Fig. 1

a MACs phenotype characterization by double staining with DiI-Ac-LDL uptake (on the left; red) and lectin UEA-1-FITC binding (in the middle, green). Merged images showed DiI-Ac-LDL/lectin double-positive MACs (on the right, yellow) (magnification 20×). b FACS quantification of the cell surface markers in MACs. The picture shows results (expression of each surface marker; mean ± SD) typically obtained from six separate experiments. Isotype controls are shown. CD14 and CD45 positivity clearly support the monocytic nature of the cells we have obtained. Growing body of evidence suggests that MACs closely resemble to M2-like macrophages which are characterized by anti-inflammatory features as well as to play pro-angiogenic functions [27]. Observations on surface expression of CD34, which may be lost—though not necessarily—during culture, are conflicting. In our study, flow cytometric analysis showed that cells were strongly positive for CD14 and CD45 with weaker expression of the hematopoietic lineage CD34 (which declines through cell culture passages). Our cells also expressed monocyte markers associated with endothelial cell features such as vascular endothelial growth factor receptor-2 (VEGFR2, also known as KDR) and platelet endothelial cell adhesion molecule-1 (PECAM-1, also known as CD31), suggesting MACs could be considered a sort of educated monocytes [28]. Several groups have reported the coexpression of endothelial markers by these cells. It has been suggested that the detection of endothelial markers might results from contamination with microparticles deriving from other elements in the culture (i.e. platelets) or by passive transfer of platelet microparticles containing CD31 leading to false-positive events by FACS quantification. Testing for CD42 (which, in our hands, was negative) allowed us to exclude contamination with and/or passive transfer of microparticles