Immunolocalization of endocan during the endothelial- mesenchymal transition process L.M. Carrillo,1 E. Arciniegas,1 H. Rojas,2 R. Ramírez1 1Servicio Autónomo Instituto de Biomedicina, Facultad de Medicina, Universidad Central de Venezuela, Caracas; 2Instituto de Inmunología, Facultad de Medicina, Universidad Central de Venezuela, Caracas, Venezuela Abstract Endocan is a dermatan sulfate proteoglycan (DSPG) that has been observed in the cyto- plasm of endothelial cells of small and large vessels in lung, kidney, liver, colon, ovary and brain tumors. This DSPG has been implicated in the regulation of cellular activities such as adhesion, migration, and proliferation. Given the important roles played by endocan in such processes, we sought to determine whether this DSPG is present in the chicken embryo aortic wall in embryonic days 12 and 14, when intimal thickening and endothelial transfor- mation are notorious. Immunolabeling of seri- al paraffin cross-sections revealed endocan immunoreactivity at the endothelium and some mesenchymal cells constituting the inti- mal thickening but not in the cells arranged in lamellar layers. We also investigated whether endocan was present in monolayers of primary embryonic aortic endothelial cells attached to fibronectin when they were deprived of serum and stimulated with epidermal growth factor. Immunofluorescence determined that in the epidermal growth factor (EGF) condition where separating, detaching, and migrating cells were observed, endocan appeared organ- ized in arrays typical of focal complexes in the leading edge of these cells. In serum-free medium condition in which the endothelial cells displayed a cobblestone appearance, endocan appeared mainly delineating the mar- gin of many cells. This study demonstrates for the first time the presence of endocan during the aortic wall remodeling, and provides evi- dence that suggests a possible contribution of this DSPG in the endothelial-mesenchymal transition (EndoMT) process. European Journal of Histochemistry 2011; volume 55:e13 Introduction Endothelial-mesenchymal transition (EndoMT) is a complex process by which cer- tain endothelial cell subsets lose endothelial characteristics and transform into mesenchy- mal or smooth muscle cells (SMCs).1 This process is now recognized as a phenomenon that occurs during cardiac fibrosis2 and neoin- timal thickening formation observed in ather- osclerosis and restenosis.1,3 In addition to this role, recent evidence has also demonstrated that EndoMT is critical in heart and vascular development,1,4 hypertrophic scarring,5 kidney fibrosis,6 diabetic nephropathy7 and during cancer progression.8 EndoMT process entails growth factor and cytokines synthesis and secretion, activation of receptor tyrosine kinases (RTKs) and non-RTKs, cytoskeleton reorganization, disassembly of the adherence junctions or loss of endothelial cell-cell con- tacts, change in cell shape and polarity, pro- teases secretion, extracellular matrix (ECM) remodeling, cell separation or detachment from the substratum, and cell migration and differentiation.1,9 In addition to permanent interaction with other cells, endothelial cells also need to inter- act with the ECM. It is now well established that most ECM molecules participate in tissue remodeling by promoting cell adhesion provok- ing cytoskeletal organization, and regulating cell spreading, polarization, detachment, migration and differentiation in response to mechanical stimuli and/or cross talk of a vari- ety of growth factors and cytokines.10 Some of the most studied ECM molecules in the vessel wall belong to the family of proteoglycans (PGs), which are actually recognized by its role in several cellular processes either via interac- tions with ECM receptors like integrins or via signaling through receptor tyrosine kinases (RTKs).11 Some particularly significant studies on the developing of chick aorta show that large PGs containing EGF-like domains such as versican and aggrecan have different spatial and temporal expression patterns, suggesting a possible role for these molecules during inti- mal thickening formation and EndoMT.11,12 Endocan, also called endothelial cell-specific molecule-1, is a small dermatan sulfate proteo- glycan (DSPG) that does not seem to belong to the small leucine-rich PGs (SLRPG) family and is mainly expressed by endothelial cells and regulated in vivo by inflammatory cytokines such as vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2), and tumor necrosis factor-α (TNF-α).13-16 This DSPG consist of a core protein that contains an N-terminal rich in cysteine residues, a puta- tive EGF-like domain, whose functional signif- icance is not yet fully understood, and a single Correspondence: Dr. Enrique Arciniegas, Servicio Autónomo Instituto de Biomedicina, Facultad de Medicina, Universidad Central de Venezuela, Caracas 1010, Venezuela. Tel. +58.212.6054050 - Fax: +58.212.8611258. E-mail: earciniegasbeta@yahoo.com Key words: endocan, chicken embryo, aorta, focal complexes, EGF-like domain. Acknowledgments: we thank Eng. Antonio Salgado and Mr. Wilman Clark for the photo- graphic contribution (Area de Informática, SAIB). This study was supported by Consejo de Desarrollo Científico y Humanístico, Universidad Central de Venezuela. Caracas (Grant no. PI-09- 7319-2008). Received for publication: 22 November 2010. Accepted for publication: 7 March 2011. This work is licensed under a Creative Commons Attribution 3.0 License (by-nc 3.0). ©Copyright L.M. Carrillo et al., 2011 Licensee PAGEPress, Italy European Journal of Histochemistry 2011; 55:e13 doi:10.4081/ejh.2011.e13 chain of dermatan sulfate (DS), which is cova- lently linked to the core protein.15 Interestingly, a marked expression of endocan has been observed in breast, brain, lung, liver, kidney, ovary tumors, thus suggesting an important role of endocan in angiogenesis and in the development and progression of cancer.16 Nevertheless, the factors regulating the syn- thesis and secretion as well as their functions, have not yet been fully elucidated. In view of the above observations, the aim of this study was to investigate immunohisto- chemically whether endocan is present in the chicken embryo aortic wall in embryonic days 12 and 14 (days E12 and E14; stages 38 and 40), when intimal thickening and endothelial transformation are notorious. We also investi- gated the presence of endocan in monolayers of primary embryonic aortic endothelial cells attached to fibronectin (FN) and stimulated with epidermal growth factor (EGF). Materials and Methods Tissue extraction Fertilized chicken eggs (White leghorn) were obtained from a local hatchery (Granja Avícola Agropollito C.A., Paracotos, Estado Miranda, Venezuela) and incubated at 37ºC and 60% humidity for 12 and 14 days (stages 38 and 40). Embryos were staged according to Hamburger and Hamilton.17 The aortae were [European Journal of Histochemistry 2011; 55:e13] [page 73]