Differential regulation of aldose reductase expression during macrophage polarization depends on hyperglycemia.

Aldose reductase (AR; gene AKR1B1) is the rate-limiting enzyme of the polyol pathway and has been associated with diabetes and atherosclerosis. Here, we sought to identify the mechanisms underlying differential AR expression in human atherosclerotic plaque macrophages. In vitro, M1-polarized human monocyte-derived macrophages expressed significantly higher levels of AKR1B1 mRNA and AR protein compared with M2-polarized macrophages. AR activity was significantly higher in M1 macrophages. AKR1B1 mRNA expression correlated positively with the M1 marker TNF(r = 0.430,P = 0.006) and negatively with the M2 marker MRC1 (r = -0.443,P = 0.044). Increased AR expression in M1 macrophages depended on hyperglycemia. Concomitantly, expression of SLC2A1 (coding for the Glc transporter GLUT-1) was significantly higher in M1 than in M2 macrophages. Pharmacological inhibition of GLUT-1 using STF-32 completely abrogated Glc-induced AR up-regulation in M1 macrophages. When analyzing AR expression in post-mortem coronary artery plaque macrophages, a history of diabetes was associated with a significantly increased proportion of CD68(+)AR(++)macrophages, supporting the in vivo relevance of our in vitro findings. We demonstrate that the phenotype of atherosclerotic plaque macrophages may be affected by cardiovascular risk factors such as hyperglycemia. Our data illustrate the complex interplay between systemic and local factors in atherogenesis.

CXCL4-induced plaque macrophages can be specifically identified by co-expression of MMP7+S100A8+ in vitro and in vivo.

Macrophage heterogeneity in human atherosclerotic plaques has been recognized; however, markers for unequivocal identification of some subtypes are lacking. We found that the platelet chemokine CXCL4 induces a unique macrophage phenotype, which we proposed to call 'M4'. Here, we sought to identify suitable markers that identify M4 macrophages in vitro and in vivo. Using a stringent algorithm, we identified a set of potential markers from transcriptomic data derived from polarized macrophages. We specifically focused on matrix metalloproteinase (MMP)7 and S100A8, the co-expression of which has not been described in any macrophage type thus far. We found dose- and time-dependent MMP7 and S100A8 expression in M4 macrophages at the gene and protein levels. CXCL4-induced up-regulation of both MMP7 and S100A8 was curbed in the presence of heparin, which binds to CXCL4 and glycosaminoglycans, most likely representing the macrophage receptor for CXCL4. Immunofluorescence of post-mortem atherosclerotic coronary arteries identified CD68(+)MMP7(+), CD68(+)MMP7(-), CD68(+)S100A8(+) and CD68(+)S100A8(-) macrophages. A small proportion of MMP7(+)S100A8(+) macrophages most likely represent M4 macrophages. In summary, we have identified co-expression of MMP7 and S100A8 to be a marker combination exclusively found in M4 macrophages. This finding may allow further dissection of the role of M4 macrophages in atherosclerosis and other pathologic conditions.

An in vitro model to study heterogeneity of human macrophage differentiation and polarization.

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the phenotypic and functional differences between murine and human monocyte-derived macrophages. Therefore, we here describe an in vitro model to generate and study primary human macrophages. Briefly, after density gradient centrifugation of peripheral blood drawn from a forearm vein, monocytes are isolated from peripheral blood mononuclear cells using negative magnetic bead isolation. These monocytes are then cultured for six days under specific conditions to induce different types of macrophage differentiation or polarization. The model is easy to use and circumvents the problems caused by species-specific differences between mouse and man. Furthermore, it is closer to the in vivo conditions than the use of immortalized cell lines. In conclusion, the model described here is suitable to study macrophage biology, identify disease mechanisms and novel therapeutic targets. Even though not fully replacing experiments with animals or human tissues obtained post mortem, the model described here allows identification and validation of disease mechanisms and therapeutic targets that may be highly relevant to various human diseases.