Non‐invasive placentation in the marsupials Macropus eugenii (Macropodidae) and Trichosurus vulpecula (Phalangeridae) involves redistribution of uterine Desmoglein‐2

In mammalian pregnancy, the uterus is remodeled to become receptive to embryonic implantation. Since non‐invasive placentation in marsupials is likely derived from invasive placentation, and is underpinned by intra‐uterine conflict between mother and embryo, species with non‐invasive placentation may employ a variety of molecular mechanisms to maintain an intact uterine epithelium and to prevent embryonic invasion. Identifying such modifications to the uterine epithelium of marsupial species with non‐invasive placentation is key to understanding how conflict is mediated during pregnancy in different mammalian groups. Desmoglein‐2, involved in maintaining lateral cell–cell adhesion of the uterine epithelium, is redistributed before implantation to facilitate embryo invasion in mammals with invasive placentation. We identified localization patterns of this cell adhesion molecule throughout pregnancy in two marsupial species with non‐invasive placentation, the tammar wallaby (Macropus eugenii; Macropodidae), and the brushtail possum (Trichosurus vulpecula; Phalangeridae). Interestingly, Desmoglein‐2 redistribution also occurs in both M. eugenii and T. vulpecula, suggesting that cell adhesion, and thus integrity of the uterine epithelium, is reduced during implantation regardless of placental type, and may be an important component of uterine remodeling. Desmoglein‐2 also localizes to the mesenchymal stromal cells of M. eugenii and to epithelial cell nuclei in T. vulpecula, suggesting its involvement in cellular processes that are independent of adhesion and may compensate for reduced lateral adhesion in the uterine epithelium. We conclude that non‐invasive placentation in marsupials involves diverse and complementary strategies to maintain an intact epithelial barrier.     

RNAi-mediated inhibition of the desmosomal cadherin (desmoglein 3) impairs epithelial cell proliferation

Objectives: Desmoglein 3 (Dsg3) is a desmosomal adhesion protein expressed in basal and immediate suprabasal layers of skin. Importance of Dsg3 in cell-cell adhesion and maintenance of tissue integrity is illustrated by findings of keratinocyte dissociation in the autoimmune disease, pemphigus vulgaris, where autoantibodies target Dsg3 on keratinocyte surfaces and cause Dsg3 depletion from desmosomes. However, recognition of possible participation of involvement of Dsg3 in cell proliferation remains controversial. Currently, available evidence suggests that Dsg3 may have both anti- and pro-proliferative roles in keratinocytes. The aim of this study was to use RNA interference (RNAi) strategy to investigate effects of silencing Dsg3 in cell-cell adhesion and cell proliferation in two cell lines, HaCaT and MDCK. Materials and methods: Cells were transfected with siRNA, and knockdown of Dsg3 was assessed by western blotting, fluorescence-activated cell sorting and confocal microscopy. Cell-cell adhesion was analysed using the hanging drop/fragmentation assay, and cell proliferation by colony forming efficiency, BrdU incorporation, cell counts and organotypic culture. Results: Silencing Dsg3 caused defects in cell-cell adhesion and concomitant reduction in cell proliferation in both HaCaT and MDCK cells. Conclusion: These findings suggest that Dsg3 depletion by RNAi reduces cell proliferation, which is likely to be secondary to a defect in cell-cell adhesion, an essential function required for cell differentiation and morphogenesis.     

Imaging and Force Spectroscopy on Desmoglein 1 Using Atomic Force Microscopy Reveal Multivalent Ca2+-Dependent, Low-Affinity Trans-Interaction

Desmoglein 1 is a desmosomal member of the cadherin family expressed in stratified epithelia. Desmoglein 1 is the target adhesion molecule of severe blistering skin diseases such as pemphigus or bullous impetigo. However, despite this enormous pathological relevance, the molecular binding properties of desmoglein 1 are largely unknown. Using atomic force microscopic imaging, we found that desmoglein 1 molecules displayed Ca²⁺-dependent conformational changes of the extracellular domains. By single-molecule force-distance cycles, we provide evidence that desmoglein 1 undergoes Ca²⁺-dependent (K _d = 0.8 mm Ca²⁺) homophilic trans-interaction, which is highly relevant for the contribution of desmoglein 1 homophilic binding to keratinocyte cohesion in distinct epidermal layers. Moreover, while the single-unit unbinding force is comparable to other cadherins (∼40 pN at retrace velocity of 300 nm/s), apparent differences with respect to multivalency of interaction and lifetime of single bonds (0.17 s) were observed. Thus, besides the biophysical characterization of desmoglein 1, a main outcome of the study is that desmoglein 1 differs from other members of the cadherin family in terms of some molecular binding properties. Jens Waschke, Carlos Menendez-Castro, and Paola Bruggeman contributed equally to this study.     

The lanceolate hair rat phenotype results from a missense mutation in a calcium coordinating site of the desmoglein 4 gene

Desmosomal cadherins are essential cell adhesion molecules present throughout the epidermis and other organs, whose major function is to provide mechanical integrity and stability to epithelial cells in a wide variety of tissues. We recently identified a novel desmoglein family member, Desmoglein 4 (Dsg4), using a positional cloning approach in two families with localized autosomal recessive hypotrichosis (LAH) and in the lanceolate hair (lah) mouse. In this study, we report cloning and identification of the rat Dsg4 gene, in which we discovered a missense mutation in a naturally occurring lanceolate hair (lah) rat mutant. Phenotypic analysis of lah/lah mutant rats revealed a striking hair shaft defect with the appearance of a lance head within defective hair shafts. The mutation disrupts a critical calcium binding site bridging the second and third extracellular domains of Dsg4, likely disrupting extracellular interactions of the protein.     

Nanorobotic Investigation Identifies Novel Visual,Structural and Functional Correlates of Autoimmune Pathology in a Blistering Skin Disease Model

There remain major gaps in our knowledge regarding the detailed mechanisms by which autoantibodies mediate damage at the tissue level. We have undertaken novel strategies at the interface of engineering and clinical medicine to integrate nanoscale visual and structural data using nanorobotic atomic force microscopy with cell functional analyses to reveal previously unattainable details of autoimmune processes in real-time. Pemphigus vulgaris is a life-threatening autoimmune blistering skin condition in which there is disruption of desmosomal cell-cell adhesion structures that are associated with the presence of antibodies directed against specific epithelial proteins including Desmoglein (Dsg) 3. We demonstrate that pathogenic (blister-forming) anti-Dsg3 antibodies, distinct from non-pathogenic (non-blister forming) anti-Dsg3 antibodies, alter the structural and functional properties of keratinocytes in two sequential steps - an initial loss of cell adhesion and a later induction of apoptosis-related signaling pathways, but not full apoptotic cell death. We propose a “2-Hit” model for autoimmune disruption associated with skin-specific pathogenic autoantibodies. These data provide unprecedented details of autoimmune processes at the tissue level and offer a novel conceptual framework for understanding the action of self-reactive antibodies.     

Toxin in bullous impetigo and staphylococcal scalded-skin syndrome targets desmoglein 1

Exfoliative toxin A, produced by Staphylococcus aureus, causes blisters in bullous impetigo and its more generalized form, staphylococcal scalded-skin syndrome. The toxin shows exquisite specificity in causing loss of cell adhesion only in the superficial epidermis. Although exfoliative toxin A has the structure of a serine protease, a target protein has not been identified. Desmoglein (Dsg) 1, a desmosomal cadherin that mediates cell-cell adhesion, may be the target of exfoliative toxin A, because it is the target of autoantibodies in pemphigus foliaceus, in which blisters form with identical tissue specificity and histology. We show here that exfoliative toxin A cleaved mouse and human Dsg1, but not closely related cadherins such as Dsg3. We demonstrate this specific cleavage in cell culture, in neonatal mouse skin and with recombinant Dsg1, and conclude that Dsg1 is the specific receptor for exfoliative toxin A cleavage. This unique proteolytic attack on the desmosome causes a blister just below the stratum corneum, which forms the epidermal barrier, presumably allowing the bacteria in bullous impetigo to proliferate and spread beneath this barrier.     

Desmoglein 3, via an interaction with E-cadherin, is associated with activation of Src

Background

Desmoglein 3 (Dsg3), a desmosomal adhesion protein, is expressed in basal and immediate suprabasal layers of skin and across the entire stratified squamous epithelium of oral mucosa. However, increasing evidence suggests that the role of Dsg3 may involve more than just cell-cell adhesion.

Methodology/Principal Findings

To determine possible additional roles of Dsg3 during epithelial cell adhesion we used overexpression of full-length human Dsg3 cDNA, and RNAi-mediated knockdown of this molecule in various epithelial cell types. Overexpression of Dsg3 resulted in a reduced level of E-cadherin but a colocalisation with the E-cadherin-catenin complex of the adherens junctions. Concomitantly these transfected cells exhibited marked migratory capacity and the formation of filopodial protrusions. These latter events are consistent with Src activation and, indeed, Src-specific inhibition reversed these phenotypes. Moreover Dsg3 knockdown, which also reversed the decreased level of E-cadherin, partially blocked Src phosphorylation.

Conclusions/Significance

Our data are consistent with the possibility that Dsg3, as an up-stream regulator of Src activity, helps regulate adherens junction formation.     

Coordinated expression of desmoglein 1 and desmocollin 1 regulates intercellular adhesion

Desmoglein 1 (Dsg1) is a component of desmosomes present in the upper epidermis and can be targeted by autoimmune antibodies or bacterial toxins, resulting in skin blistering diseases. These defects in tissue integrity are believed to result from compromised desmosomal adhesion; yet, previous attempts to directly test the adhesive roles of desmosomal cadherins using normally non-adherent L cells have yielded mixed results. Here, two complementary approaches were used to better resolve the molecular determinants for Dsg1-mediated adhesion: (1) a tetracycline-inducible system was used to modulate the levels of Dsg1 expressed in L cell lines containing desmocollin 1 (Dsc1) and plakoglobin (PG) and (2) a retroviral gene delivery system was used to introduce Dsg1 into normal human epidermal keratinocytes (NHEK). By increasing Dsg1 expression relative to Dsc1 and PG, we were able to demonstrate that the ratio of Dsg1:Dsc1 is a critical determinant of desmosomal adhesion in fibroblasts. The distribution of Dsg1 was organized at areas of cell-cell contact in the multicellular aggregates that formed in these suspension cultures. Similarly, the introduction of Dsg1 into NHEKs was capable of increasing the aggregation of single cell suspensions and further enhanced the adhesive strength of intact epithelial sheets. Endogenous Dsc1 levels were also increased in NHEKs containing Dsg1, providing further support for the coordination of these two desmosomal cadherins in regulating adhesive structures. These Dsg1-mediated effects on intercellular adhesion were directly related to the presence of an intact extracellular domain as ETA, a toxin that specifically cleaves this desmosomal cadherin, inhibited adhesion in both fibroblasts and keratinocytes. Collectively, these observations demonstrate that Dsg1 promotes the formation of intercellular adhesion complexes and suggest that the relative level of Dsg and Dsc expressed at the cell surface regulates this adhesive process.     

Loss of desmoglein 2 suggests essential functions for early embryonic development and proliferation of embryonal stem cells

Desmoglein 2 (Dsg2) is a Ca(2+)-dependent adhesion molecule of desmosomes and is synthesized in all desmosome-bearing tissues from their earliest appearance onward. To examine the function of Dsg2, its gene was inactivated by homologous recombination in embryonal stem (ES) cells for the generation of knockout mice. DSG2 -/- mice and a considerable number of DSG2 +/- mice died at or shortly after implantation. On the other hand, DSG2 -/- blastocysts developed an apparently normal trophectoderm layer, the first tissue known to produce desmosomes, and hatched properly. Immunofluorescence analyses of these blastocysts showed, however, that the distribution of the desmosomal plaque protein desmoplakin was disturbed, whereas the adherens junction proteins E-cadherin and beta-catenin appeared to be unaffected. Unexpectedly, we found that Dsg2 seems to be essential for the inner cell mass and the ES cell population derived there from. We present evidence that Dsg2, which is located in desmoplakin-negative wild-type ES cells in non-desmosomal junctions, is needed for normal ES cell proliferation. Our observations thus reveal that important Dsg2 functions are desmosome-independent during early development and are needed for ES cell and early embryo survival.     

Desmoglein 3 acting as an upstream regulator of Rho GTPases, Rac-1/Cdc42 in the regulation of actin organisation and dynamics

Desmoglein 3 (Dsg3), a member of the desmoglein sub-family, serves as an adhesion molecule in desmosomes. Our previous study showed that overexpression of human Dsg3 in several epithelial lines induces formation of membrane protrusions, a phenotype suggestive of Rho GTPase activation. Here we examined the interaction between Dsg3 and actin in detail and showed that endogenous Dsg3 colocalises and interacts with actin, particularly the junctional actin in a Rac1-dependent manner. Ablation of Rac1 activity by dominant negative Rac1 mutant (N17Rac1) or the Rac1 specific inhibitor (NSC23766) directly disrupts the interaction between Dsg3 and actin. Assembly of the junctional actin at the cell borders is accompanied with enhanced levels of Dsg3, while inhibition of Dsg3 by RNAi results in profound changes in the organisation of actin cytoskeleton. In accordance, overexpression of Dsg3 results in a remarkable increase of Rac1 and Cdc42 activities and to a lesser extent, RhoA. The enhancements in Rho GTPases are accompanied by the pronounced actin-based membrane structures such as lamellipodia and filopodia, enhanced rate of actin turnover and cell polarisation. Together, our results reveal an important novel function for Dsg3 in promoting actin dynamics through regulating Rac1 and Cdc42 activation in epithelial cells.     

A monoclonal antibody to the desmosomal glycoprotein desmoglein I binds the same polypeptide as human autoantibodies in pemphigus foliaceus

Recent studies have demonstrated that antibodies from about half of patients with pemphigus foliaceus (PF) bind to a 160 kd polypeptide ("PF antigen") in sodium dodecyl sulfate (SDS) extracts of normal human epidermis. Desmoglein (DG) I, a glycoprotein enriched in desmosomal cores, is approximately the same m.w. as PF antigen. To demonstrate that PF autoantibodies bind to DG I, we used a monoclonal IgG antibody (MmDGI-1) that was raised against bovine muzzle desmosomal cores, and that specifically binds DG I. Double immunofluorescence labeling was performed on the same section of normal human skin with PF antibodies, detected by fluorescein-conjugated goat anti-human IgG, and MmDGI-1, detected by rhodamine-conjugated goat anti-mouse IgG. The pattern of reactivity with both antibodies was identical. Immunoblotting studies on proteins extracted from normal human epidermis and separated by SDS-polyacrylamide gel electrophoresis demonstrated that PF antibodies and MmDGI-1 bound co-migrating polypeptide bands of approximate m.w. 160,000. To confirm that these were identical polypeptides, we performed immunoblots of these epidermal extracts that were separated by two-dimensional gel electrophoreses (isoelectric focusing followed by SDS-PAGE). PF antibodies and MmDGI-1 bound identical spots with pI approximately 5.4 to 5.7 and m.w. approximately 160,000. These studies demonstrate that autoantibodies from certain patients with PF, a disorder of cell adhesion, bind to DG I, a desmosomal core glycoprotein.     

Increased expression of Dsg2 in malignant skin carcinomas: A tissue-microarray based study

Desmoglein 2 (Dsg2), a transmembrane cadherin of the desmosomal cell-cell adhesion structure, is downregulated with epithelial differentiation. We recently demonstrated that overexpression of Dsg2 in epidermal keratinocytes deregulates multiple signaling pathways associated with increased growth rate, anchorage-independent cell survival, and the development of skin tumors. While changes in Dsg2 expression have been observed in neoplastic lesions, the correlation of expression levels and localization of Dsg2 and the state of tumor development has not been fully established. Here we generated a highly sensitive Dsg2 antibody (Ab10) and characterized that antibody along with a previously developed Dsg2 specific antibody 10D2. Using these antibodies in immunostaining of tissue microarrays, we show a dramatic upregulation of Dsg2 expression in certain human epithelial malignancies including basal cell carcinomas (BCC; n = 12), squamous cell carcinomas (SCC; n = 57), carcinomas of sebaceous and sweat glands (n = 12), and adenocarcinomas (n = 3). Dsg2 expression was completely absent in malignant fibrosarcomas (n = 16) and melanomas (n = 15). While Dsg2 expression was consistently strong in BCC, it varied in SCC with a minor correlation between a decrease of Dsg2 expression and tumor differentiation. In summary, we have identified Dsg2 as a potential novel marker for epithelial-derived malignancies.Key words: carcinogenesis, desmoglein, desmosome, skin     

Direct Ca2+-dependent Heterophilic Interaction between Desmosomal Cadherins, Desmoglein and Desmocollin, Contributes to Cell–Cell Adhesion

Human fibrosarcoma cells, HT-1080, feature extensive adherens junctions, lack mature desmosomes, and express a single known desmosomal protein, Desmoglein 2 (Dsg2). Transfection of these cells with bovine Desmocollin 1a (Dsc1a) caused dramatic changes in the subcellular distribution of endogenous Dsg2. Both cadherins clustered in the areas of the adherens junctions, whereas only a minor portion of Dsg2 was seen in these areas in the parental cells. Deletion mapping showed that intact extracellular cadherin-like repeats of Dsc1a (Arg¹-Thr¹⁷⁰) are required for the translocation of Dsg2. Deletion of the intracellular C-domain that mediates the interaction of Dsc1a with plakoglobin, or the CSI region that is involved in the binding to desmoplakin, had no effect. Coimmunoprecipitation experiments of cell lysates stably expressing Dsc1a with anti-Dsc or -Dsg antibodies demonstrate that the desmosomal cadherins, Dsg2 and Dsc1a, are involved in a direct Ca²⁺-dependent interaction. This conclusion was further supported by the results of solid phase binding experiments. These showed that the Dsc1a fragment containing cadherin-like repeats 1 and 2 binds directly to the extracellular portion of Dsg in a Ca²⁺-dependent manner. The contribution of the Dsg/ Dsc interaction to cell–cell adhesion was tested by coculturing HT-1080 cells expressing Dsc1a with HT-1080 cells lacking Dsc but expressing myc-tagged plakoglobin (MPg). In the latter cells, MPg and the endogenous Dsg form stable complexes. The observed specific coimmunoprecipitation of MPg by anti-Dsc antibodies in coculture indicates that an intercellular interaction between Dsc1 and Dsg is involved in cell–cell adhesion.     

The human gene (DSG2) coding for HDGC, a second member of the desmoglein subfamily of the desmosomal cadherins, is, like DSG1 coding for desmoglein DGI, assigned to chromosome 18.

Desmoglein is a transmembrane glycoprotein of the cadherin superfamily present in the desmosomal junction in vertebrate epithelial cells. At least two variants of desmoglein are differentially expressed in human tissues: DGI, a characteristic desmosomal protein; and HDGC, which is, for example, expressed in the simple epithelium of the colon. Using a PCR assay, we were able to assign DSG2, the gene coding for desmoglein HDGC, to chromosome 18, the same chromosomal localization to which we have previously assigned DSG1 coding for desmoglein DGI.     

Dominant autoimmune epitopes recognized by pemphigus antibodies map to the N-terminal adhesive region of desmogleins.

Desmoglein (Dsg) is a cadherin-type adhesion molecule found in desmosomes. Dsg1 and Dsg3 are the target Ags in the autoimmune blistering diseases pemphigus foliaceus (PF) and pemphigus vulgaris (PV), respectively. To map conformational epitopes of Dsg1 and Dsg3 in PF and PV, we generated Dsg1- and Dsg3-domain-swapped molecules and point-mutated Dsg3 molecules with Dsg1-specific residues by baculovirus expression. The swapped domains were portions of the N-terminal extracellular domains of Dsg1 (1-496) and Dsg3 (1-566), which have similar structures but distinct epitopes. The binding of autoantibodies to the mutant molecules was assessed by competition ELISAs. Domain-swapped molecules containing the N-terminal 161 residues of Dsg1 and Dsg3 yielded >50% competition in 30/43 (69.8%) PF sera and 31/40 (77.5%) PV sera, respectively. Furthermore, removal of Abs against the 161 N-terminal residues of Dsg1 by immunoadsorption eliminated the ability of PF sera to induce cutaneous blisters in neonatal mice. Within these N-terminal regions, most of the epitopes were mapped to residues 26-87 of Dsg1 and 25-88 of Dsg3. Furthermore, a point-mutated Dsg3 molecule containing Dsg1-specific amino acid substitutions (His(25), Cys(28), Ala(29)) reacted with anti-Dsg1 IgG, thus defining one of the epitopes of Dsg1. Using the predicted three-dimensional structure of classic cadherins as a model, these findings suggest that the dominant autoimmune epitopes in both PF and PV are found in the N-terminal adhesive surfaces of Dsgs.     

Characterization of desmoglein‐3 epitope region peptides as synthetic antigens: analysis of their in vitro T cell stimulating efficacy,cytotoxicity, stability,and their conformational features

Desmoglein‐3 (Dsg3) adhesion protein is the main target of autoantibodies and autoreactive T cells in Pemphigus vulgaris (PV) autoimmune skin disorder. Several mapping studies of Dsg3 T cell epitope regions were performed, and based on those data, we designed and synthesized four peptide series corresponding to Dsg3 T cell epitope regions. Each peptide series consists of a 17mer full‐length peptide (Dsg3/189–205, Dsg3/206–222, Dsg3/342–358, and Dsg3/761–777) and its N‐terminally truncated derivatives, resulting in 15 peptides altogether. The peptides were prepared on solid phase and were chemically characterized. In order to establish a structure–activity relationship, the solution conformation of the synthetic peptides has been investigated using electronic circular dichroism spectroscopy. The in vitro T cell stimulating efficacy of the peptides has been determined on peripheral blood mononuclear cells isolated from whole blood of PV patients and also from healthy donors. After 20 h of stimulation, the interferon (IFN)‐γ content of the supernatants was measured by enzyme‐linked immunosorbent assay. In the in vitro conditions, peptides were stable and non‐cytotoxic. The in vitro IFN‐γ production profile of healthy donors and PV patients, induced by peptides as synthetic antigens, was markedly different. The most unambiguous differences were observed after stimulation with 17mer peptide Dsg3/342–358, and three truncated derivatives from two other peptide series, namely, peptides Dsg3/192–205, Dsg3/763–777, and Dsg3/764–777. Comparative analysis of in vitro activity and the capability of oligopeptides to form ordered or unordered secondary structure showed that peptides bearing high solvent sensibility and backbone flexibility were the most capable to distinguish between healthy and PV donors. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Desmoglein 2 can undergo Ca2+-dependent interactions with both desmosomal and classical cadherins including E-cadherin and N-cadherin

Desmoglein (Dsg) 2 is a ubiquitously expressed desmosomal cadherin. Particularly, it is present in all cell types forming desmosomes, including epithelial cells and cardiac myocytes and is upregulated in the autoimmune skin disease pemphigus. Thus, we here characterized the binding properties of Dsg2 in more detail using atomic force microscopy (AFM). Dsg2 exhibits homophilic interactions and also heterophilic interactions with the desmosomal cadherin desmocollin (Dsc) 2, and further with the classical cadherins E-cadherin (E-Cad) and N-cadherin (N-Cad), which may be relevant for cross talk between desmosomes and adherens junctions in epithelia and cardiac myocytes. We found that all homo- and heterophilic interactions were Ca²⁺-dependent. All binding forces observed are in the same force range, i.e., 30 to 40 pN, except for the Dsg2/E-Cad unbinding force, which with 45 pN is significantly higher. To further characterize the nature of the interactions, we used tryptophan, a critical amino acid required for trans-interaction, and a tandem peptide (TP) designed to cross-link Dsg isoforms. TP was sufficient to prevent the tryptophan-induced loss of Dsg2 interaction with the desmosomal cadherins Dsg2 and Dsc2; however, not with the classical cadherins E-Cad and N-Cad, indicating that the interaction modes of Dsg2 with desmosomal and classical cadherins differ. TP rescued the tryptophan-induced loss of Dsg2 binding on living enterocytes, suggesting that interaction with desmosomal cadherins may be more relevant. In summary, the data suggest that the ubiquitous desmosomal cadherin Dsg2 enables the cross talk with adherens junctions by interacting with multiple binding partners with implications for proper adhesive function in healthy and diseased states.     

Phenotypic characterization of human umbilical vein endothelial (ECV304) and urinary carcinoma (T24) cells: Endothelial versus epithelial features

ECV304 cells reported as originating from human umbilical vein endothelial cells by spontaneous transformation have been used as a model cell line for endothelia over the last decade. Recently, deoxyribonucleic acid fingerprinting revealed an identical genotype for ECV304 and T24 cells (urinary bladder carcinoma cell line). In order to resolve the apparent discrepancy between the identical genotype and the fact that ECV304 cells phenotypically show important endothelial characteristics, a comparative study was performed. Immortalized porcine brain microvascular endothelial cells/C1-2, and Madin Darby canine kidney cells were included as typical endothelial and epithelial cells, respectively. Various methods, such as confocal laser scanning microscopy. Western blot, and protein activity tests, were used to study the cell lines. ECV304 and T24 cells differ in criteria, such as growth behavior, cytoarchitecture, tight junction arrangement. transmembrane electrical resistance, and activity of gamma-glutamyltransferase. Several endothelial markers (von Willebrand factor, uptake of low-density lipoprotein, vimentin) could clearly be identified in ECV304, but not in T24 cells. Desmoglein and cytokeratin, both known as epithelial markers, were found in ECV304 as well as in T24 tells. However, differences were found for the two cell lines with respect to the type of cytokeratin: in ECV304 cells mainly cytokeratin 18 (45 kDa) is found, whereas in T24 cells cytokeratin 8 (52 kDa) is predominant. As we could demonstrate, the ECV304 cell line exposes many endothelial features which, in view of the scarcity of suitable endothelial cell lines, still make it an attractive in vitro model for endothelia.