## 4 HUVEC Identification
### Cellular Markers
#### 4.2.1 Protein Markers  
_Platelet endothelial cell adhesion molecule (PECAM-1, endoCAM or CD31)_. CD31 is a glycoprotein of the immunoglobulin (Ig) superfamily. This protein is found in the EC membrane, near the intercellular junctions. Its main function is to regulate the EC-EC and EC-leukocyte adhesion [84]. It is a universal endothelial marker, used for microcapillary count in tumor samples [49]. However, CD31 can also be expressed by monocytes and some leukocyte populations [85]. It can be identified by flow cytometry or by immunofluorescence [86, 87]. _CD36_. Also known as platelet glycoprotein IV, this receptor is an 88 kD transmembrane glycoprotein with a large extracellular domain and serves as an adhesion receptor for thrombospondin-1, among others. Originally found in platelets and monocytes, it is also expressed in microvascular cells, but not in large capillary vessels [88, 89]. Likewise, it has been identified not only in the endothelium, but also in monocytes and certain types of epidermal cells [89]. _CD34_. The CD34 human progenitor cell antigen is a 115 kD monomeric glycoprotein that is selectively expressed in hematopoietic progenitor cells of the spinal cord, EC, and in numerous non-hematopoietic tissues, including skin [90], leukemic cells and stem cells [91]. Several studies have described the expression of CD34 in EC of capillaries, arteries, veins, arterioles and venules, as well as in smooth muscle cells of the aorta and some fibroblasts. This marker is commonly used to identify EC by immunohistochemistry [49, 91]. _CD144_. Also known as VE-cadherin, it belongs to a group of transmembrane glycoproteins that promote calcium-dependent cell-cell hemophilic contact. VE-cadherin is specifically found at interendothelial junctions [92], necessary for vascular genesis and repair of vascular disease [93]. This protein is also used as an endothelium marker [49], guarantees the maturity of the cell-cell junctions and the homogeneity of HUVEC [57]. _Intercellular adhesion molecule 1 (ICAM-1) and Vascular cell adhesion molecule 1 (VCAM-1)_. Both molecules are members of the immunoglobulin superfamily and are involved in EC adhesion. ICAM-1 mediates leukocyte adhesion to the endothelium via lymphocyte function-associated antigen 1 (LFA-1), and to monocytes and neutrophils via macrophage-1 antigen (Mac-1). VCAM-1 mediates adhesion of mononuclear cells to the endothelium via two ligands of the integrin family, called integrin-\(\alpha\)4\(\beta\)1 and activated integrin-\(\alpha\)4\(\beta\)7 (or VLA-4) [94]. These endothelial markers can be identified using monoclonal antibodies: Anti-CD54 for ICAM-1 and anti-CD106 for VCAM-1 [11, 49, 95]. Figure 4: Protein markers in HUVEC. Schematic illustration shows the protein markers that can be identified in HUVEC using antibodies. Endothelin-I (ET-1). ET-1 is a potent vasoconstrotrictor peptide with both paracrine and local effects. ET1 levels have been found to be increased in certain pathological conditions, such as hypertension, uremia and cardiogenic shock [96]. It is the only endothelin produced by EC. ET-1 immunoreactivity is detected in EC supernatants by radioimmunoassay or by enzyme-linked immunoabsorption assay (ELISA) [49]. E-selectin and P-selectin (E and P). E and P selectins are adhesion molecules found in EC. They belong to the family of calcium-dependent lectins responsible for supporting initial leukocyte adhesion and extravasation through carbohydrate recognition [97], and mediating leukocyte adhesion under conditions of shear stress [98]. Adhesion is mediated by the expression of integrins in leukocytes and carbohydrate-containing counter-receptors expressed by EC [84]. E-selectin, considered a cell activation marker, mediates the initial low affinity interaction between leukocytes and the vascular endothelium, a process known as rolling. This protein is expressed exclusively in EC when stimulated with inflammatory cytokines [97]; however, it can promote the adhesion of leukocytes on multiple cell types [99].