Brief Note appeared mainly distributed delineating the margins of many cells (Figure 4 a,b,c). No immunolabeling was observed when a non-immune serum was used as negative con- trol (not shown). Discussion The current study reveals the existence of both endocan-immunoreactive endothelial and mesenchymal cells in the intimal thickenings observed in chicken embryo aortae on days E12 and E14. This study also provides evidence that allow us to suggest a possible contribution of this DSPG in the EndoMT process. Endocan is a DSPG expressed specifically by endothelial cells and has been implicated in several pathophysiological processes as well as in the regulation of cellular activities such as adhesion, migration and proliferation.14,16 By immunohistochemistry, endocan has been observed in the cytoplasm of endothelial cells of small and large vessels in lung, kidney, liver, colon, ovary and brain tumors.16 Thus, it is of interest that a strong endocan staining was detected in the endothelium and some migrat- ing mesenchymal cells that conform the spon- taneous intimal thickening but not in those cells organized in lamellar layers. This implies that endocan would be participating in the EndoMT and therefore contributing to the aor- tic wall remodeling. Importantly, spontaneous aortic and pulmonary intimal thickenings have been demonstrated during chicken embryo development and related to blood pressure alterations that occur rapid and exponentially from day E3 to day E10 of development accom- panied by the local production and activation of different growth factors such as FGF-2, TGFβ and IGFII.19,20 It might thus be speculated that the presence of endocan in the embryonic aor- tic wall would occur in response to hemody- namic changes and production of certain growth factors, considering that endocan expression, synthesis or secretion is highly regulated by VEGF and FGF-2,14,16 and that TGFβ stimulates the synthesis and secretion of the core protein of some PGs.21,22 Also, this presence may be interpreted as a sign of endothelial activation. In this study, we also found that the spatial distribution and organization of endocan observed in SFM condition was altered when the monolayers were stimulated with EGF. Specifically, endocan appeared delineating the margins of cells in arrays typical of focal com- plexes in the leading edges as well as sur- rounding the MTOC and aligned with MTs of the cells that were separating, detaching and migrating toward cell-free areas. We believe that this peculiar localization of endocan Figure 3. Representative CLSM fluorescence images of integrin-β3 and p-FAK in monolay- ers of endothelial cells attached to FN after 10 hr in culture in the presence of EGF. a) Integrin-β3 is seen delineating the margin of some cells and organized into linear streaks and a punctate pattern at the leading edge of the migrating cells. b) p-FAK is seen at the lead- ing edge in arrays typical of focal complexes. a) Scale bar = 25 μm. b) scale bar = 10 μm. Figure 4. Representative CLSM fluorescence images of endocan, integrin-β3 and p-FAK in monolayers attached to FN after 10 h in culture in SFM. a) and b) Endocan and inte- grin-β3 are seen delineating the margin of many cells that displayed a cobblestone appearance. c) p-FAK is seen delineating the margin of cells and organized into linear streaks, typical of focal adhesions. a), b) scale bars = 25 μm; c) scale bar = 50 μm. [page 76] [European Journal of Histochemistry 2011; 55:e13]