Desmosomes undergo dynamic architectural changes during assembly and maturation.

Desmosomes are macromolecular cell-cell junctions critical for maintaining adhesion and resisting mechanical stress in epithelial tissue. Desmosome assembly and the relationship between maturity and molecular architecture are not well understood. To address this, we employed a calcium switch assay to synchronize assembly followed by quantification of desmosome nanoscale organization using direct Stochastic Optical Reconstruction Microscopy (dSTORM). We found that the organization of the desmoplakin rod/C-terminal junction changed over the course of maturation, as indicated by a decrease in the plaque-to-plaque distance, while the plaque length increased. In contrast, the desmoplakin N-terminal domain and plakoglobin organization (plaque-to-plaque distance) were constant throughout maturation. This structural rearrangement of desmoplakin was concurrent with desmosome maturation measured by E-cadherin exclusion and increased adhesive strength. Using two-color dSTORM, we showed that while the number of individual E-cadherin containing junctions went down with the increasing time in high Ca2+, they maintained a wider desmoplakin rod/C-terminal plaque-to-plaque distance. This indicates that the maturation state of individual desmosomes can be identified by their architectural organization. We confirmed these architectural changes in another model of desmosome assembly, cell migration. Desmosomes in migrating cells, closest to the scratch where they are assembling, were shorter, E-cadherin enriched, and had wider desmoplakin rod/C-terminal plaque-to-plaque distances compared to desmosomes away from the wound edge. Key results were demonstrated in three cell lines representing simple, transitional, and stratified epithelia. Together, these data suggest that there is a set of architectural programs for desmosome maturation, and we hypothesize that desmoplakin architecture may be a contributing mechanism to regulating adhesive strength.

Single-Cell Analysis of Highly Metastatic Circulating Tumor Cells by Combining a Self-Folding Induced Release Reaction with a Cell Capture Microchip.

Nondestructive analysis of the single-cell molecular phenotype of circulating tumor cells (CTCs) is of great significance to the precise diagnosis and treatment of cancer but is also a huge challenge. To address this issue, here, we develop a facile analysis system that integrates CTCs' capture and molecular phenotype analysis. An isothermal nucleic acid amplification technique named self-folding induced release reaction (sFiR), which has high-efficiency signal amplification capabilities and can run under physiological conditions, is first developed to meet the high requirements for sensitivity and nondestructivity. By combining the sFiR with immune recognition and a single cell capture microchip, the molecular phenotype analysis of a single CTC is realized. As a model, nondestructive analysis of junction plakoglobin (JUP), an overexpressed membrane protein that is closely related to the metastasis of CTCs, is successfully achieved. Results reveal that this sFiR-based analysis system can clearly distinguish the expression of JUP in different cancer cell lines and can present quantitative information on the expression of JUP. Furthermore, the captured and analyzed CTCs maintain their basic physiological activity and can be used for drug sensitivity testing. Considering the excellent performance and ease of operation of the system, it can provide technical support for CTC-based cancer liquid biopsy and drug development.

Advanced Methods for the Investigation of Cell Contact Dynamics in Endothelial Cells Using Florescence-Based Live Cell Imaging.

Endothelial cells of the vascular system are dynamic cells whose molecular adaptability is decisive for the adjustment of homeostasis and organ perfusion. Advanced microscopic techniques, automation processing, and image analysis software was shown to improve the understanding of vascular biology. In this work, we describe advanced methods that allow investigating the dynamics of endothelial cell contacts. The development of viral vectors has contributed significantly to the genetic manipulation of endothelial cells. We used the Gibson assembly as a quick and cheap cloning system for introducing sequences into the lentiviral-based pFUGW vector. Furthermore, classical fluorescence tags such as mCherry and EGFP were compared with self-labeling tags such as Halo and SNAP for their suitability to study junction dynamics in cultured endothelium, and found the self-labeling tags as useful tools. Using such combinations, we found maintained cell junction integrity during shear stress-induced junction remodeling using VE-cadherin-EGFP. Remodeling was accompanied by VE-cadherin plaque formation, indicating that this process is mediated by the for-mation of the actin-driven junction-associated intermittent lamellipodia, JAIL. The combined methods including the Gibson assembly, lentiviral mediated gene transfer, spinning disk-based live cell imaging, and software for quantification allow analyses of the endothelial cell junction dynamics under static and under shear stress conditions.

Plakoglobin immunolocalization as a diagnostic test for arrhythmogenic right ventricular cardiomyopathy.

BACKGROUND: A recent study using an anti-plakoglobin antibody and immunofluorescence methods in endomyocardial tissue specimens found that a marked reduction in plakoglobin staining was highly sensitive and specific for the diagnosis of arrhythmogenic right ventricular cardiomyopathy (ARVC). The purpose of our study was to determine the diagnostic utility of plakoglobin immunolocalization using more standard immunoperoxidase methods suitable for clinical laboratories. METHODS: Between January 2007 and October 2010, all patients at our center with suspected ARVC underwent noninvasive and genetic testing, right ventricular (RV) angiography, electrophysiologic studies, and endomyocardial biopsy from the RV septum. Several studies using anti-plakoglobin antibodies were performed using standard immunoperoxidase methods at concentrations of 1:50,000 and 1:75,000 after serial dilutions. RESULTS: Among 16 patients, nine patients fulfilled the clinical criteria for ARVC, and seven patients were found to have other cardiac diagnoses. In the initial study (1:50,000) only one of nine ARVC patients showed reduced plakoglobin signal while the others had normal staining. On repeat staining (1:75,000), reduced signal was observed in three of five of the ARVC patients compared to none in controls (four patients did not have adequate tissue for the repeat experiment). CONCLUSION: These results confirm that abnormal plakoglobin staining can differentiate biopsies from patients with ARVC from those with other myopathies, but with low sensitivity. Further, each specimen must be studied at a particular concentration due to variable antibody reactivity. The necessity for such fine-tuning of the reaction, as well as the subjectivity involved in interpretation of the results, would make this method difficult to utilize in routine hospital laboratories.

Immune expression of E-cadherin and α, ß and γ-Catenin adhesion molecules and prognosis for upper urinary tract urothelial carcinomas.

INTRODUCTION: Cell adhesion molecules (CAM) are required for maintaining a normal epithelial phenotype, and abnormalities in CAM expression have been related to cancer progression, including bladder urothelial carcinomas. There is only one study that correlates E-cadherin and Α-, Β- and y-catenin expression with prognosis of upper tract urothelial carcinomas. Our aim is to study the pattern of immune expression of these CAMs in urothelial carcinomas from the renal pelvis and ureter in patients who have been treated surgically. Our goal is to correlate these expression levels and characteristics with well-known prognostic parameters for disease-free survival. MATERIALS AND METHODS: We evaluated specimens from 20 patients with urothelial carcinomas of the renal pelvis and ureter who were treated with nephroureterectomy or ureterectomy between June 1997 and January 2007. CAM expression was evaluated by immunohistochemistry in a tissue microarray and correlated with histopathological characteristics and patient outcomes after a mean follow-up of 55 months. RESULTS: We observed a relationship between E-cadherin expression and disease recurrence. Disease recurrence occurred in 87.5% of patients with strong E-cadherin expression. Only 50.0% of patients with moderate expression and 0% of patients with weak or no expression of E-cadherin had disease recurrence (p = 0.014). There was also a difference in disease-free survival. Patients with strong E-cadherin expression had a mean disease-free survival rate of 49.1 months, compared to 83.9 months for patients with moderate expression (p = 0.011). Additionally, an absence of Α-catenin expression was associated with tumors that were larger than 3 cm (p = 0.003). CONCLUSIONS: We demonstrated for the first time that immune expression of E-cadherin is related to tumor recurrence and disease-free survival rates, and the absence of Α-catenin expression is related to tumor size in upper tract urothelial carcinomas.

Immune expression of E-cadherin and α, β and γ-Catenin adhesion molecules and prognosis for upper urinary tract urothelial carcinomas

INTRODUCTION: Cell adhesion molecules (CAM) are required for maintaining a normal epithelial phenotype, and abnormalities in CAM expression have been related to cancer progression, including bladder urothelial carcinomas. There is only one study that correlates E-cadherin and α-, β- and γ-catenin expression with prognosis of upper tract urothelial carcinomas. Our aim is to study the pattern of immune expression of these CAMs in urothelial carcinomas from the renal pelvis and ureter in patients who have been treated surgically. Our goal is to correlate these expression levels and characteristics with well-known prognostic parameters for disease-free survival. MATERIALS AND METHODS: We evaluated specimens from 20 patients with urothelial carcinomas of the renal pelvis and ureter who were treated with nephroureterectomy or ureterectomy between June 1997 and January 2007. CAM expression was evaluated by immunohistochemistry in a tissue microarray and correlated with histopathological characteristics and patient outcomes after a mean follow-up of 55 months. RESULTS: We observed a relationship between E-cadherin expression and disease recurrence. Disease recurrence occurred in 87.5% of patients with strong E-cadherin expression. Only 50.0% of patients with moderate expression and 0% of patients with weak or no expression of E-cadherin had disease recurrence (p = 0.014). There was also a difference in disease-free survival. Patients with strong E-cadherin expression had a mean disease-free survival rate of 49.1 months, compared to 83.9 months for patients with moderate expression (p = 0.011). Additionally, an absence of α-catenin expression was associated with tumors that were larger than 3 cm (p = 0.003). CONCLUSIONS: We demonstrated for the first time that immune expression of E-cadherin is related to tumor recurrence and disease-free survival rates, and the absence of α-catenin expression is related to tumor size in upper tract urothelial carcinomas.

Assessment of desmosomal components (desmoglein 1-3, plakoglobin) in cardia mucosa in relation to gastroesophageal reflux disease and Helicobacter pylori infection.

Gastroesophageal reflux disease is associated with impaired epithelial barrier function and abnormal expression of proteins forming cell-cell contacts by tight junctions and desmosomes in distal esophageal squamous mucosa. Although gastroesophageal reflux disease and Helicobacter pylori are both associated with chronic inflammation of the adjacent cardia mucosa, it is not known whether these lead to derangements of the desmosomal complexes. Here, we assessed the expression of 4 proteins (plakoglobin and desmoglein 1, 2, and 3) forming epithelial desmosomal complexes by quantitative reverse transcription polymerase chain reaction and immunohistochemistry in biopsies from 67 patients with gastroesophageal reflux disease and 23 gastroesophageal reflux disease-negative controls. Plakoglobin and desmoglein 2 were ubiquitously expressed in all samples, whereas desmoglein 1 and 3 were not expressed in cardia mucosa. Gastroesophageal reflux disease was specifically associated with elevated transcript levels of desmoglein 2 and plakoglobin. These were significantly increased from 2.0- to 2.7-fold in patients with gastroesophageal reflux disease compared with controls (P < .01), and significantly increased immunohistochemical scores for both proteins were observed (P < .05) as well. The combined presence of gastroesophageal reflux disease and Helicobacter pylori infection had no additional effect on desmosomal gene expression. Taken together, the up-regulation of plakoglobin and desmoglein 2 in cardia mucosa of patients with gastroesophageal reflux disease supports the concept that the "transition zone" between distal esophagus and proximal stomach is affected by gastroesophageal reflux disease as well, and architectural and molecular changes in the desmosomal compartment contribute to the pathogenesis of gastroesophageal reflux disease in the cardia mucosa.

The survival effect of E-cadherin and catenins in colorectal carcinomas.

AIM: The E-cadherin/catenin complex plays an important role in epithelial tissue architecture. Decreased expression of cell adhesion molecules (E-cadherin, α-, ß- and γ-catenin) have been reported to correlate with invasive behaviour. The aim of this study was to investigate the relation between the expression of adhesion molecules and clinicopathological characteristics and survival in colorectal carcinoma. METHOD: The expression of adhesion molecules were studied by immunohistochemistry in 138 colorectal carcinomas. RESULTS: The mean age of the patients was 65 years (range: 21-89 years). In primary carcinomas, a reduction in membranous expression of E-cadherin, α-catenin, ß-catenin, γ-catenin was demonstrated (70%, 68%, 73%, 77%, respectively). Nuclear expression of ß-catenin was found in eight (5%) patients. Decreased membranous ß- and γ-catenin expression significantly correlated with tumour differentiation (P = 0.013, P = 0.03, respectively). There was a significant association between advanced stage of the tumour and decreased membranous α-catenin expression (P = 0.012). Decreased E-cadherin and ß-catenin membranous expression correlated with short survival following curative resection of the primary tumour (P = 0.04, P = 0.03, respectively). CONCLUSION: The decreased membranous expression of E-cadherin and ß-catenin and increased cytoplasmic expression of ß-catenin might be used as a prognostic marker to monitor patients with colorectal cancer.

beta- and gamma-catenin expression in oral dysplasia.

Cell-cell and cell-matrix interactions regulate important cellular functions; they involve a number of specialised molecules and the corresponding receptors, among which a key role is played by cadherins and the associated catenins. Deregulation of these molecules has been associated with tumour progression in many human malignancies. While catenins expression has been extensively studied in many human cancers, including oral carcinoma (OSCC), less is known about their expression in oral epithelial dysplasia. The objective of this study was to evaluate the expression of these proteins in a large group of displastic lesions of the oral mucosa and their relation with subsequent malignant transformation. Expression of beta- and gamma-catenin was investigated by immunohistochemistry using specific monoclonal antibodies in 49 cases of oral epithelial dysplasia and 10 samples of normal oral mucosa. The presence and absence of beta- and gamma-catenin staining was expressed differently in relation to dysplasia grade; while the degree of dysplasia became more severe, we observed a statistically significant loss and/or reduction of catenins expression, the loss of the exclusive membranous expression and a cytoplasmic delocalisation. Progression to OSCC occurred in 10 out of our 49 cases (20.4%); all of them, except one, showed a concurrent and concordantly located beta- and gamma-catenin staining even, if no statistically significant differences were found between cases progressed to invasive OSCC or not. Catenins physiology alterations may be involved in the transformation process; however, the role of catenins expression as possible prognostic markers in precancerous oral lesions seems to be limited. Nonetheless, further studies on larger series of samples are necessary in order to clarify the role of catenins expression in oral carcinogenesis from both a biological and clinical point of view.

Different effects of adenosine and calcium channel blockade on myocardial no-reflow after acute myocardial infarction and reperfusion.

BACKGROUND: Adenosine and calcium channel blockers have been used in the treatment of angiographic no-reflow directly after angioplasty for acute myocardial infarction (AMI). However, their effects on tissue perfusion after AMI and reperfusion are undefined. The present study was designed to compare the effect of adenosine with that of the calcium channel blockers diltiazem and verapamil on myocardial no-reflow. MATERIALS AND METHODS: Coronary ligation area and area of no-reflow were determined with both myocardial contrast echocardiography in vivo and histopathological evaluation in 44 Yorkshire mini-swines randomized into five study groups: ten in control, eight in adenosine-treated, nine in diltiazem-treated, nine in verapamil-treated and eight in sham-operated. An acute myocardial infarction and reperfusion model was created with 3-h occlusion of the left anterior descending coronary artery followed by 1-h reperfusion. RESULTS: Compared with the control group, adenosine significantly decreased the area of no-reflow measured with both methods from 78.5 and 82.3% to 20.7 and 21.5% of ligation area, respectively (both P < 0.01), reduced necrosis area, maintained VE-cadherin, beta-catenin and gamma-catenin levels in reflow myocardium (P < 0.05-0.01). Although diltiazem and verapamil also significantly decreased the area of no-reflow, they failed to significantly modify necrosis area, VE-cadherin, beta-catenin and gamma-catenin levels. CONCLUSIONS: These findings support the concept that adenosine can reduce both structural and functional no-reflow, while calcium channel blockade can only reduce functional no-reflow.

Aberrant retinal tight junction and adherens junction protein expression in an animal model of autosomal dominant Retinitis pigmentosa: the Rho(-/-) mouse.

Retinitis pigmentosa (RP) comprises a heterogeneous group of inherited diseases that are characterised by primary degeneration of rod photoreceptors and secondary degeneration of cone photoreceptors in the retina. Additional pathological changes include vascular changes and invasion of the inner retina by retinal pigment epithelial (RPE) cells. RP represents a major cause of progressive retinal disease worldwide. Using a mouse model of autosomal dominant Retinitis pigmentosa (adRP) with retinopathy induced by targeted disruption of the rhodopsin gene Rho(-/-), we have analysed the levels of expression of a range of tight and adherens junction associated proteins, in order to further elucidate the pathogenic mechanisms occurring at an early stage of this condition. Using western blot analysis and indirect immunostaining of retinal cryosections from 6-week-old mice from a C-129 background we have determined changes, if any, in the levels of expression and localisation of a series of tight and adherens junction associated proteins, including Zonula Occludens-1 (ZO-1), occludin, N-Cadherin, p120-Catenin, alpha-Catenin, gamma-Catenin, beta-Catenin, and E-Cadherin. We have found an up-regulation of the tight junction and adherens junction associated protein Zonula Occludens-1 (ZO-1) in the neural retina of 6-week-old Rho(-/-) knockout mice compared with 6-week-old Wild-Type (WT) mice. Following immunohistochemistry, however, it appears, that ZO-1, beta-Catenin and p120-Catenin expression at the Outer Limiting Membrane (OLM) of the Rho(-/-) retina is compromised, in part, compared to WT animals of the same age. We hypothesise that these retinal changes following photoreceptor cell death may contribute to the pathogenesis of adRP. Our findings of changes in the levels of expression of ZO-1 and associated adherens junction proteins beta-Catenin and p120-Catenin at the OLM in 6-week-old Rho(-/-) mice provide evidence for tight junction and adherens junction associated protein modifications in an animal model of autosomal dominant RP (adRP).

Role of vimentin in smooth muscle force development.

Vimentin intermediate filaments undergo spatial reorganization in cultured smooth muscle cells in response to contractile activation; however, the role of vimentin in the physiological properties of smooth muscle has not been well elucidated. Tracheal smooth muscle strips were loaded with antisense oligonucleotides (ODNs) against vimentin and then cultured for 2 days to allow for protein degradation. Treatment with vimentin antisense, but not sense, ODNs suppressed vimentin protein expression; neither vimentin antisense nor sense ODNs affected protein levels of desmin and actin. Force development in response to ACh stimulation or KCl depolarization was lower in vimentin-deficient tissues than in vimentin sense ODN- or non-ODN-treated muscle strips. Passive tension was also depressed in vimentin-depleted muscle tissues. Vimentin downregulation did not attenuate increases in myosin light chain (MLC) phosphorylation in response to contractile stimulation or basal MLC phosphorylation. In vimentin sense ODN-treated or non-ODN-treated smooth muscle strips, the desmosomal protein plakoglobin was primarily localized in the cell periphery. The membrane-associated localization of plakoglobin was reduced in vimentin-depleted muscle tissues. These studies suggest that vimentin filaments play an important role in mediating active force development and passive tension, which are not regulated by MLC phosphorylation. Vimentin downregulation impairs the structural organization of desmosomes, which may be associated with the decrease in force development.

Immunohistochemical study of the expression of adhesion molecules in ovarian serous neoplasms.

To clarify possible roles of adhesion molecules including E-cadherin, beta- and gamma-catenin, CD44s, CD44v6, CD56, and CD99 in ovarian serous neoplasms, an immunohistochemical study was undertaken for 23 benign, 40 borderline, and 95 malignant ovarian serous neoplasms using tissue microarray (TMA). Significantly reduced expression of E-cadherin, and overexpression of CD44s, CD56, and CD99 were more frequently observed in adenocarcinomas than in benign and borderline tumors. Expression of CD44v6 and nuclear beta- and gamma-catenin were detected only in borderline tumors and adenocarcinomas. Reduced expression of E-cadherin was also correlated with high tumor grade (P=0.03), presence of peritoneal seeding (P=0.03), and low overall survival rate (P=0.02). Overexpression of CD44s was significantly associated with high tumor grade (P=0.04), advanced stage (P=0.03), and low overall survival rate (P=0.02). CD56 was increasingly expressed in the case of advanced stage (P=0.005) and peritoneal seeding (P=0.001). Nuclear staining for gamma-catenin was correlated with tumor progression (P=0.004) and advanced International Federation of Gynecology and Obstetrics (FIGO) stage (P=0.02). Only CD44s expression and stage were correlated with overall survival in multivariate study. These results suggest that although E-cadherin, CD44s, CD56, and nuclear gamma-catenin immuno-expression seem to be useful prognostic markers for serous neoplasm of the ovary, CD44s expression and FIGO stage are independent prognostic factors.

Expression of E-cadherin, cadherin-11, alpha-, beta- and gamma-catenins in nephroblastomas: relationship with clinicopathological parameters, prognostic factors and outcome.

AIM: This study was undertaken to determine the expression of cell adhesion molecules E-cadherin, cadherin-11, and alpha-, beta- and gamma-catenins in nephroblastomas and to correlate this expression with pathological features and known prognostic factors. METHODS: Immunohistochemistry was performed on 140 cases of nephroblastoma following heat-induced epitope retrieval and using the streptavidin-biotin technique. RESULTS: E-cadherin was expressed in 75 cases (54%), cadherin-11 in 128 cases (91%), alpha-catenin in 93 cases (66%), beta-catenin in 133 cases (95%) and gamma-catenin in 22 cases (16%). Nuclear localisation of beta-catenin was not demonstrated. There was a statistically significant relationship between the administration of preoperative chemotherapy and the expression of E-cadherin, alpha- and gamma-catenin, respectively. These proteins were more frequently expressed in tumours treated with preoperative chemotherapy. Those tumours that expressed all four proteins (E-cadherin, alpha-, beta- and gamma-catenin) showed a statistically significant association with the administration of preoperative chemotherapy, in contrast to tumours that did not express all four proteins. CONCLUSION: Nephroblastomas show a heterogeneous distribution of staining for E-cadherin, cadherin-11, alpha-, beta- and gamma-catenins. Tumours treated with preoperative chemotherapy are more likely to express these molecules. The expression status of E-cadherin, cadherin-11 and the catenins in this cohort does not appear to be of prognostic value.

Expression of E-cadherin and alpha-, beta-, gamma-catenins in patients with bladder cancer: identification of gamma-catenin as a new prognostic marker of neoplastic progression in T1 superficial urothelial tumors.

Loss of intercellular adhesion facilitates tumor invasion. To clarify the relation between altered expression of cell adhesion molecules and progression of T1 superficial bladder tumors, 101 cases (71 T1 tumors, 30 T2/T3 tumors) were examined immunohistochemically for E-cadherin and alpha-, beta-, and gamma-catenins. A highly significant correlation was observed between the decreased expression of all molecules and increased TNM stage (P < .001). Univariate analysis, performed in cases of T1 tumors, revealed association of abnormal E-cadherin with beta-catenin diminution. Survival curves were established with the Kaplan-Meier method and analyzed according to clinical and histopathologic parameters using the log-rank test. Cox multivariate analysis revealed only gamma-catenin as an independent predictor of progression-free survival in patients with stage T1 bladder urothelial tumors. The characterization of T1 tumors that will progress could lead to the identification of patients who might benefit from surgery to avoid vesical muscle invasion and, consequently, metastasis.

Prognostic significance of E-cadherin-catenin complex in epithelial ovarian cancer.

OBJECTIVE: To clarify the prognostic role of E-cadherin and beta- and gamma-catenins, and their relation to CD44 in epithelial ovarian carcinoma. METHODS: The expression of E-cadherin and beta- and gamma-catenins was analysed immunohistochemically in 305 primary epithelial ovarian cancers and 44 metastases, and related to CD44 expression, clinicopathological factors, and the patients' survival. RESULTS: Reduced cell surface expression of E-cadherin, beta-catenin, and gamma-catenin was particularly frequent in serous and endometrioid histological types. Reduced cell surface expression of E-cadherin and beta-catenin was also associated with poor differentiation. Nuclear positivity of beta-catenin was associated with high CD44 expression, endometrioid histology, and local stage of the tumour, whereas nuclear gamma-catenin expression was associated with serous histology and poor differentiation. In the univariate analysis, preserved cell surface beta-catenin expression in the whole study material and nuclear expression of beta- and gamma-catenins in the subgroup of endometrioid ovarian cancers were predictors of better 10 year disease related survival. Preserved cell surface expression of E-cadherin and beta-catenin predicted favourable recurrence-free survival. These statistical significances were not retained in multivariate analysis. CONCLUSIONS: The correlation between nuclear beta-catenin and CD44 indicates that beta-catenin may regulate the transcription of CD44 in epithelial ovarian cancer. E-cadherin-catenin complex members are associated with the prognosis of patients with epithelial ovarian cancer, but these univariate associations were not strong enough to compete for significance with the traditional clinicopathological factors.

The area composita of adhering junctions connecting heart muscle cells of vertebrates. I. Molecular definition in intercalated disks of cardiomyocytes by immunoelectron microscopy of desmosomal proteins.

Among sarcomeric muscles the cardiac muscle cells are unique by, inter alia, a systemic and extended cell-cell contact structure, the intercalated disk (ID), comprising frequent and closely spaced arrays of plaque-coated cell-cell adhering junctions (AJs). As some of these junctions may look somewhat like desmosomes and others like fasciae adhaerentes, the dogma has emerged in the literature that IDs contain - like epithelial cells - both kinds of AJs formed by - for the most - mutually exclusive molecular ensembles. This, however, is not the case. In comprehensive immunoelectron microscopic studies of mammalian (human, bovine, rat, mouse) and non-mammalian (chicken, amphibia, fishes) heart muscle tissues, we have localized major constituents of the desmosomal plaques of polar epithelia, desmoplakin, plakophilin-2 and plakoglobin, as well as the desmosomal cadherins, desmoglein Dsg2 and desmocollin Dsc2, in both kinds of ID AJs, independent of the specific morphological appearance. The desmosomal molecules are not restricted to the desmosome-like-looking junctions but can also be detected in junctions appearing similar to the zonula or fascia adhaerens structures. These AJs of cardiac ID are therefore subsumed under the collective term area composita. We discuss our results with respect to the importance of ID junction molecules for the formation, maintenance and function of the heart, particularly in relation to recent findings that deletions of - or mutations in - genes encoding such proteins can cause severe, sometimes lethal damages.

Aprotinin preserves cellular junctions and reduces myocardial edema after regional ischemia and cardioplegic arrest.

BACKGROUND: Cardiac surgery with cardiopulmonary bypass (CPB) and cardioplegic arrest has been associated with myocardial edema attributable to vascular permeability, which is regulated in part by thrombin-induced alterations in cellular junctions. Aprotinin has been demonstrated to prevent activation of the thrombin protease-activated receptor, and we hypothesized that aprotinin preserves myocardial cellular junctions and prevents myocardial edema in a porcine model of regional ischemia and cardioplegic arrest. METHODS AND RESULTS: Fourteen pigs were subjected to 30 minutes of regional ischemia, followed by 60 minutes of CPB, with 45 minutes of crystalloid cardioplegia, then 90 minutes of post-CPB reperfusion. The treatment group (n=7) was administered aprotinin (40,000 kallikrein inhibitor units [KIU]/kg loading dose, 40,000 KIU/kg pump prime, and 10,000 KIU/kg per hour continuous infusion). Control animals (n=7) received normal saline. Myocardial vascular endothelial (VE)-cadherin, beta-catenin and gamma-catenin, and associated mitogen-activated protein kinase (MAPK) pathways were assessed by immunoblot and immunoprecipitation. Histologic analysis of the cellular junctions was done by immunofluorescence. Myocardial tissue water content was measured. VE-cadherin, beta-catenin, and gamma-catenin levels were significantly greater in the aprotinin group (all P<0.05). Immunfluorescence confirmed that aprotinin prevented loss of coronary endothelial adherens junction continuity. Aprotinin reduced tyrosine phosphorylation in myocardial tissue sections. Phospho-p38 activity was approximately 30% lower in the aprotinin group (P=0.007). The aprotinin group demonstrated decreased myocardial tissue water content (81.2+/-0.5% versus 83.5+/-0.3%; P=0.01) and reduced intravenous fluid requirements (2.9+/-0.2 L versus 4.0+/-0.4 L; P=0.03). CONCLUSIONS: Aprotinin preserves adherens junctions after regional ischemia and cardioplegic arrest through a mechanism potentially involving the p38 MAPK pathway, resulting in preservation of the VE barrier and reduced myocardial tissue edema.

Identification of two molecular groups of seminomas by using expression and tissue microarrays.

Highly effective tailored clinical management of testicular germ cell tumors is based on the identification of two major histologic subtypes: seminomatous and nonseminomatous germ cell tumors. Expression array analysis of these two histologic subtypes using hierarchical clustering reveals two tumor groups, one composed solely of seminomas and the other containing embryonal carcinomas and seminomas. Supervised analysis between these groups identified 55 significantly dysregulated genes (false discovery rate = 2.3). The genes with the highest overexpression in the first group compared with the second included SLC43A1 (POV1), NET-7, IGF2, and JUP; down-regulated genes included GRB7, PFKP, and CDC6. In situ hybridization of SLC43A1 mRNA showed significantly increased signal intensity in the seminomas. At the protein level, expression of the immunohistochemical markers cytokeratins (pan-cytokeratin staining), placental-like alkaline phosphatase, anti-cytokeratin clone 5.2, CD30, anion exchanger 1/3, junction plakoglobulin (JUP), and POU domain, class 5, transcription factor 1 (octomer-binding transcription factor 3/4) was significantly different between seminomas and embryonal tumors. Hierarchical clustering based on a refined protein expression profile identified two groups, the first consisting solely of seminomas the other of seminomas and embryonal carcinomas. No histomorphologic differences were observed between the two seminoma groups such as the presence or absence of lymphocytes or extent of stromal elements. In summary, using independent methodologies and samples, we have identified two groups of seminomas. One group of seminomas has a molecular profile similar to embryonal carcinoma. The findings in the current study may help explain aberrant immunoprofiles seen with some seminomas.