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Background: Mitral valve (MV) disease including myxomatous degeneration is the most common form of valvular heart disease with an age-dependent frequency. Genetic evidence indicates that mutations of the human transcription factor FOXC1 are associated with MV defects, including MV regurgitation. In this study, we sought to determine whether murine Foxc1 and its closely related factor, Foxc2 , are required in valvular endothelial cells (VECs) for the maintenance of MV leaflets, including VEC junctions and the stratified trilaminar ECM (extracellular matrix)., Methods: Adult mice carrying tamoxifen-inducible, vascular endothelial cell (EC), and lymphatic EC-specific, compound Foxc1;Foxc2 mutations (ie, EC- Foxc -DKO and lymphatic EC- Foxc -DKO mice, respectively) were used to study the function of Foxc1 and Foxc2 in the maintenance of MVs. The EC and lymphatic EC mutations of Foxc1/c2 were induced at 7 to 8 weeks of age by tamoxifen treatment, and abnormalities in the MVs of these mutant mice were assessed via whole-mount immunostaining, immunohistochemistry/RNAscope, Movat pentachrome/Masson Trichrome staining, and Evans blue injection., Results: EC deletions of Foxc1 and Foxc2 in mice resulted in abnormally extended and thicker MVs by causing defects in the regulation of ECM organization with increased proteoglycan and decreased collagen. Notably, reticular adherens junctions were found in VECs of control MV leaflets, and these reticular structures were severely disrupted in EC- Foxc -DKO mice. PROX1 (prospero homeobox protein 1), a key regulator in a subset of VECs on the fibrosa side of MVs, was downregulated in EC- Foxc1/c2 mutant VECs. Furthermore, we determined the precise location of lymphatic vessels in murine MVs, and these lymphatic vessels were aberrantly expanded and dysfunctional in EC- Foxc1/c2 mutant MVs. Lymphatic EC deletion of Foxc1/c2 also resulted in similar structural/ECM abnormalities as seen in EC- Foxc1/c2 mutant MVs., Conclusions: Our results indicate that Foxc1 and Foxc2 are required for maintaining the integrity of the MV, including VEC junctions, ECM organization, and lymphatic vessel formation/function to prevent myxomatous MV degeneration., Competing Interests: None.

Clinically, Porcine circovirus type 2 (PCV2) often causes disease through coinfection with other bacterial pathogens, including Glaesserella parasuis (G. parasuis), which causes high morbidity and mortality. However, the mechanism of PCV2 and G. parasuis serotype 4 (GPS4) co-infection is still not fully understood. In this study, swine tracheal epithelial cells (STEC) were used as a barrier model, and our results showed that PCV2 infection increased the adhesion of GPS4 to STEC, while decreasing the levels of ZO-1, Occludin and increasing tracheal epithelial permeability, and ultimately facilitated GPS4 translocation. Snail1 is a transcriptional repressor, and has been known to induce epithelial-to-mesenchymal transition (EMT) during development or in cancer metastasis. Importantly, we found that Snail1, as a transcriptional repressor, was crucial in destroying the tracheal epithelial barrier induced by PCV2, GPS4, PCV2 and GPS4 coinfection. For the first time, we found that PCV2, GPS4, PCV2 and GPS4 coinfection cross-activates TGF-β and p38/MAPK signaling pathways to upregulate the expression of Snail1, down-regulate the levels of ZO-1 and Occludin, and thus disrupt the integrity of tracheal epithelial barrier then promoting GPS4 translocation. Finally, PCV2 and GPS4 co-infection also can activate TGF-β and p38/MAPK signaling pathways in vivo and upregulate Snail1, ultimately down-regulating the expression of ZO-1 and Occludin. Our study elucidates how PCV2 infection promotes GPS4 to breach the tracheal epithelial barrier and aggravate clinical manifestations., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Cell communication and signal transduction rely heavily on the charge on the cell surface. The cell surface is negatively charged, with glycoproteins on the cell membrane providing a large percentage of the charge. Sialic acid is found on the outermost side of glycan chains and contributes to glycoprotein's negative charge. Sialic acid is highly expressed in tumor cells and plays an important role in tumor metastasis and immune escape by interacting with extracellular ligands. However, the specific effect of negative charge changes on glycoproteins is still poorly understood. In this study, we used 9-azido sialic acid (9Az-Sia) to create artificial epitopes on glycoproteins via metabolic glycan labeling, and we attached charged groups such as amino and carboxyl to 9Az-Sia via a click reaction with dibenzocyclooctyne (DBCO). The charge of glycoproteins was changed by metabolic glycan labeling and click modification. The results showed that the migration and invasion ability of the MDA-MB-231 cell labeled with 9Az-Sia was significantly reduced after the modification with amino groups rather than carboxyl groups. Epithelial-mesenchymal transition (EMT) is the biological process of metastatic tumor cells, with an increasing ability of tumor cells to migrate and invade. In particular, the expression of adhesion molecules increased in the amine-linked group, whereas the expression of matrix metalloproteinases (MMPs) increased significantly, which is not identical to EMT characteristics. In vivo experiments have demonstrated that the loss of negative charge on glycoproteins has an inhibitory effect on tumors. In conclusion, modifying the positive charge on the surface of glycoproteins can inhibit tumor cell metastasis and has great potential for tumor therapy.

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.)

Thanatochemistry also known as chemistry of death and is used to determine post mortem interval (PMI). It is arguably one of the critical steps in forensic investigation. Recent addition of analyzing biochemical changes along with the traditional methods have gained importance, as they help us to record very early changes in the tissue specimens. In this view, our study aimed to correlate both histological changes and enzymatic changes in gingival tissue samples at intervals of immediate, 1 h, 5 h, 24 h and 48 h after death. Histologic changes noted were loss of epithelial architecture, chromatin clumping, nuclear vacuolation, karryopyknosis, eosinophilia and wide intercellular junctions. Two enzymes which differentiate between the autolytic phase (acid phosphatase) and putrefactive phase (ammonia) of decomposition were evaluated using UV spectrometer. Results in our study demonstrated there were variations as in gradual increase in ammonia levels (1.13±0.24-26.6±2.09) and gradual decrease in acid phosphatase levels (5.61±0.67-1.25±0.53) at different time intervals till 48 h. The cellular changes in gingival tissue could also be related to time. The result of our study helps us to identify potential of enzymatic changes which when correlated with histological reports helps us to predict the time of death accurately. Replicating this experiment in various known taphonomic conditions and other enzymes could highlight the usefulness of gingival tissue samples in determining time of death., Competing Interests: Funding and conflict statement The authors did not receive any external funding towards this study. The authors also state that there is no conflict of interest among them for this study., (Copyright © 2021. Published by Elsevier B.V.)

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).)

Organs consist of multiple cell types that ensure proper architecture and function. How different cell types coexist and interact to maintain their homeostasis in vivo remains elusive. The skin epidermis comprises mostly epithelial cells, but also harbours Langerhans cells (LCs) and dendritic epidermal T cells (DETCs). Whether and how distributions of LCs and DETCs are regulated during homeostasis is unclear. Here, by tracking individual cells in the skin of live adult mice over time, we show that LCs and DETCs actively maintain a non-random spatial distribution despite continuous turnover of neighbouring basal epithelial cells. Moreover, the density of epithelial cells regulates the composition of LCs and DETCs in the epidermis. Finally, LCs require the GTPase Rac1 to maintain their positional stability, density and tiling pattern reminiscent of neuronal self-avoidance. We propose that these cellular mechanisms provide the epidermis with an optimal response to environmental insults.

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.)

Respiratory viruses such as influenza and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are a constant threat to public health given their ability to cause global pandemics. Infection with either virus may lead to aberrant host responses, such as excessive immune cell recruitment and activation, dysregulated inflammation, and coagulopathy. These may contribute to the development of lung edema and respiratory failure. An increasing amount of evidence suggests that lung endothelial cells play a critical role in the pathogenesis of both viruses. In this review, we discuss how infection with influenza or SARS-CoV-2 may induce endothelial dysfunction. We compare the effects of infection of these two viruses, how they may contribute to pathogenesis, and discuss the implications for potential treatment. Understanding the differences between the effects of these two viruses on lung endothelial cells will provide important insight to guide the development of therapeutics., Competing Interests: W.L.L. is a co-inventor on a patent for the use of Vasculotide for influenza and previously (2016–2018) served on the Scientific Advisory Board for Vasomune. E.L. has no disclosures.

Background and Aims: As the incidence of nonalcoholic steatohepatitis (NASH) continues to rise, understanding how normal liver functions are affected during disease is required before developing novel therapeutics which could reduce morbidity and mortality. However, very little is understood about how the transport of proteins and cells from the liver by the lymphatic vasculature is affected by inflammatory mediators or during disease., Methods: To answer these questions, we utilized a well-validated mouse model of NASH and exposure to highly oxidized low density lipoprotein (oxLDL). In addition to single cell sequencing, multiplexed immunofluorescence and metabolomic analysis of liver lymphatic endothelial cells (LEC)s we evaluated lymphatic permeability and transport both in vitro and in vivo., Results: Confirming similarities between human and mouse liver lymphatic vasculature in NASH, we found that the lymphatic vasculature expands as disease progresses and results in the downregulation of genes important to lymphatic identity and function. We also demonstrate, in mice with NASH, that fluorescein isothiocyanate (FITC) dextran does not accumulate in the liver draining lymph node upon intrahepatic injection, a defect that was rescued with therapeutic administration of the lymphatic growth factor, recombinant vascular endothelial growth factor C (rVEGFC). Similarly, exposure to oxLDL reduced the amount of FITC-dextran in the portal draining lymph node and through an LEC monolayer. We provide evidence that the mechanism by which oxLDL impacts lymphatic permeability is via a reduction in Prox1 expression which decreases lymphatic specific gene expression, impedes LEC metabolism and reorganizes the highly permeable lymphatic cell-cell junctions which are a defining feature of lymphatic capillaries., Conclusions: We identify oxLDL as a major contributor to decreased lymphatic permeability in the liver, a change which is consistent with decreased protein homeostasis and increased inflammation during chronic liver disease., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

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.)

Plasticity of cancer invasion and metastasis depends on the ability of cancer cells to switch between collective and single-cell dissemination, controlled by cadherin-mediated cell-cell junctions. In clinical samples, E-cadherin-expressing and -deficient tumours both invade collectively and metastasize equally, implicating additional mechanisms controlling cell-cell cooperation and individualization. Here, using spatially defined organotypic culture, intravital microscopy of mammary tumours in mice and in silico modelling, we identify cell density regulation by three-dimensional tissue boundaries to physically control collective movement irrespective of the composition and stability of cell-cell junctions. Deregulation of adherens junctions by downregulation of E-cadherin and p120-catenin resulted in a transition from coordinated to uncoordinated collective movement along extracellular boundaries, whereas single-cell escape depended on locally free tissue space. These results indicate that cadherins and extracellular matrix confinement cooperate to determine unjamming transitions and stepwise epithelial fluidization towards, ultimately, cell individualization.

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.

Background and Aims: The Wnt/β-catenin signaling pathway has a well-described role in liver pathobiology. Its suppression was recently shown to decrease bile acid (BA) synthesis, thus preventing the development of cholestatic liver injury and fibrosis after bile duct ligation (BDL)., Approach and Results: To generalize these observations, we suppressed β-catenin in Mdr2 knockout (KO) mice, which develop sclerosing cholangitis due to regurgitation of BA from leaky ducts. When β-catenin was knocked down (KD) in KO for 2 weeks, hepatic and biliary injury were exacerbated in comparison to KO given placebo, as shown by serum biochemistry, ductular reaction, inflammation, and fibrosis. Simultaneously, KO/KD livers displayed increased oxidative stress and senescence and an impaired regenerative response. Although the total liver BA levels were similar between KO/KD and KO, there was significant dysregulation of BA transporters and BA detoxification/synthesis enzymes in KO/KD compared with KO alone. Multiphoton intravital microscopy revealed a mixing of blood and bile in the sinusoids, and validated the presence of increased serum BA in KO/KD mice. Although hepatocyte junctions were intact, KO/KD livers had significant canalicular defects, which resulted from loss of hepatocyte polarity. Thus, in contrast to the protective effect of β-catenin KD in BDL model, β-catenin KD in Mdr2 KO aggravated rather than alleviated injury by interfering with expression of BA transporters, hepatocyte polarity, canalicular structure, and the regenerative response., Conclusions: The resulting imbalance between ongoing injury and restitution led to worsening of the Mdr2 KO phenotype, suggesting caution in targeting β-catenin globally for all cholestatic conditions., (© 2019 by the American Association for the Study of Liver Diseases.)

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.)

Altered intestinal epithelial integrity is an important susceptibility trait in inflammatory bowel disease (IBD), and early life stressors are reported to contribute to this disease susceptibility in adulthood. To identify disease mechanisms associated with early-life trauma that exacerbate IBD in adulthood, we used a "double-hit" neonatal inflammation (NI) and adult inflammation (AI) model that exhibits more severe mucosal injury in the colon later in life. In this study, we explore the underlying mechanisms of this aggravated injury. In rats exposed to both NI and AI, we found sustained increases in colonic permeability accompanied by significantly attenuated expression of the epithelial junction protein E-cadherin. Quantitative RT-PCR revealed a decreased Cdh1 (gene of E-cadherin) mRNA expression in NI + AI rats compared with NI or AI rats. Next, we performed microRNA microarrays to identify potential regulators of E-cadherin in NI + AI rats. We confirmed the overexpression of miR-155, a predicted regulator of E-cadherin, and selected it for further analysis based on reported significance in human IBD. Using ingenuity pathway analysis software, the targets and related canonical pathway of miR-155 were analyzed. Mechanistic studies identified histone hyperacetylation at the Mir155 promoter in NI + AI rats, concomitant with elevated RNA polymerase II binding. In vitro, E-cadherin knockdown markedly increased epithelial cell permeability, as did overexpression of miR-155 mimics, which significantly suppressed E-cadherin protein. In vivo, NI + AI colonic permeability was significantly reversed with administration of miR-155 inhibitor rectally. Our collective findings indicate that early-life inflammatory stressors trigger a significant and sustained epithelial injury by suppressing E-cadherin through epigenetic mechanisms., (Copyright © 2020 by The American Association of Immunologists, Inc.)

Endothelial cells form the inner lining of all blood vessels and play a vital role in regulating vascular permeability. This applies to the circulation in general and also to specific capillary beds including the renal glomerular capillaries. Endothelial dysfunction, including increased permeability, is a key component of diabetes-induced organ damage. Endothelial cells together with their glycocalyx, grown on porous membranes, provide an excellent model to study endothelial permeability properties. Here we describe the measurement of two characteristics of glomerular endothelial cell (GEnC) monolayers: electrical resistance and macromolecular passage. Trans-endothelial electrical resistance provides a measure of small-pore pathways across the endothelium and provides an index of monolayer confluence and cell-cell junction integrity. Measurement of macromolecular passage provides an index of large-pore pathways and use of labeled albumin provides direct relevance to the clinically important parameter of albuminuria. The combination of the two approaches provides a fantastic tool to elucidate endothelial barrier function in vitro including in response to cytokines, pathological stimuli, and potential therapeutic agents.

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.

Chlamydia trachomatis is the most prevalent sexually transmitted bacterial pathogen and the leading cause of preventable blindness in the developing world. C. trachomatis invades the epithelium of the conjunctiva and genital tract and replicates within an intracellular membrane-bound compartment termed the inclusion. To invade and replicate in mammalian cells, Chlamydia remodels epithelial surfaces by reorganizing the cytoskeleton and cell-cell adhesions, reprograms membrane trafficking, and modulates cell signaling to dampen innate immune responses. If the infection ascends to the upper female genital tract, it can result in pelvic inflammatory disease and tissue scarring. C. trachomatis infections are associated with infertility, ectopic pregnancies, the fibrotic disorder endometriosis, and potentially cancers of the cervix and uterus. Unfortunately, the molecular mechanisms by which this clinically important human pathogen subverts host cellular functions and causes disease have remained relatively poorly understood because of the dearth of molecular genetic tools to study Chlamydiae and limitations of both in vivo and in vitro infection models. In this review, we discuss recent advances in the experimental molecular tool kit available to dissect C. trachomatis infections with a special focus on Chlamydia -induced epithelial barrier disruption by regulating the structure, function, and dynamics of epithelial cell-cell junctions., Competing Interests: Competing interests: RHV is a member of the scientific advisory board of Meridian Biosciences. LD declares that he has no competing interests.No competing interests were disclosed.No competing interests were disclosed.No competing interests were disclosed.

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.)

Lanthanum could cause cognitive impairment in children and rodent animals. The normal blood-brain barrier (BBB) integrity is essential for protecting the brain from systemic toxins and maintaining the homeostasis for proper neuronal function. BBB dysfunction has been implicated as a potential mechanism of heavy metal-induced neurotoxicity. The present study was aimed to investigate effects of lanthanum on BBB integrity and endothelial junctional complexes in the cerebral cortex of young rats. Animals were exposed to lanthanum chloride (LaCl 3 ) through drinking water under 0, 0.25, 0.5, and 1.0% concentrations from postnatal day 0 until 30 days after weaning. LaCl 3 -exposure increased BBB permeability, caused ultrastructure changes in cerebral capillaries, and reduced protein expression of claudin-5, occludin, and VE-cadherin. Due to the critical role of matrix metalloproteinases (MMPs) in BBB integrity, we further examined alterations in MMPs activity and expression. Enhanced gelatinase activity and upregulated MMP-9 expression were observed after LaCl 3 -exposure, concurrently with decreased expression of endogenous inhibitor tissue inhibitors of metalloproteinase (TIMP)-1. Taken together, this study demonstrated that postnatal lanthanum exposure caused leakage of BBB in young rats, partially attributed to upregulation of MMP-9 and reduction of junctional proteins expression.

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.)

Merkel cell carcinoma (MCC) is an aggressive skin cancer with a high propensity for recurrence and metastasis. Merkel cell polyomavirus (MCPyV) is recognised as the causative factor in the majority of MCC cases. The MCPyV small tumour antigen (ST) is considered to be the main viral transforming factor, however potential mechanisms linking ST expression to the highly metastatic nature of MCC are yet to be fully elucidated. Metastasis is a complex process, with several discrete steps required for the formation of secondary tumour sites. One essential trait that underpins the ability of cancer cells to metastasise is how they interact with adjoining tumour cells and the surrounding extracellular matrix. Here we demonstrate that MCPyV ST expression disrupts the integrity of cell-cell junctions, thereby enhancing cell dissociation and implicate the cellular sheddases, A disintegrin and metalloproteinase (ADAM) 10 and 17 proteins in this process. Inhibition of ADAM 10 and 17 activity reduced MCPyV ST-induced cell dissociation and motility, attributing their function as critical to the MCPyV-induced metastatic processes. Consistent with these data, we confirm that ADAM 10 and 17 are upregulated in MCPyV-positive primary MCC tumours. These novel findings implicate cellular sheddases as key host cell factors contributing to virus-mediated cellular transformation and metastasis. Notably, ADAM protein expression may be a novel biomarker of MCC prognosis and given the current interest in cellular sheddase inhibitors for cancer therapeutics, it highlights ADAM 10 and 17 activity as a novel opportunity for targeted interventions for disseminated MCC., Competing Interests: The authors have declared that no competing interests exist.

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.)

Oral mucositis is the most frequently occurring early side effect of head-and-neck cancer radiotherapy. Systemic dermatan sulfate (DS) treatment revealed a significant radioprotective potential in a preclinical model of oral mucositis. This study was initiated to elucidate the mechanistic effects of DS in the same model. Irradiation comprised daily fractionated irradiation (5 × 3 Gy/week) over two weeks, either alone (IR) or in combination with daily dermatan sulfate treatment of 4 mg/kg (IR + DS). Groups of mice ( n = 5) were sacrificed every second day over the course of 14 days in both experimental arms, their tongues excised and evaluated. The response to irradiation with and without DS was analyzed on a morphological (cell numbers, epithelial thickness) as well as on a functional (proliferation and expression of inflammation, hypoxia and epithelial junction markers) level. The mucoprotective activity of DS can be attributed to a combination of various effects, comprising increased expression of epithelial junctions, reduced inflammation and reduced hypoxia. No DS-mediated effect on proliferation was observed. DS demonstrated a significant mucositis-ameliorating activity and could provide a promising strategy for mucositis treatment, based on targeting specific, radiation-induced, mucositis-associated signaling without stimulating proliferation.

Centromere-binding protein F (CENP-F) is a very large and complex protein with many and varied binding partners including components of the microtubule network. Numerous CENP-F functions impacting diverse cellular behaviors have been identified. Importantly, emerging data have shown that CENP-F loss- or gain-of-function has critical effects on human development and disease. Still, it must be noted that data at the single cardiac myocyte level examining the impact of CENP-F loss-of-function on fundamental cellular behavior is missing. To address this gap in our knowledge, we analyzed basic cell structure and function in cardiac myocytes devoid of CENP-F. We found many diverse structural abnormalities including disruption of the microtubule network impacting critical characteristics of the cardiac myocyte. This is the first report linking microtubule network malfunction to cardiomyopathy. Importantly, we also present data demonstrating a direct link between a CENP-F single nucleotide polymorphism (snp) and human cardiac disease. In a proximate sense, these data examining CENP-F function explain the cellular basis underlying heart disease in this genetic model and, in a larger sense, they will hopefully provide a platform upon which the field can explore diverse cellular outcomes in wide-ranging areas of research on this critical protein.

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.)

Background: The morbidity of sepsis induced acute kidney injury remains unacceptable high and the mechanisms of that disease remains unclear. For urine backleak and intercellular tight junction among tubular epithelial cells (TECs) destruction often occur during sepsis induced acute kidney injury, we examined whether lipopolysaccharide could damage intercellular tight junction among TECs and associated mechanisms in our present study. Methods: HK-2 cells were cultured, transfected with different SiRNAs and stimulated with LPS and PYR-41. Transepithelial Permeability Assay and Transepithelial Electrical Resistance Assay were used to evaluate intercellular tight junction destruction and Western Blot and Immunofluorescence were used to evaluate proteins expression. Results: Transepithelial Permeability increased significantly (P<0.05) and Transepithelial Electrical Resistance reduced remarkably (P<0.05) of the monolayer TECs stimulated with LPS. The expression of JAM-3 and RhoT1 decreased significantly (P<0.05) in TECs stimulated with LPS, while the level of SMAD-4 increased significantly (P<0.05). Downregulation of the expression of SMAD-4 with RNA interference could increase the expression of JAM-3 in LPS treated TECs. Moreover, upregulation of RhoT1 level by decreased the degradation of RhoT1 could decrease the expression of SMAD-4 and increase the JAM-3 level in TECs treated with LPS, while downregulation of RhoT1 level with RNA interference had the opposite effects. Conclusion: LPS mediates intercellular tight junction destruction among TECs and RhoT1/SMAD-4/JAM-3 is a pivotal pathway to mediate the phenomenon., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Objectives: Tumor-derived exosomes (TDE) have been shown to participate in different steps of the dissemination of cancer cells. However, the role of salivary adenoid cystic carcinoma-derived (SACC-derived) exosomes had not been documented in SACC. The study aims to explore the functions of SACC-derived TDE in SACC progression and investigate potential mechanisms., Methods: Salivary adenoid cystic carcinoma cell line SACC-83 was used to generate TDE. Afterward, SACC-83 or HUVECs were cocultured with or without TDE. Tumor migration, tumor invasion, and endothelial permeability were examined by wound healing assay, tumor invasion assay, endothelial permeability assay, and tumor cell transendothelial migration assay, respectively. Moreover, the expression levels of cell junction-related proteins were examined by qRT-PCR and Western blot., Results: Salivary adenoid cystic carcinoma -83-derived exosomes were taken up by their host cells. Meanwhile, TDE increased migration and invasion capacity of SACC-83 cells and enhanced endothelial cell permeability. Furthermore, we demonstrated that the expression of cell junction-related proteins (Claudins and ZO-1) was downregulated, which is presumably involved in the TDE-mediated promotion of migration, invasion, and metastasis., Conclusion: The results suggested that SACC cell-derived exosomes were loaded with individual components that could enhance invasiveness and induce microenvironment changes, thus promoting SACC aggression., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

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.

Diabetic retinopathy is an important cause of blindness in adults, and is characterized by progressive loss of vascular cells and slow dissolution of inter-vascular junctions, which result in vascular leakage and retinal oedema. Later stages of the disease are characterized by inflammatory cell infiltration, tissue destruction and neovascularization. Here we identify soluble epoxide hydrolase (sEH) as a key enzyme that initiates pericyte loss and breakdown of endothelial barrier function by generating the diol 19,20-dihydroxydocosapentaenoic acid, derived from docosahexaenoic acid. The expression of sEH and the accumulation of 19,20-dihydroxydocosapentaenoic acid were increased in diabetic mouse retinas and in the retinas and vitreous humour of patients with diabetes. Mechanistically, the diol targeted the cell membrane to alter the localization of cholesterol-binding proteins, and prevented the association of presenilin 1 with N-cadherin and VE-cadherin, thereby compromising pericyte-endothelial cell interactions and inter-endothelial cell junctions. Treating diabetic mice with a specific sEH inhibitor prevented the pericyte loss and vascular permeability that are characteristic of non-proliferative diabetic retinopathy. Conversely, overexpression of sEH in the retinal Müller glial cells of non-diabetic mice resulted in similar vessel abnormalities to those seen in diabetic mice with retinopathy. Thus, increased expression of sEH is a key determinant in the pathogenesis of diabetic retinopathy, and inhibition of sEH can prevent progression of the disease.

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.)

ASPP2 regulates cell polarity and cell-cell adhesion by binding to, and co-localizing with PAR3 at tight junctions. Here we show a novel role of ASPP2 in suppressing gastric cancer (GC) invasiveness. Immunoprecipitation and immunofluorescence analyses showed that ASPP2 promoted the recruitment of PAR3 to cell-cell junctions in GC cells. Diminished expression of ASPP2 and loss of junctional PAR3 localization were significantly associated with diffuse-type histology, deeper invasion depth, positive peritoneal dissemination and worse prognosis in primary GC. ASPP2 suppressed migration and invasion of GC cells in vitro and peritoneal dissemination of GC cells in vivo in a mouse model. ASPP2 suppressed epithelial-mesenchymal transition (EMT) induced by TGF-β1-Smad2/3 signaling in GC cells through suppression of the degradation of Smad7, a negative regulator of TGF-β1-Smad2/3 signaling, by interacting with the E3 ubiquitin ligase ITCH. In conclusion, ASPP2 suppresses invasion, peritoneal dissemination and TGF-β1-induced EMT by inhibiting Smad7 degradation mediated by ITCH., (Copyright © 2017 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.)