Upregulation of transmembrane endothelial junction proteins in human cerebral cavernous malformations

OBJECT: Cerebral cavernous malformations (CCMs) are among the most prevalent cerebrovascular malformations, and endothelial cells seem to play a major role in the disease. However, the underlying mechanisms, including endothelial intercellular communication, have not yet been fully elucidated. In this article, the authors focus on the endothelial junction proteins CD31, VE-cadherin, and occludin as important factors for functional cell-cell contacts known as vascular adhesion molecules and adherence and tight junctions. METHODS: Thirteen human CCM specimens and 6 control tissue specimens were cryopreserved and examined for the presence of VE-cadherin, occludin, and CD31 by immunofluorescence staining. Protein quantification was performed by triplicate measurements using western blot analysis. RESULTS: Immunofluorescent analyses of the CCM sections revealed a discontinuous pattern of dilated microvessels and capillaries as well as increased expression of occludin, VE-cadherin, and CD31 in the intima and in the enclosed parenchymal tissue compared with controls. Protein quantification confirmed these findings by showing upregulation of the levels of these proteins up to 2-6 times. CONCLUSIONS: A protocol enabling the molecular and morphological examination of the intercellular contact proteins in human CCM was validated. The abnormal and discontinuous pattern in these endothelial cell-contact proteins compared with control tissue explains the loose intercellular junctions that are considered to be one of the causes of CCM-associated bleeding or transendothelial oozing of erythrocytes. Despite the small number of specimens, this study demonstrates for the first time a quantitative analysis of endothelial junction proteins in human CCM

Apoptosis screening of human chromosome 21 proteins reveals novel cell death regulators

The functional analysis of chromosome 21 (Chr21) proteins is of great medical relevance. This refers, in particular, to the trisomy of human Chr21, which results in Down’s syndrome, a complex developmental and neurodegenerative disease. In a previous study we analyzed 89 human Chr21 genes for the subcellular localization of their encoded proteins using a transfected-cell array technique. In the present study, the results of the follow-up investigation are presented in which 52 human Chr21 genes were over-expressed in HEK cells using the transfected-cell array platform, and the effect of this protein over-expression on the induction of apoptosis has been analyzed. We found that the over-expression of two Chr21 proteins (claudin-14 and -8) induced cell death independent of the classic caspase-mediated apoptosis. Our results strongly suggest the functional involvement of claudins in the control of the cell cycle and regulation of the cell death induction mechanism

Divergent expression of claudin -1, -3, -4, -5 and -7 in developing human lung

<p>Abstract</p> <p>Background</p> <p>Claudins are the main components of tight junctions, structures which are associated with cell polarity and permeability. The aim of this study was to analyze the expression of claudins 1, 3, 4, 5, and 7 in developing human lung tissues from 12 to 40 weeks of gestation.</p> <p>Methods</p> <p>47 cases were analyzed by immunohistochemisty for claudins 1, 3, 4, 5 and 7. 23 cases were also investigated by quantitative RT-PCR for claudin-1, -3 and -4.</p> <p>Results</p> <p>Claudin-1 was expressed in epithelium of bronchi and large bronchioles from week 12 onwards but it was not detected in epithelium of developing alveoli. Claudin-3, -4 and -7 were strongly expressed in bronchial epithelium from week 12 to week 40, and they were also expressed in alveoli from week 16 to week 40. Claudin-5 was expressed strongly during all periods in endothelial cells. It was expressed also in epithelium of bronchi from week 12 to week 40, and in alveoli during the canalicular period. RT-PCR analyses revealed detectable amounts of RNAs for claudins 1, 3 and 4 in all cases studied.</p> <p>Conclusion</p> <p>Claudin-1, -3, -4, -5, and -7 are expressed in developing human lung from week 12 to week 40 with distinct locations and in divergent quantities. The expression of claudin-1 was restricted to the bronchial epithelium, whereas claudin-3, -4 and -7 were positive also in alveolar epithelium as well as in the bronchial epithelium. All claudins studied are linked to the development of airways, whereas claudin-3, -4, -5 and -7, but not claudin-1, are involved in the development of acinus and the differentiation of alveolar epithelial cells.</p

The claudin gene family: expression in normal and neoplastic tissues

BACKGROUND: The claudin (CLDN) genes encode a family of proteins important in tight junction formation and function. Recently, it has become apparent that CLDN gene expression is frequently altered in several human cancers. However, the exact patterns of CLDN expression in various cancers is unknown, as only a limited number of CLDN genes have been investigated in a few tumors. METHODS: We identified all the human CLDN genes from Genbank and we used the large public SAGE database to ascertain the gene expression of all 21 CLDN in 266 normal and neoplastic tissues. Using real-time RT-PCR, we also surveyed a subset of 13 CLDN genes in 24 normal and 24 neoplastic tissues. RESULTS: We show that claudins represent a family of highly related proteins, with claudin-16, and -23 being the most different from the others. From in silico analysis and RT-PCR data, we find that most claudin genes appear decreased in cancer, while CLDN3, CLDN4, and CLDN7 are elevated in several malignancies such as those originating from the pancreas, bladder, thyroid, fallopian tubes, ovary, stomach, colon, breast, uterus, and the prostate. Interestingly, CLDN5 is highly expressed in vascular endothelial cells, providing a possible target for antiangiogenic therapy. CLDN18 might represent a biomarker for gastric cancer. CONCLUSION: Our study confirms previously known CLDN gene expression patterns and identifies new ones, which may have applications in the detection, prognosis and therapy of several human cancers. In particular we identify several malignancies that express CLDN3 and CLDN4. These cancers may represent ideal candidates for a novel therapy being developed based on CPE, a toxin that specifically binds claudin-3 and claudin-4

Enhancement of metastatic ability by ectopic expression of ST6GalNAcI on a gastric cancer cell line in a mouse model

ST6GalNAcI is a sialyltransferase responsible for the synthesis of sialyl Tn (sTn) antigen which is expressed in a variety of adenocarcinomas including gastric cancer especially in advanced cases, but the roles of ST6GalNAcI and sTn in cancer progression are largely unknown. We generated sTn-expressing human gastric cancer cells by ectopic expression of ST6GalNAcI to evaluate metastatic ability of these cells and prognostic effect of ST6GalNAcI and sTn in a mouse model, and identified sTn carrier proteins to gain insight into the function of ST6GalNAcI and sTn in gastric cancer progression. A green fluorescent protein-tagged human gastric cancer cell line was transfected with ST6GalNAcI to produce sTn-expressing cells, which were transplanted into nude mice. STn-positive gastric cancer cells showed higher intraperitoneal metastatic ability in comparison with sTn-negative control, resulting in shortened survival time of the mice, which was mitigated by anti-sTn antibody administration. Then, sTn-carrying proteins were immunoprecipitated from culture supernatants and lysates of these cells, and identified MUC1 and CD44 as major sTn carriers. It was confirmed that MUC1 carries sTn also in human advanced gastric cancer tissues. Identification of sTn carrier proteins will help understand mechanisms of metastatic phenotype acquisition of gastric cancer cells by ST6GalNAcI and sTn

A Claudin-9–Based Ion Permeability Barrier Is Essential for Hearing

Hereditary hearing loss is one of the most common birth defects, yet the majority of genes required for audition is thought to remain unidentified. Ethylnitrosourea (ENU)–mutagenesis has been a valuable approach for generating new animal models of deafness and discovering previously unrecognized gene functions. Here we report on the characterization of a new ENU–induced mouse mutant (nmf329) that exhibits recessively inherited deafness. We found a widespread loss of sensory hair cells in the hearing organs of nmf329 mice after the second week of life. Positional cloning revealed that the nmf329 strain carries a missense mutation in the claudin-9 gene, which encodes a tight junction protein with unknown biological function. In an epithelial cell line, heterologous expression of wild-type claudin-9 reduced the paracellular permeability to Na+ and K+, and the nmf329 mutation eliminated this ion barrier function without affecting the plasma membrane localization of claudin-9. In the nmf329 mouse line, the perilymphatic K+ concentration was found to be elevated, suggesting that the cochlear tight junctions were dysfunctional. Furthermore, the hair-cell loss in the claudin-9–defective cochlea was rescued in vitro when the explanted hearing organs were cultured in a low-K+ milieu and in vivo when the endocochlear K+-driving force was diminished by deletion of the pou3f4 gene. Overall, our data indicate that claudin-9 is required for the preservation of sensory cells in the hearing organ because claudin-9–defective tight junctions fail to shield the basolateral side of hair cells from the K+-rich endolymph. In the tight-junction complexes of hair cells, claudin-9 is localized specifically to a subdomain that is underneath more apical tight-junction strands formed by other claudins. Thus, the analysis of claudin-9 mutant mice suggests that even the deeper (subapical) tight-junction strands have biologically important ion barrier function

Chitosan encapsulation modulates the effect of capsaicin on the tight junctions of MDCK cells

Capsaicin has known pharmacological effects including the ability to reversibly open cellular tight junctions, among others. The aim of this study was to develop a strategy to enhance the paracellular transport of a substance with low permeability (FITC-dextran) across an epithelial cell monolayer via reversible opening of cellular tight junctions using a nanosystem comprised by capsaicin and of chitosan. We compared the biophysical properties of free capsaicin and capsaicin-loaded chitosan nanocapsules, including their cytotoxicity towards epithelial MDCK-C7 cells and their effect on the integrity of tight junctions, membrane permeability and cellular uptake. The cytotoxic response of MDCK-C7 cells to capsaicin at a concentration of 500 μM, which was evident for the free compound, is not observable following its encapsulation. The interaction between nanocapsules and the tight junctions of MDCK-C7 cells was investigated by impedance spectroscopy, digital holographic microscopy and structured illumination fluorescence microscopy. The nanocapsules modulated the interaction between capsaicin and tight junctions as shown by the different time profile of trans-epithelial electrical resistance and the enhanced permeability of monolayers incubated with FITC-dextran. Structured illumination fluorescence microscopy showed that the nanocapsules were internalized by MDCK-C7 cells. The capsaicin-loaded nanocapsules could be further developed as drug nanocarriers with enhanced epithelial permeability