortal intima is enhanced in atherosclerosis as is the secretion of their soluble forms into patients' plasma.