Cell-cell contacts in the human cell line ECV304 exhibit both endothelial and epithelial characteristics

Abstract

Endothelial cells separate the intra- and extravascular space and regulate transport processes between these compartments. Since intercellular junctions are required for these specific cell functions, the cell-cell contacts in the permanent cell line ECV304 were systematically analyzed and compared with human umbilical vein endothelial cells (HUVECs) in primary culture and with the epithelial Madin Darby Canine Kidney (MDCK) cell line. Filter-grown ECV304 cells generate a distinct electrical resistance and a permeability barrier between cell culture compartments. Electron microscopy of ECV304 cells revealed lateral membrane interdigitations, typically found in endothelial cells in vivo, with direct membrane contact sites, which prevented the diffusion of lanthanum. By immunoblot and immunofluorescence analysis, the expression and cellular localization of the tight junction and adherens-type junction proteins occludin, ZO-1, symplekin, beta-catenin, and plakoglobin were analyzed. ECV304 cells display further characteristics of endothelial cells, including the expresssion of thrombomodulin and of the vitronectin receptor CD51, as well as the secretion of plasminogen activator inhibitor 1 (PAI-1) and endothelin. However, ECV304 cells also express proteins characteristically found in epithelial cells, including E-cadherin and the desmosomal proteins desmoplakin, desmocollin, and desmoglein; occasionally desmosomal structures can be identified by electron microscopy. In conclusion, ECV304 cells express many endothelial markers and form specialized intercellular junctions that display some epithelial features. Thus this reportedly endothelial-derived permanent human cell line may be dedifferentiated toward an epithelial phenotype.

An Epithelial Cell Line That Can Stimulate Alloproliferation of Resting CD4+ T Cells, But Not After IFN-γ Stimulation

“…EaHy.926, a hybridoma of primary HUVEC and a human airway epithelial cell line (20), has been previously shown to behave similarly to primary endothelial cells in vitro, expressing high levels of the endothelial specific marker, CD31, as well as retaining the ability to up-regulate HLA-DR and cause proliferation of allospecific T cells in vitro (16,21). In contrast, the second cell line we have transfected is a bladder epithelial carcinoma T24 (previously misidentified as a spontaneously transformed endothelial cell line ECV.304) (22). This cell line does not express HLA-class II determinants after IFN-␥ stimulation for several days, while responding with up-regulation of other molecules influenced by IFN-␥ treatment (21).…”

Productive Dengue Virus Infection of Human Endothelial Cells Is Directed by Heparan Sulfate-Containing Proteoglycan Receptors

“…One report, using a continuous ECV304 cell line, suggested that dengue virus interacted with 3 undefined cellular proteins (78). However, these interactions have not been studied further or analyzed in primary human endothelial cells (2,12,41,44).…”

Dynamic regulation of metabolism and respiration by endogenously produced nitric oxide protects against oxidative stress

“…ECV304 cells maintain typical cobblestone morphology, form lateral membrane interdigitations that prevent the diffusion of lanthanum, and respond to classical and selective endothelial stimulants such as ionomycin, ADP, vascular endothelial growth factor, bradykinin, angiotensin, and oxidized low density lipoprotein (13)(14)(15)(16)(17)(18)(19)(20). The ECV304 cells express classical endothelial receptors such as thrombomodulin and vitronectin receptor CD51, and are capable of secreting plasminogen activator inhibitor-1 and endothelin (21). However, during transformation the cells lost the ability to generate nitric oxide and have acquired phenotypic expression of epithelial proteins such as E-cadherin and desmosomal proteins (21,22).…”

“…The ECV304 cells express classical endothelial receptors such as thrombomodulin and vitronectin receptor CD51, and are capable of secreting plasminogen activator inhibitor-1 and endothelin (21). However, during transformation the cells lost the ability to generate nitric oxide and have acquired phenotypic expression of epithelial proteins such as E-cadherin and desmosomal proteins (21,22). Therefore, although these cells express all of the classical endothelial markers in culture, they have differentiated toward an epithelium-like phenotype (22).…”