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Background: An immature intestine is a high-risk factor for necrotizing enterocolitis (NEC), which is a serious intestinal disease in newborns. The regulation of developmentally regulated GTP-binding protein 1 (DRG1) during organ development suggests a potential role of DRG1 in the maturation process of the intestine., Aim: To illustrate the function of DRG1 during the pathogenesis of NEC., Methods: DRG1 expression in the intestine was measured using immunohistochemistry and q-PCR. Immunoprecipitation coupled with mass spectrometry was used to identify the interacting proteins of DRG1. The biological functions of the potential interactors were annotated with the Database for Annotation, Visualization and Integrated Discovery. Caco2 and FHs74Int cells with stable DRG1 silencing or overexpression were used to investigate the influence of DRG1 on cell junctions and intestinal barrier permeability and to elucidate the downstream mechanism., Results: DRG1 was constitutively expressed during the intestinal maturation process but significantly decreased in the ileum in the context of NEC. Protein interaction analysis revealed that DRG1 was closely correlated with cell junctions. DRG1 deficiency destabilized the E-cadherin and occludin proteins near the cell membrane and increased the permeability of the epithelial cell monolayer, while DRG1 overexpression prevented lipopolysaccharide-induced disruption of E-cadherin and occludin expression and cell monolayer integrity. Further investigation suggested that DRG1 maintained cell junctions, especially adherens junctions, by regulating RAC1 activity, and RAC1 inhibition with NSC23766 attenuated intestinal injury and led to improved barrier integrity in experimental NEC., Conclusions: Our findings illustrate the mechanism underlying the effect of DRG1 deficiency on epithelial cell permeability regulation and provide evidence supporting the application of RAC1 inhibitors for protection against NEC., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC part of Springer Nature.)

Rationale: Congenital biliary atresia (BA) is a destructive obliterative cholangiopathy of neonates that affects both intrahepatic and extrahepatic bile ducts. However, the cause of BA is largely unknown. Methods: We explored the cell junctions and polarity complexes in early biopsy BA livers by immunofluorescence staining and western blot. Cdc42, as a key cell junction and polarity regulator, was found dramatically decreased in BA livers. Therefore, in order to investigate the role of Cdc42 in BA development, we constructed liver-specific and tamoxifen induced cholangiocyte-specific Cdc42 deleted transgenic mice. We further evaluated the role of bile acid in aggravating biliary damage in Cdc42 insufficient mouse liver. Results: We found a dramatic defect in the assembly of cell junctions and polarity complexes in both cholangiocytes and hepatocytes in BA livers. This defect was characterized by the disordered location of cell junction proteins, including ZO1, β-catenin, E-cadherin and claudin-3. Cdc42 and its active form, Cdc42-GTP, which serves as a small Rho GTPase to orchestrate the assembly of polarity complexes with Par6/Par3/αPKC, were substantially reduced in BA livers. Selective Cdc42 deficiency in fetal mouse cholangiocytes resulted in histological changes similar to those found in human BA livers, including obstruction in both the intra- and extrahepatic bile ducts, epithelial atrophy, and the disruption of cell junction and polarity complexes. A reduction in bile acids notably improved the histology and serological indices in Cdc42-mutant mice. Conclusion: Our results illustrate that BA is closely correlated with the impaired assembly of cell junction and polarity complexes in liver cells, which is likely caused by Cdc42 insufficiency and aggravated by bile acid corrosion., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Tunneling nanotubes (TNTs) are recognized long membrane nanotubes connecting distance cells. In the last decade, growing evidence has shown that these subcellular structures mediate the specific transfer of cellular materials, pathogens, and electrical signals between cells. As intercellular bridges, they play a unique role in embryonic development, collective cell migration, injured cell recovery, cancer treatment resistance, and pathogen propagation. Although TNTs have been considered as potential drug targets for treatment, there is still a long way to go to translate the research findings into clinical practice. Herein, we emphasize the heterogeneous nature of TNTs by systemically summarizing the current knowledge on their morphology, structure, and biogenesis in different types of cells. Furthermore, we address the communication efficiency and biological outcomes of TNT-dependent transport related to diseases. Finally, we discuss the opportunities and challenges of TNTs as an exciting therapeutic approach by focusing on the development of efficient and safe drugs targeting TNTs.

Epithelial cells are typically connected through different types of cell junctions that are localized from the apical membrane to the basal surface. In this way, epithelium cells form the first barrier against pathogenic microorganisms and prevent their entry into internal organs and the circulatory system. Recent studies demonstrate that bacterial pathogens disrupt epithelial cell junctions through targeting junctional proteins by secreted virulence factors. In this review, we discuss the diverse strategies used by common bacterial pathogens, including Pseudomonas aeruginosa, Helicobacter pylori, and enteropathogenic Escherichia coli, to disrupt epithelial cell junctions during infection. We also discuss the potential of targeting the pathogenic mechanisms in the treatment of pathogen-associated diseases., (© 2020 The Authors. Journal of Clinical Laboratory Analysis Published by Wiley Periodicals LLC.)

An understanding of polychlorinated biphenyl (PCB) congener-specific effects on cell membrane and intercellular communication is important within the studies of PCB absorption, organ-related PCB accumulation and exertion of toxic responses. Toxic potential of PCBs is linked to various deleterious effects on human health, including neurotoxicity, immunotoxicity, reproductive toxicity and genotoxicity and, recently in 2016 International Agency for Research on Cancer (IARC) has upgraded the classification of PCBs to Group 1 "Carcinogenic to humans." Proposed mechanisms of aforementioned PCBs adverse effects at cellular membrane level are: (i) downregulation of gap junction intercellular communication and/or connexins; (ii) compromised membrane integrity; and (iii) altered tight junction barrier function. This study, based on an extensive literature survey, shows the progress in scientific research of each of these three levels with the aim of pointing out the earliest toxic events of PCBs, which can result in serious cell/tissue/organ damage., (© 2020 John Wiley & Sons, Ltd.)

Collective metastasis is defined as the cohesive migration and metastasis of multicellular tumor cell clusters. Disrupting various cell adhesion genes markedly reduces cluster formation and colonization efficiency, yet the downstream signals transmitted by clustering remain largely unknown. Here, we use mouse and human breast cancer models to identify a collective signal generated by tumor cell clusters supporting metastatic colonization. We show that tumor cell clusters produce the growth factor epigen and concentrate it within nanolumina-intercellular compartments sealed by cell-cell junctions and lined with microvilli-like protrusions. Epigen knockdown profoundly reduces metastatic outgrowth and switches clusters from a proliferative to a collective migratory state. Tumor cell clusters from basal-like 2, but not mesenchymal-like, triple-negative breast cancer cell lines have increased epigen expression, sealed nanolumina, and impaired outgrowth upon nanolumenal junction disruption. We propose that nanolumenal signaling could offer a therapeutic target for aggressive metastatic breast cancers., Competing Interests: Declaration of Interests E.D.W. and K.J.C. are inventors on a pending patent application related to this work., (Copyright © 2020 Elsevier Inc. All rights reserved.)

This study explored the impact of gold nanoparticles on the metabolic activity and morphology of human pulmonary endothelial cell monolayers. We developed a gold nanoparticle library of three different sizes and two surface chemistries that include anionic citrate and the cationic polyelectrolyte poly(allylamine hydrochloride). The nanoparticles were characterized in cell culture medium to assess how their physical properties are altered after exposure to biological fluids. A bovine serum albumin pretreatment protocol was developed to stabilize the nanoparticles in cell culture medium. Results of this study show that an 18 h exposure of human pulmonary artery endothelial cells to the different nanoparticles modestly affects cellular metabolic activity. However, nanoparticle exposure perturbs the cortical actin networks and induces the formation of intercellular gaps. In particular, exposure to the poly(allylamine hydrochloride)-coated particles reduces the area of cell-cell junctions-a change that correlates with increased leakiness of endothelial barriers. The presence of excess polyelectrolyte capping agents in the supernatant of poly(allylamine hydrochloride)-coated nanoparticles significantly impacts endothelial morphology. Pretreatment of the particle supernatant with bovine serum albumin mitigates the negative effects of free or bound polyelectrolytes on endothelial cell monolayers.

Background: Ten-eleven translocation (Tet) methyl-cytosine dioxygenases (including Tet1/2/3)-mediated 5mC oxidation and DNA demethylation play important roles in embryonic development and adult tissue homeostasis. The expression of Tet2 and Tet3 genes are relatively abundant in the adult murine kidneys while Tet1 gene is expressed at a low level. Although Tet3 has been shown to suppress kidney fibrosis, the role of Tet2 in kidney physiology as well as renal ischemia-reperfusion (IR) injury is still largely unknown., Results: Tet2 -/- mice displayed normal kidney morphology and renal function as WT mice while the expression of genes associated with tight junction and adherens junction was impaired. At 24 h post-renal IR, Tet2 -/- mice showed higher SCr and BUN levels, more severe tubular damage, and elevated expression of Kim1 and Ngal genes in the kidney in comparison with WT mice. Moreover, the transcriptomic analysis revealed augmented inflammatory response in the kidneys of Tet2 -/- mice., Conclusions: Tet2 is dispensable for kidney development and function at baseline condition while protects against renal IR injury possibly through repressing inflammatory response. Our findings suggest that Tet2 may be a potential target for the intervention of IR-induced acute kidney injury (AKI).

Cellular communication network (CCN) proteins are matricellular proteins that coordinate signaling among extracellular matrix, secreted proteins, and cell surface receptors. Their specific in vivo function is context-dependent, but they play profound roles in pathological conditions, such as fibrosis and cancers. Anti-CCN therapies are in clinical consideration. Only recently, however, has the function of these complex molecules begun to emerge. This review summarizes and interprets our current knowledge regarding these fascinating molecules and provides experimental evidence for their utility as therapeutic targets.

Although it is known that psoriatic dermal-derived mesenchymal stem cells (DMSCs) dysregulate keratinocyte proliferation, the biological activity profile of keratinocytes influenced by psoriatic DMSCs remain unknown. In the present study, we assessed the impact of psoriatic DMSCs on keratinocyte proliferation, differentiation, and glucose metabolism in normal human epidermal keratinocytes co-cultured with or without psoriatic DMSCs. Co-culture of normal human epidermal keratinocytes with psoriatic DMSCs downregulated expression levels of proteins associated with cell junction assembly (alpha-actinin-1, catenin beta-1, poliovirus receptor-related protein 4 and procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2), while upregulating proteins associated with keratinocyte proliferation and differentiation (involucrin, isoform 2 of Histone-binding protein, isoform 3 of Telomeric repeat-binding factor 2 and keratin 13). Moreover, co-culture of normal human epidermal keratinocytes with psoriatic DMSCs stimulated keratinocyte proliferation and glycolysis, but reduced keratinocyte junctions. Taken together, these results demonstrate that psoriatic DMSCs increase keratinocyte proliferation and glycolysis, and reduce cell junctions, suggesting a pathogenic role of psoriatic DMSCs in epidermal hyperplasia, aberrant differentiation, and reduction in turnover time of keratinocytes in psoriasis.

Cell junctions serve as a protective barrier for cells and provide an important channel for information transmission between cells and the surrounding environment. Viruses are parasites that invade and commandeer components of host cells in order to survive and replicate, and they have evolved various mechanisms to alter cell junctions to facilitate viral infection. In this review, we examined the current state of knowledge on the action of viruses on host cell junctions. The existing evidence suggests that targeting the molecules involved in the virus-cell junction interaction can prevent the spread of viral diseases., (© 2020 The Authors. Thoracic Cancer published by China Lung Oncology Group and John Wiley & Sons Australia, Ltd.)

Background: Membrane contact sites are fundamental for transmission and translation of signals in multicellular organisms. The junctional membrane complexes in the cardiac dyads, where transverse (T) tubules are juxtaposed to the sarcoplasmic reticulum, are a prime example. T-tubule uncoupling and remodeling are well-known features of cardiac disease and heart failure. Even subtle alterations in the association between T-tubules and the junctional sarcoplasmic reticulum can cause serious cardiac disorders. NEXN (nexilin) has been identified as an actin-binding protein, and multiple mutations in the NEXN gene are associated with cardiac diseases, but the precise role of NEXN in heart function and disease is still unknown., Methods: Nexn global and cardiomyocyte-specific knockout mice were generated. Comprehensive phenotypic and RNA sequencing and mass spectrometry analyses were performed. Heart tissue samples and isolated single cardiomyocytes were analyzed by electron and confocal microscopy., Results: Global and cardiomyocyte-specific loss of Nexn in mice resulted in a rapidly progressive dilated cardiomyopathy. In vivo and in vitro analyses revealed that NEXN interacted with junctional sarcoplasmic reticulum proteins, was essential for optimal calcium transients, and was required for initiation of T-tubule invagination and formation., Conclusions: These results demonstrated that NEXN is a pivotal component of the junctional membrane complex and is required for initiation and formation of T-tubules, thus providing insight into mechanisms underlying cardiomyopathy in patients with mutations in NEXN.

Mechanisms of transmesothelial invasion of ovarian cancer are still poorly understood. Here we examined whether this phenomenon may be determined by an expression of intercellular junctions in peritoneal mesothelial cells (PMCs). Analysis of ovarian tumors showed that cancer cells are localized below an intact layer of PMCs. The PMCs located near the invaded cancer cells displayed low expression of connexin 43, E-cadherin, occludin, and desmoglein, as well as expressed SA-β-Gal, a marker of senescence. Experiments in vitro showed that senescent PMCs exhibited decreased levels of the four tested intercellular junctions, and that the invasion of ovarian cancer cells through the PMCs increased proportionally to the admixture of senescent cells. Intervention studies showed that the expression of connexin 43, E-cadherin, occludin, and desmoglein in senescent PMCs could be restored upon the blockade of p38 MAPK, NF-κB, AKT, JNK, HGF, and TGF-β1. When these molecules were neutralized, the efficiency of the transmesothelial cancer cell invasion was diminished. Collectively, our findings show that the integrity of the peritoneal mesothelium, which is determined by the expression of junctional proteins, is critical for the invasion of ovarian cancer. They also indicate a mechanism by which senescent PMCs may promote the invasive potential of cancer cells.

New Findings: What is the central question of this study? Can a single bone marrow mononuclear cell (BMMC) transplant into the subcapsular region of kidney improve cellular communication and adhesion, while restoring renal tissue cytoarchitecture and function during renovascular hypertension? What is the main finding and its importance? The BMMC transplantation restored connexin 40 expression and led to recovery of N- and E-cadherin levels within 15 days. It was observed, for the first time, that BMMC transplantation restores expression of nephrin, a component of the glomerular filtration barrier related to podocytes and the glomerular basal membrane., Abstract: Stem cell therapy has emerged as a potential treatment for renal diseases owing to the regenerative potential of stem cells. However, a better understanding of the morphological and functional changes of damaged renal cells in the presence of transplanted stem cells is needed. The aim of this study was to investigate cell-cell communication and adhesion in renal parenchyma, with analysis of fibrosis, to evaluate renal morphology and function after bone marrow mononuclear cell (BMMC) transplantation in two-kidney-one-clip rats. The BMMC therapy significantly decreased blood pressure and renin expression, improved renal morphology and restored the glomerular filtration barrier, with remodelling of podocytes. In addition, there was a reduction in fibrosis, and connexin 40 and nephrin expression were significantly increased after 7 and 15 days of transplantation. Plasma creatinine, urea and total protein levels were restored, and proteinuria was reduced. Furthermore, N- and E-cadherin expression was increased soon after BMMC therapy. Green fluorescent protein-positive BMMCs were found in the renal cortex 24 and 48 h after transplantation into the renal subcapsule, and at 7 and 15 days after transplantation, these cells were observed throughout the renal medulla, indicating cellular migration. Therefore, these data suggest that transplanted BMMCs improve cell-cell communication and adhesion between damaged cells, which is accompanied by a recovery of renal morphology and function., (© 2019 The Authors. Experimental Physiology © 2019 The Physiological Society.)

The transient receptor potential vanilloid 4 (TRPV4) channel is a mechanosensor in endothelial cells (EC) that regulates cyclic strain-induced reorientation and flow-mediated nitric oxide production. We have recently demonstrated that TRPV4 expression is reduced in tumor EC and tumors grown in TRPV4KO mice exhibited enhanced growth and immature leaky vessels. However, the mechanism by which TRPV4 regulates tumor vascular integrity and metastasis is not known. Here, we demonstrate that VE-cadherin expression at the cell-cell contacts is significantly reduced in TRPV4-deficient tumor EC and TRPV4KO EC. In vivo angiogenesis assays with Matrigel of varying stiffness (700-900 Pa) revealed a significant stiffness-dependent reduction in VE-cadherin-positive vessels in Matrigel plugs from TRPV4KO mice compared with WT mice, despite an increase in vessel growth. Further, syngeneic Lewis Lung Carcinomatumor experiments demonstrated a significant decrease in VE-cadherin positive vessels in TRPV4KO tumors compared with WT. Functionally, enhanced tumor cell metastasis to the lung was observed in TRPV4KO mice. Our findings demonstrate that TRPV4 channels regulate tumor vessel integrity by maintaining VE-cadherin expression at cell-cell contacts and identifies TRPV4 as a novel target for metastasis., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Intercellular communication is essential for the development and maintenance of multicellular organisms. Tunneling nanotubes (TNTs) are a recently recognized means of long and short distance communication between a wide variety of cell types. TNTs are transient filamentous membrane protrusions that connect cytoplasm of neighboring or distant cells. Cytoskeleton fiber-mediated transport of various cargoes occurs through these tubules. These cargoes range from small ions to whole organelles. TNTs have been shown to contribute not only to embryonic development and maintenance of homeostasis, but also to the spread of infectious particles and resistance to therapies. These functions in the development and progression of cancer and infectious disease have sparked increasing scrutiny of TNTs, as their contribution to disease progression lends them a promising therapeutic target. Herein, we summarize the current knowledge of TNT structure and formation as well as the role of TNTs in pathology, focusing on viral, prion, and malignant disease. We then discuss the therapeutic possibilities of TNTs in light of their varied functions. Despite recent progress in the growing field of TNT research, more studies are needed to precisely understand the role of TNTs in pathological conditions and to develop novel therapeutic strategies., (© 2018 Wiley Periodicals, Inc.)

Irrespective of its diverse etiologies, acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) leads to increased permeability of the alveolar-capillary barrier, which in turn promotes edema formation and respiratory failure. We investigated the mechanism of ALI/ARDS lung hyperpermeability triggered by pulmonary exposure of mice to the highly toxic plant-derived toxin ricin. One prominent hallmark of ricin-mediated pulmonary intoxication is the rapid and massive influx of neutrophils to the lungs, where they contribute to the developing inflammation yet may also cause tissue damage, thereby promoting ricin-mediated morbidity. Here we show that pulmonary exposure of mice to ricin results in the rapid diminution of the junction proteins VE-cadherin, claudin 5, and connexin 43, belonging, respectively, to the adherens, tight, and gap junction protein families. Depletion of neutrophils in ricin-intoxicated mice attenuated the damage caused to these junction proteins, alleviated pulmonary edema, and significantly postponed the time to death of the intoxicated mice. Inhibition of matrix metalloproteinase (MMP) activity recapitulated the response to neutrophil depletion observed in ricin-intoxicated mice and was associated with decreased insult to the junction proteins and alveolar-capillary barrier. However, neutrophil-mediated MMP activity was not the sole mechanism responsible for pulmonary hyperpermeability, as exemplified by the ricin-mediated disruption of claudin 18, via a neutrophil-independent mechanism involving tyrosine phosphorylation. This in-depth study of the early stage mechanisms governing pulmonary tissue integrity during ALI/ARDS is expected to facilitate the tailoring of novel therapeutic approaches for the treatment of these diseases.

Objective- Maintenance of lymphatic permeability is essential for normal lymphatic function during adulthood, but the precise signaling pathways that control lymphatic junctions during development are not fully elucidated. The G s -coupled AM (adrenomedullin) signaling pathway is required for embryonic lymphangiogenesis and the maintenance of lymphatic junctions during adulthood. Thus, we sought to elucidate the downstream effectors mediating junctional stabilization in lymphatic endothelial cells. Approach and Results- We knocked-down both Rap1A and Rap1B isoforms in human neonatal dermal lymphatic cells (human lymphatic endothelial cells) and genetically deleted the mRap1 gene in lymphatic endothelial cells by producing 2 independent, conditional Rap1a/b knockout mouse lines. Rap1A/B knockdown caused disrupted junctional formation with hyperpermeability and impaired AM-induced lymphatic junctional tightening, as well as rescue of histamine-induced junctional disruption. Less than 60% of lymphatic- Rap1a/b knockout embryos survived to E13.5 exhibiting interstitial edema, blood-filled lymphatics, disrupted lymphovenous valves, and defective lymphangiogenesis. Consistently, inducible lymphatic- Rap1a/b deletion in adult animals prevented AM-rescue of histamine-induced lymphatic leakage and dilation. Conclusions- Rap1 (Ras-related protein) serves as the dominant effector downstream of AM to stabilize lymphatic junctions. Rap1 is required for maintaining lymphatic permeability and driving normal lymphatic development.

Despite an enhanced permeability and retention effect typical of many solid tumors, drug penetration is not always sufficient. Possible strategies for the drug delivery improvement are a modification of the tumor cell-to-cell junctions and usage of cell membrane permeabilization proteins. In this review we discuss epithelial cell junctions as targets for a combined anticancer therapy and propose new possible sources of such agents. We suggest considering viral and bacterial pathogens disrupting epithelial layers as plentiful sources of new therapeutic agents for increasing tumor permeability for other effector agents. We also observe the application of pore forming proteins and peptides of different origin for cytoplasmic delivery of anti-cancer agents and consider the main obstacles of their use in vivo., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Intraventricular hemorrhage (IVH) is the most common cause of pediatric hydrocephalus in North America but remains poorly understood. Cell junction-mediated ventricular zone (VZ) disruption and astrogliosis are associated with the pathogenesis of congenital, nonhemorrhagic hydrocephalus. Recently, our group demonstrated that VZ disruption is also present in preterm infants with IVH. On the basis of this observation, we hypothesized that blood triggers the loss of VZ cell junction integrity and related cytopathology. In order to test this hypothesis, we developed an in vitro model of IVH by applying syngeneic blood to cultured VZ cells obtained from newborn mice. Following blood treatment, cells were assayed for N-cadherin-dependent adherens junctions, ciliated ependymal cells, and markers of glial activation using immunohistochemistry and immunoblotting. After 24-48 hours of exposure to blood, VZ cell junctions were disrupted as determined by a significant reduction in N-cadherin expression (p < 0.05). This was also associated with significant decrease in multiciliated cells and increase in glial fibrillary acid protein-expressing cells (p < 0.05). These observations suggest that, in vitro, blood triggers VZ cell loss and glial activation in a pattern that mirrors the cytopathology of human IVH and supports the relevance of this in vitro model to define injury mechanisms.

We used the nanometer-wide tubules of the transverse tubular (t)-system of human skeletal muscle fibers as sensitive sensors for the quantitative monitoring of the Ca 2+ -handling properties in the narrow junctional cytoplasmic space sandwiched between the tubular membrane and the sarcoplasmic reticulum cisternae in single muscle fibers. The t-system sealed with a Ca 2+ -sensitive dye trapped in it is sensitive to changes in ryanodine receptor (RyR) Ca 2+ leak, the store operated calcium entry flux, plasma membrane Ca pump, and sodium-calcium exchanger activities, thus making the sealed t-system a nanodomain Ca 2+ sensor of Ca 2+ dynamics in the junctional space. The sensor was used to assess the basal Ca 2+ -handling properties of human muscle fibers obtained by needle biopsy from control subjects and from people with a malignant hyperthermia (MH) causative RyR variant. Using this approach we show that the muscle fibers from MH-susceptible individuals display leakier RyRs and a greater capacity to extrude Ca 2+ across the t-system membrane compared with fibers from controls. This study provides a quantitative way to assess the effect of RyR variants on junctional membrane Ca 2+ handling under defined ionic conditions., Competing Interests: The authors declare no conflict of interest.

Vascular disruption is the underlying cause of cerebral hemorrhage, including intracerebral, subarachnoid and intraventricular hemorrhage. The disease etiology also involves cerebral hemorrhage-induced blood-brain barrier (BBB) disruption, which contributes an important component to brain injury after the initial cerebral hemorrhage. BBB loss drives vasogenic edema, allows leukocyte extravasation and may lead to the entry of potentially neurotoxic and vasoactive compounds into brain. This review summarizes current information on changes in brain endothelial junction proteins in response to cerebral hemorrhage (and clot-related factors), the mechanisms underlying junction modification and potential therapeutic targets to limit BBB disruption and, potentially, hemorrhage occurrence. It also addresses advances in the tools that are now available for assessing changes in junctions after cerebral hemorrhage and the potential importance of such junction changes. Recent studies suggest post-translational modification, conformational change and intracellular trafficking of junctional proteins may alter barrier properties. Understanding how cerebral hemorrhage alters BBB properties beyond changes in tight junction protein loss may provide important therapeutic insights to prevent BBB dysfunction and restore normal function.

Loss of endothelial integrity promotes capillary leakage in numerous diseases, including sepsis, but there are no effective therapies for preserving endothelial barrier function. Angiopoietin-2 (ANGPT2) is a context-dependent regulator of vascular leakage that signals via both endothelial TEK receptor tyrosine kinase (TIE2) and integrins. Here, we show that antibodies against β1-integrin decrease LPS-induced vascular leakage in murine endotoxemia, as either a preventative or an intervention therapy. β1-integrin inhibiting antibodies bound to the vascular endothelium in vivo improved the integrity of endothelial cell-cell junctions and protected mice from endotoxemia-associated cardiac failure, without affecting endothelial inflammation, serum proinflammatory cytokine levels, or TIE receptor signaling. Moreover, conditional deletion of a single allele of endothelial β1-integrin protected mice from LPS-induced vascular leakage. In endothelial monolayers, the inflammatory agents thrombin, lipopolysaccharide (LPS), and IL-1β decreased junctional vascular endothelial (VE)-cadherin and induced actin stress fibers via β1- and α5-integrins and ANGPT2. Additionally, β1-integrin inhibiting antibodies prevented inflammation-induced endothelial cell contractility and monolayer permeability. Mechanistically, the inflammatory agents stimulated ANGPT2-dependent translocation of α5β1-integrin into tensin-1-positive fibrillar adhesions, which destabilized the endothelial monolayer. Thus, β1-integrin promotes endothelial barrier disruption during inflammation, and targeting β1-integrin signaling could serve as a novel means of blocking pathological vascular leak., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Objective: During vascular development, integrin-mediated signaling regulates the formation and stabilization of cell-cell junctions, which are required for endothelial cell (EC) apical-basal polarity and proper deposition of the vascular basement membrane. Parvins are actin-binding proteins that facilitate the interaction of integrins with the actin cytoskeleton. The endothelium expresses 2 parvin isoforms: α-pv (α-parvin) and β-pv (β-parvin). Recently, we have shown that α-pv is critical for vessel growth and vessel stability at late embryonic developmental stages. The role of parvins during early embryonic development is unknown., Approach and Results: To investigate the role of endothelial parvins in the developing vasculature, we generated mice with ECs lacking both parvin isoforms by deleting α-pv in ECs in global β-pv -/- mice (α-pv ΔEC ;β-pv -/- mice). Here, we show that α-pv ΔEC ;β-pv -/- mice die around embryonic day 11.5 and exhibit hemorrhages, immature capillary beds, and severe vascular defects in the central nervous system, including reduced vessel branching, increased vessel diameter, and balloon-like hemorrhagic clusters of ECs. Vessels in α-pv ΔEC ;β-pv -/- embryos display disorganized cell-cell junctions, impaired endothelial apical-basal polarity, and discontinuous basement membranes. These vascular defects are accompanied by defective pericyte-vessel interaction., Conclusions: Our results show that parvins are critical for the organization of endothelial cell-cell junctions, the establishment of endothelial apical-basal polarity, and the integrity of the basement membrane., (© 2018 American Heart Association, Inc.)

Recent studies have linked impairment of intestinal epithelial function in inflammatory bowel disease to the disturbance of endoplasmic reticulum homeostasis (ER) in response to stress. Most studies are on goblet and Paneth cells, which are considered more susceptible to stress due to their role in the protection of intestinal epithelium against microbes and harmful substances. However, studies on the role of inflammation-induced ER stress in absorptive intestinal cells are scarce. In this study, we show, using Caco-2 cells as a model of intestinal epithelial barrier, that inducing ER stress using a cocktail mixture of pro-inflammatory mediators [TNFα (50ng/ml), MCP1 (50ng/ml), and IL-1β (25ng/ml)] as observed in IBD patients induces ER stress and leads to significant changes in key proteins of the apical (sucrase-isomaltase (SI), dipeptidyl-peptidase (DPPIV), and ezrin) and basolateral (E-cadherin, zonula occludens (ZO-1), and connexin-43) membranes. Aberrant trafficking of SI, DPPIV was observed as early as 8h post-inflammation-induced ER stress and even in the absence of loss of intestinal cell integrity. The observed effect was associated with a re-localization of ezrin, ZO-1, and connexin-43, key differentiation and junction proteins. Collectively, this study shows that disruption of the trafficking of key digestive enzymes of the intestinal epithelium occur in response to inflammation induced ER stress before the loss of monolayer integrity., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Pemphigus is a severe autoimmune blistering disease mediated by pathogenic anti-desmoglein antibodies leading to an inter keratinocyte disjunction. Rituximab is a monoclonal antibody that binds to the CD-20 antigen of B lymphocytes, which causes B-cell depletion and a subsequent reduction in pathogenic autoantibodies. Its therapeutic role in pemphigus has been progressively growing with increasing evidence of successful outcomes. Rituximab was initially off-labeled used as an alternative in patients with recalcitrant or relapsing pemphigus and in patients with contraindications to systemic corticosteroids. Recently, a large randomized clinical trial has shown that first-line use of rituximab combined with short-term prednisone regimen was both more effective and potentially safer than a standard regimen of high doses of corticosteroids in patients with moderate to severe pemphigus.

Chronic sleep loss in the rat increases blood-brain barrier permeability to Evans blue and FITC-dextrans in almost the whole brain and sleep recovery during short periods restores normal blood-brain barrier permeability. Sleep loss increases vesicle density in hippocampal endothelial cells and decreases tight junction protein expression. However, at the ultrastructural level the effect of chronic sleep loss on interendothelial junctions is unknown. In this study we characterised the ultrastructure of interendothelial junctions in the hippocampus, the expression of tight junction proteins, and quantified blood-brain barrier permeability to fluorescein-sodium after chronic sleep restriction. Male Wistar rats were sleep restricted using the modified multiple platform method during 10 days, with a daily schedule of 20-h sleep deprivation plus 4-h sleep recovery at their home-cages. At the 10th day hippocampal samples were obtained immediately at the end of the 20-h sleep deprivation period, and after 40 and 120 min of sleep recovery. Samples were processed for transmission electron microscopy and western blot. Chronic sleep restriction increased blood-brain barrier permeability to fluorescein-sodium, and decreased interendothelial junction complexity by increasing the frequency of less mature end-to-end and simply overlap junctions, even after sleep recovery, as compared to intact controls. Chronic sleep loss also induced the formation of clefts between narrow zones of adjacent endothelial cell membranes in the hippocampus. The expression of claudin-5 and actin decreased after chronic sleep loss as compared to intact animals. Therefore, it seems that chronic sleep loss disrupts interendothelial junctions that leads to blood-brain barrier hyperpermeability in the hippocampus., (© 2017 The Authors Journal of Microscopy © 2017 Royal Microscopical Society.)

Dipeptidyl peptidase-4 (DPP-4) inhibitors are relatively new class of anti-diabetic drugs. Some protective effects of DPP-4 on cardiovascular disease have been described independently from glucose-lowering effect. However, the detailed mechanisms by which DPP-4 inhibitors exert on endothelial cells remain elusive. The purpose of this research was to determine the effects of DPP-4 inhibitor on endothelial barrier function. Human umbilical vein endothelial cells (HUVECs) were cultured and exposed to hypoxia in the presence or absence of Diprotin A, a DPP-4 inhibitor. Immunocytochemistry of vascular endothelial (VE-) cadherin showed that jagged VE-cadherin staining pattern induced by hypoxia was restored by treatment with Diprotin A. The increased level of cleaved β-catenin in response to hypoxia was significantly attenuated by Diprotin A, suggesting that DPP-4 inhibition protects endothelial adherens junctions from hypoxia. Subsequently, we found that Diprotin A inhibited hypoxia-induced translocation of NF-κB from cytoplasm to nucleus through decreasing TNF-α expression level. Furthermore, the tube formation assay showed that Diprotin A significantly restored hypoxia-induced decrease in number of tubes by HUVECs. These results suggest that DPP-4 inhibitior protects HUVECs from hypoxia-induced barrier impairment., (Copyright © 2017 The Authors. Production and hosting by Elsevier B.V. All rights reserved.)

Colonization and disruption of the epithelium is a major infection mechanism of mucosal pathogens. The epithelium counteracts infection by exfoliating damaged cells while maintaining the mucosal barrier function. The sexually transmitted bacterium Neisseria gonorrhoeae (GC) infects the female reproductive tract primarily from the endocervix, causing gonorrhea. However, the mechanism by which GC overcome the mucosal barrier remains elusive. Using a new human tissue model, we demonstrate that GC can penetrate into the human endocervix by inducing the exfoliation of columnar epithelial cells. We found that GC colonization causes endocervical epithelial cells to shed. The shedding results from the disassembly of the apical junctions that seal the epithelial barrier. Apical junction disruption and epithelial exfoliation increase GC penetration into the endocervical epithelium without reducing bacterial adherence to and invasion into epithelial cells. Both epithelial exfoliation and junction disruption require the activation and accumulation of non-muscle myosin II (NMII) at the apical surface and GC adherent sites. GC inoculation activates NMII by elevating the levels of the cytoplasmic Ca2+ and NMII regulatory light chain phosphorylation. Piliation of GC promotes, but the expression of a GC opacity-associated protein variant, OpaH that binds to the host surface proteins CEACAMs, inhibits GC-induced NMII activation and reorganization and Ca2+ flux. The inhibitory effects of OpaH lead to reductions in junction disruption, epithelial exfoliation, and GC penetration. Therefore, GC phase variation can modulate infection in the human endocervix by manipulating the activity of NMII and epithelial exfoliation.

Urothelial bladder cancer is a major cause of morbidity and mortality worldwide, causing an estimated 150 000 deaths per year. Whilst non-muscle-invasive bladder tumours can be effectively treated, with high survival rates, many tumours recur, and some will progress to muscle-invasive disease with a much poorer long-term prognosis. Thus, there is a pressing need to understand the molecular transitions occurring within the progression of bladder cancer to an invasive disease. Tumour invasion is often associated with a down-regulation of E-cadherin expression concomitant with a suppression of cell:cell junctions, and decreased levels of E-cadherin expression have been reported in higher grade urothelial bladder tumours. We find that expression of E-cadherin in a panel of bladder cancer cell lines correlated with the presence of cell:cell junctions and the level of PAK5 expression. Interestingly, exogenous PAK5 has recently been described to be associated with cell:cell junctions and we now find that endogenous PAK5 is localised to cell junctions and interacts with an E-cadherin complex. Moreover, depletion of PAK5 expression significantly reduced junctional integrity. These data suggest a role for PAK5 in maintaining junctional stability and we find that, in both our own patient samples and a commercially available dataset, PAK5mRNA levels are reduced in human bladder cancer compared with normal controls. Taken together, the present study proposes that PAK5 expression levels could be used as a novel prognostic marker for bladder cancer progression., (© 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.)

This review presents an extensively integrated model of the cardiac intercalated disc (ID), a highly orchestrated structure that connects adjacent cardiomyocytes. Classically, three main structures are distinguished: gap junctions (GJs) metabolically and electrically connect cytoplasm of adjacent cardiomyocytes; adherens junctions (AJs) connect the actin cytoskeleton of adjacent cells; and desmosomes function as cell anchors and connect intermediate filaments. Furthermore, ion channels reside in the ID. Mutations in ID proteins have been associated with cardiac arrhythmias such as Brugada syndrome and arrhythmogenic cardiomyopathy. However, rather than being independent, all ID components work together intensively by multifunctional proteins such as ZO-1, Ankyrin G, and β-catenin, integrating mechanical and electrical functions. GJs form a plaque surrounded by the perinexus in which free connexons reside; the connexome integrates NaV channels, the desmosome and GJs; and the area composita hosts AJs and desmosomes, also integrated as adhering junctions. Furthermore, the transitional junction connects sarcomeres to the plasma membrane. Lastly, this review integrates all these findings in comprehensible figures, illustrating the interdependencies of ID proteins., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2017. For permissions, please email: journals.permissions@oup.com.)

In this study, we demonstrated that hypoxic conditions stimulated an increase in tunneling nanotube (TNT) formation in chemoresistant ovarian cancer cells (SKOV3, C200).We found that suppressing the mTOR pathway using either everolimus or metformin led to suppression of TNT formation in vitro, verifying TNTs as a potential target for cancer-directed therapy. Additionally, TNT formation was detected in co-cultures including between platinum-resistant SKOV3 cells, between SKOV3 cells and platinum-chemosensitive A2780 cells, and between SKOV3 cells cultured with benign ovarian epithelial (IOSE) cells; these findings indicate that TNTs are novel conduits for malignant cell interactions and tumor cell interactions with other cells in the microenvironment. When chemoresistant C200 and parent chemosensitive A2780 cells were co-cultured, chemoresistant cells displayed a higher likelihood of TNT formation to each other than to chemosensitive malignant or benign epithelial cells. Hypoxia-induced TNT formation represents a potential mechanism for intercellular communication in ovarian cancer and other forms of invasive refractory cancers., Competing Interests: The authors have no conflicts of interest to declare.

Chorioamnionitis is an acute inflammatory reaction associated with the premature rupture of the fetal membranes. It is caused mainly by invasion of bacteria from the vaginal tract that can penetrate the intact membranes and invade the amnion cavity and the decidua. Tight junctions (TJs) and adherent junctions (AJs) are intercellular junctions crucial for epithelia adhesion and permeability regulation in a wide variety of tissues and organs. Our aim is to investigate if TJ and AJ molecules are involved in human chorioamnionitis. We studied the protein expression (by immunohistochemistry and western blotting) and the mRNA levels (by RT-PCR) of some junction proteins such as Zonula Occludens-1 (ZO-1), occludin, VE-cadherin and β-catenin in fetal membranes from women with chorioamnionitis compared to those membranes derived from idiopathic pregnancies. Western blotting and immunohistochemical data established that occludin expression was decreased in amnion with chorioamnionitis compared to amnion from idiopathic pregnancies. Samples tested for ZO-1, VE-cadherin and β-catenin (proteins and mRNAs) showed no differences between idiopathic and pathological membranes. One of the most relevant results is the decrease of occludin in membranes with chorioamnionitis. Since we have previously demonstrated that some cytokines, particularly elevated in the chorioamnionitis, cause the disruption of TJs in placental villi, we suggest that the decrease of occludin in amnion may be the first change that leads to the rupture of the amniotic membrane in this pathology.

Salivary glands are vital for maintaining oral health. Head and neck radiation therapy is one of the most common causes of salivary gland hypofunction. Little is known about the structural changes that occur in salivary glands after radiation therapy. The aim of this study is to understand the structural changes that occur in post-irradiated human (submandibular gland [SMG]) as compared with untreated ones. We determined changes in epithelial polarity, presence of collagen deposition, and alteration in adipose tissue. We used formalin-fixed paraffin-embedded human SMG from two female subjects exposed to head and neck irradiation. We utilized hematoxylin and eosin staining and Masson's Trichrome staining. The immunostained tissue sections were examined using confocal microscopy. The number and size of adipocytes per tissue section were calculated using ImageJ, Prism, and SPSS software. Post-irradiated human SMG displayed high collagen deposition, disorganized cell junctions, and an increased number of adipocytes as compared with non-irradiated controls. These findings are important to improve our understanding of the individual risk and variation in radiation-related salivary gland dysfunction., (© 2016 The Histochemical Society.)

Background: Administration of silver nanoparticles (AgNPs) to mice could result in their distribution and accumulation in multiple organs, with notable prominence in liver, lungs, and kidneys. However, how AgNPs transport through blood vesicular system to reach the target organs is unclear, and the precise differences in the mechanisms of toxicity between AgNPs and silver ions still remain elusive. In the present research, the pathological changes on these target organs with a focus on inter-endothelial junction was investigated to gain a new insight of AgNPs toxicity by comparing the mechanisms of action of AgNPs and AgNO3., Methods: We investigated the in vitro cytotoxicity of either citrated-coated AgNPs (10, 75, and 110 nm) or silver nitrate (AgNO3) following 24 h incubations (1-40 μg/mL) in the presence of primary human umbilical vein endothelial cells (HUVEC). Meanwhile, we detected the effects of AgNPs on intercellular conjunction and intracellular ROS by VE-cadherin staining and 2', 7'-dichlorodihydrofluorescein diacetate (DCFH-DA) assay, respectively. To assess in vivo toxicity, we administered single or multiple intravenous injections (25 μg Ag for AgNPs and 2.5 μg Ag for AgNO3 per dose) to mice., Results: In the in vitro study, the TEM observation showed that AgNPs were taken up by endothelial cells while AgNO3 was taken up little. Meanwhile AgNPs incubation induced the elevation of intracellular ROS and down-regulation of VE-cadherin between the endothelial cells and affected the cytoskeleton actin reorganization, which could be rescued by antioxidant N-acetylcysteine. In contrast, AgNO3 caused direct cell death when the concentration was higher than 20 μg/mL and without ROS induction at lower concentration. The release of AgNPs from leaking vessels induced peripheral inflammation in the liver, lungs, and kidneys, and the severity increased in proportion to the diameter of the AgNPs used., Conclusion: It is AgNPs but not AgNO3 that were taken up by vascular endothelial cells and induced intracellular ROS elevated, which was closely related to disruption of the integrity of endothelial layer. The AgNPs-induced leakiness of endothelial cells could mediate the common peripheral inflammation in liver, kidney and lung through intravenous exposure.

The cellular prion protein PrP(c) plays important roles in proliferation, cell death and survival, differentiation and adhesion. The participation of PrP(c) in tumor growth and metastasis was pointed out, but the underlying mechanisms were not deciphered completely. In the constantly renewing intestinal epithelium, our group demonstrated a dual localization of PrP(c), which is targeted to cell-cell junctions in interaction with Src kinase and desmosomal proteins in differentiated enterocytes, but is predominantly nuclear in dividing cells. While the role of PrP(c) in the dynamics of intercellular junctions was confirmed in other biological systems, we unraveled its function in the nucleus only recently. We identified several nuclear PrP(c) partners, which comprise γ-catenin, one of its desmosomal partners, β-catenin and TCF7L2, the main effectors of the canonical Wnt pathway, and YAP, one effector of the Hippo pathway. PrP(c) up-regulates the activity of the β-catenin/TCF7L2 complex and its invalidation impairs the proliferation of intestinal progenitors. We discuss how PrP(c) could participate to oncogenic processes through its interaction with Wnt and Hippo pathway effectors, which are controlled by cell-cell junctions and Src family kinases and dysregulated during tumorigenesis. This highlights new potential mechanisms that connect PrP(c) expression and subcellular redistribution to cancer.

Oral, intestinal and genital mucosal epithelia have a barrier function to prevent paracellular penetration by viral, bacterial and other pathogens, including human immunodeficiency virus (HIV). HIV can overcome these barriers by disrupting the tight and adherens junctions of mucosal epithelia. HIV-associated disruption of epithelial junctions may also facilitate paracellular penetration and dissemination of other viral pathogens. This review focuses on possible molecular mechanisms of HIV-associated disruption of mucosal epithelial junctions and its role in HIV transmission and pathogenesis of HIV and acquired immune deficiency syndrome (AIDS).

Adenomyosis (ADS) is a common estrogen-dependent gynecological disease with unknown etiology. The RhoA/Rho-kinase (ROCK) signaling pathway is involved in various cellular functions, including migration, proliferation, and smooth muscle contraction. Here we examined the potential role of this pathway in junctional zone (JZ) contraction in women with and without ADS. We demonstrated that in the normal JZ, RhoA and ROCK-I messenger RNA (mRNA) and protein expression was significantly higher in the proliferative phase of the menstrual cycle than in the secretory phase. Expression of RhoA and ROCK-I in the JZ from women with ADS was significantly higher than in the control women and showed no significant differences across the menstrual cycle. Treatment of JZ smooth muscle cells (JZSMCs) with estrogen at 0, 1, 10, or 100 nmol/L for 24 hours resulted in increased expression of RhoA, ROCK-I, and myosin light-chain (MLC) phosphorylation (p-MLC) in a dose-dependent manner. In parallel to its effects on p-MLC, estrogen-mediated, dose-dependent contraction responses in JZSMCs. Estrogen-mediated contraction in the ADS group was significantly higher than in the controls and also showed no significant differences across the menstrual cycle. These effects were suppressed in the presence of ICI 182780 or Y27632, supporting an estrogen receptor-dependent and RhoA activation-dependent mechanism. Our results indicate that the level of RhoA and ROCK-I increases in patients with ADS and the cyclic change is lost. Estrogen may affect uterine JZ contraction of ADS by enhancing RhoA/ ROCK-I signaling., (© The Author(s) 2015.)

Although calcineurin (CN) is distributed in many cell types and functions in regulating cell functions, the precise roles ofCNremained in each type of the cells are not well understood yet. ACNinhibitor (CNI) has been used for steroid-resistant nephrotic syndrome. ACNIis assumed to ameliorate proteinuria by preventing the overproduction of T-cell cytokines. However, recent reports suggest thatCNIhas a direct effect on podocyte. It is accepted that a slit diaphragm (SD), a unique cell-cell junction of podocytes, is a critical barrier preventing a leak of plasma protein into urine. Therefore, we hypothesized thatCNIhas an effect on theSD In this study, we analyzed the expression ofCNin physiological and in the nephrotic model caused by the antibody against nephrin, a critical component of theSD We observed thatCNis expressed at theSDin normal rat and human kidney sections and has an interaction with nephrin. The staining ofCNat theSDwas reduced in the nephrotic model, whileCNactivity in glomeruli was increased. We also observed that the treatment with tacrolimus, aCNI, in this nephrotic model suppressed the redistribution ofCN, nephrin, and otherSDcomponents and ameliorated proteinuria. These observations suggested that the redistribution and the activation ofCNmay participate in the development of theSDinjury., (© 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.)

We analyzed the potential role of oxidative stress induced by mono (2-ethylhexyl) phthalate (MEHP) in adherent cell junction protein expression of prepubertal rat Sertoli cells (SC) in vitro. Five-day SC cultures were treated with MEHP (200μM) for 24h and compared to cells in basal conditions. Western blot and immunofluorescent (IF) analyses showed that MEHP induced increase of N-cadherin and catenin expression, modifying its distribution. Concomitantly, Cx-43 expression decreased significantly and delocalization of the IF signal for tight junction proteins (occludin, claudin-11 and ZO-1) occurred. Indicative of oxidative stress, MEHP induced in SC an increase of lipoperoxides, a decrease in glutathione (GSH) levels and a concomitant increase in Glutathione S-Transferases (GST) activity. Antioxidant N-acetyl-cysteine (1mM) treatment prevented GSH decrease and N-cadherin and α-catenin up-regulation induced by MEHP. Our data suggest that oxidative stress signaling is a mechanism involved in adherent cell junctions disruption induced by MEHP in SC cultures., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Fetal-onset hydrocephalus affects 1 to 3 per 1,000 live births. It is not only a disorder of cerebrospinal fluid dynamics but also a brain disorder that corrective surgery does not ameliorate. We hypothesized that cell junction abnormalities of neural stem cells (NSCs) lead to the inseparable phenomena of fetal-onset hydrocephalus and abnormal neurogenesis. We used bromodeoxyuridine labeling, immunocytochemistry, electron microscopy, and cell culture to study the telencephalon of hydrocephalic HTx rats and correlated our findings with those in human hydrocephalic and nonhydrocephalic human fetal brains (n = 12 each). Our results suggest that abnormal expression of the intercellular junction proteins N-cadherin and connexin-43 in NSC leads to 1) disruption of the ventricular and subventricular zones, loss of NSCs and neural progenitor cells; and 2) abnormalities in neurogenesis such as periventricular heterotopias and abnormal neuroblast migration. In HTx rats, the disrupted NSC and progenitor cells are shed into the cerebrospinal fluid and can be grown into neurospheres that display intercellular junction abnormalities similar to those of NSC of the disrupted ventricular zone; nevertheless, they maintain their potential for differentiating into neurons and glia. These NSCs can be used to investigate cellular and molecular mechanisms underlying this condition, thereby opening the avenue for stem cell therapy.

Arrhythmogenic cardiomyopathy (ACM) is an acquired progressive disease having an age-related penetrance and showing clinical manifestations usually during adolescence and young adulthood. It is characterized clinically by a high incidence of severe ventricular tachyarrhythmias and sudden cardiac death and pathologically by degeneration of ventricular cardiomyocytes with replacement by fibro-fatty tissue. Whereas, in the past, the disease was considered to involve only the right ventricle, more recent clinical studies have established that the left ventricle is frequently involved. ACM is an inherited disease in up to 50% of cases, with predominantly an autosomal dominant pattern of transmission, although recessive inheritance has also been described. Since most of the pathogenic mutations have been identified in genes encoding desmosomal proteins, ACM is currently defined as a disease of desmosomes. However, on the basis of the most recent description of the intercalated disc organization and of the identification of a novel ACM gene encoding for an area composita protein, ACM can be considered as a disease of the intercalated disc, rather than only as a desmosomal disease. Despite increasing knowledge of the genetic basis of ACM, we are just beginning to understand early molecular events leading to cardiomyocyte degeneration, fibrosis and fibro-fatty substitution. This review summarizes recent advances in our comprehension of the link between the molecular genetics and pathogenesis of ACM and of the novel role of cardiac intercalated discs.

Reflectance confocal microscopy (RCM) images skin noninvasively, with optical sectioning and nuclear-level resolution comparable with that of pathology. On the basis of the assessment of the dermal-epidermal junction (DEJ) and morphologic features in its vicinity, skin cancer can be diagnosed in vivo with high sensitivity and specificity. However, the current visual, qualitative approach for reading images leads to subjective variability in diagnosis. We hypothesize that machine learning-based algorithms may enable a more quantitative, objective approach. Testing and validation were performed with two algorithms that can automatically delineate the DEJ in RCM stacks of normal human skin. The test set was composed of 15 fair- and 15 dark-skin stacks (30 subjects) with expert labelings. In dark skin, in which the contrast is high owing to melanin, the algorithm produced an average error of 7.9±6.4 μm. In fair skin, the algorithm delineated the DEJ as a transition zone, with average error of 8.3±5.8 μm for the epidermis-to-transition zone boundary and 7.6±5.6 μm for the transition zone-to-dermis. Our results suggest that automated algorithms may quantitatively guide the delineation of the DEJ, to assist in objective reading of RCM images. Further development of such algorithms may guide assessment of abnormal morphological features at the DEJ.

Unidirectional zippering is a key step in neural tube closure that remains poorly understood. Here, we combine experimental and computational approaches to identify the mechanism for zippering in a basal chordate, Ciona intestinalis. We show that myosin II is activated sequentially from posterior to anterior along the neural/epidermal (Ne/Epi) boundary just ahead of the advancing zipper. This promotes rapid shortening of Ne/Epi junctions, driving the zipper forward and drawing the neural folds together. Cell contact rearrangements (Ne/Epi + Ne/Epi → Ne/Ne + Epi/Epi) just behind the zipper lower tissue resistance to zipper progression by allowing transiently stretched cells to detach and relax toward isodiametric shapes. Computer simulations show that measured differences in junction tension, timing of primary contractions, and delay before cell detachment are sufficient to explain the speed and direction of zipper progression and highlight key advantages of a sequential contraction mechanism for robust efficient zippering., (Copyright © 2015 Elsevier Inc. All rights reserved.)

An experimental ischemia (EI)-induced mouse model was used to analyze pathological and biochemical alterations in testes. Initial morphological changes were observed in Sertoli cells of EI testes at the light microscopic level. Examination of the ultrastructure using transmission electron microscopy confirmed that Sertoli cells were partially detached from the basement membrane of the seminiferous epithelium and that the cell membranes of adjacent Sertoli cells were not joined. The functional integrity of the blood-testis barrier (BTB) was assessed using the lanthanum tracer technique. Lanthanum had penetrated into the spaces between adjacent Sertoli cells in the adluminal compartment up to the lumen of the seminiferous epithelium in EI testes. Proteome analysis showed that the expression of heat shock protein (HSP) 70 was significantly upregulated in EI testes. Western blot analysis confirmed that the expression of HSP70 increased in a time-dependent manner after the EI procedure. HSP70 immunostaining was observed in spermatocytes and in round and elongated spermatids in EI testes. Our results suggest that a change in the junctions between adjacent Sertoli cells on the basal compartment is involved in the BTB disruption in EI testes. Therefore, male infertility caused by the BTB disruption could be associated with heat stress induced by ischemia.

Aims: The majority of patients diagnosed with arrhythmogenic right ventricular cardiomyopathy (ARVC) have mutations in genes encoding desmosomal proteins, raising the possibility that abnormal intercellular adhesion plays an important role in disease pathogenesis. We characterize cell mechanical properties and molecular responses to oscillatory shear stress in cardiac myocytes expressing mutant forms of the desmosomal proteins, plakoglobin and plakophilin, which are linked to ARVC in patients., Methods and Results: Cells expressing mutant plakoglobin or plakophilin showed no differences in cell-cell adhesion relative to controls, while knocking down these proteins weakened cell-cell adhesion. However, cells expressing mutant plakoglobin failed to increase the amount of immunoreactive signal for plakoglobin or N-cadherin at cell-cell junctions in response to shear stress, as seen in control cells. Cells expressing mutant plakophilin exhibited a similar attenuation in the shear-induced increase in junctional plakoglobin immunoreactive signal in response to shear stress, suggesting that the phenotype is independent of the type of mutant protein being expressed. Cells expressing mutant plakoglobin also showed greater myocyte apoptosis compared with controls. Apoptosis rates increased greatly in response to shear stress in cells expressing mutant plakoglobin, but not in controls. Abnormal responses to shear stress in cells expressing either mutant plakoglobin or plakophilin could be reversed by SB216763, a GSK3β inhibitor., Conclusions: Desmosomal mutations linked to ARVC do not significantly affect cell mechanical properties, but cause myocytes to respond abnormally to mechanical stress through a mechanism involving GSK3β. These results may help explain why patients with ARVC experience disease exacerbations following strenuous exercise., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2014. For permissions please email: journals.permissions@oup.com.)

During melanoma cell extravasation through the vascular endothelium, melanoma cells interact with endothelial cells through secretion of cytokines and by adhesion between proteins displayed on opposing cell surfaces. How these tumor cell associated signals together regulate the dynamics of intracellular signaling pathways within endothelial cells leading to endothelial cell-cell junction disruption is not well understood. Here, we used a combination of experimental and computational approaches to examine the individual and combined effects of activation of the vascular cell adhesion molecule (VCAM)-1, interleukin (IL)-8, and IL-1β signaling pathways on the integrity of vascular junctions. Our simulations predict a multifaceted interplay of signaling resulting from individual activation of VCAM-1, IL-8 and IL-1β pathways that is neither synergistic nor additive compared to all inputs turned on simultaneously. Furthermore, we show that the levels of phosphorylated proteins associated with actinomyosin contractility and junction disassembly peak prior to those related to actin remodeling. The results of this work provide insight into the dynamics of tumor-mediated endothelial junction disassembly and suggest that targeting proteins downstream of several interaction pathways may be the most effective therapeutic approach to reduce melanoma extravasation.

Weil's disease, the most severe form of leptospirosis, is characterized by jaundice, haemorrhage and renal failure. The mechanisms of jaundice caused by pathogenic Leptospira remain unclear. We therefore aimed to elucidate the mechanisms by integrating histopathological changes with serum biochemical abnormalities during the development of jaundice in a hamster model of Weil's disease. In this work, we obtained three-dimensional images of infected hamster livers using scanning electron microscope together with freeze-cracking and cross-cutting methods for sample preparation. The images displayed the corkscrew-shaped bacteria, which infiltrated the Disse's space, migrated between hepatocytes, detached the intercellular junctions and disrupted the bile canaliculi. Destruction of bile canaliculi coincided with the elevation of conjugated bilirubin, aspartate transaminase and alkaline phosphatase levels in serum, whereas serum alanine transaminase and γ-glutamyl transpeptidase levels increased slightly, but not significantly. We also found in ex vivo experiments that pathogenic, but not non-pathogenic leptospires, tend to adhere to the perijunctional region of hepatocyte couplets isolated from hamsters and initiate invasion of the intercellular junction within 1 h after co-incubation. Our results suggest that pathogenic leptospires invade the intercellular junctions of host hepatocytes, and this invasion contributes in the disruption of the junction. Subsequently, bile leaks from bile canaliculi and jaundice occurs immediately. Our findings revealed not only a novel pathogenicity of leptospires, but also a novel mechanism of jaundice induced by bacterial infection., (© 2014 The Authors. International Journal of Experimental Pathology © 2014 International Journal of Experimental Pathology.)

Epidemiologic data suggest an association between depot medroxyprogesterone acetate (DMPA), a progesterone-based hormonal contraceptive, and increased risk of HIV acquisition and transmission. DMPA is highly effective and is among the most commonly used form of hormonal contraception in areas of high HIV prevalence. Thus, defining the biological mechanisms that contribute to the potential negative synergy between DMPA and HIV is key and may facilitate the identification of alternative contraceptive strategies. Proposed mechanisms include thinning or disruption of the cervicovaginal epithelial barrier, induction of mucosal inflammation, interference with innate and adaptive soluble and cellular immune responses, and/or alterations in the vaginal microbiome. DMPA may also indirectly increase the risk of HIV by promoting genital herpes or other sexually transmitted infections. However, there is a paucity of rigorous in vitro, animal model and clinical data to support these potential mechanisms highlighting the need for future research., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)