Claudin-5, occludin, zonulin and tricellulin levels of children with attention-deficit/hyperactivity disorder

Background and objectives Accumulating studies have pointed out that gut-blood and blood-brain barrier dysfunctions due to the alterations in permeability may play a role in the pathophysiology of neurodevelopmental disorders. Tight junctions are crucial components of these barriers and some peptides including claudin-5, occludin, zonulin and tricellulin are important components of these structures. This study aimed to investigate the relationship between these molecules and attention deficit hyperactivity disorder (ADHD) in children and adolescents. Methods A total of 57 children with ADHD and 60 controls aged between 6 and 12 years were included in the study. The severity of ADHD symptoms was assessed through a parent-rated questionnaire, and Conner's Continuous Performance Test was administered to the study group. Serum levels of biochemical variables were measured using enzyme-linked immunosorbent assay kits. Biochemical parameter levels and scale scores were compared using Mann-Whitney U or Student's t tests. In addition, a multivariate analysis of covariance (MANCOVA) and a one-way analysis of covariance (ANCOVA) was performed on the outcome variables. Finally, a hierarchical regression model was conducted on the study group. Results Serum claudin-5 and tricellulin levels were significantly lower in the ADHD group compared to the control group. The difference between the groups in terms of serum claudin-5 and tricellulin levels remained significant after controlling for confounding factors such as age, gender and autistic characteristics. There was no significant difference between the groups in terms of serum zonulin and occludin levels.Conclusion These results reveal that claudin-5 and tricellulin levels vary in patients with ADHD. Alterations in these peptides may affect the brain by leading to a dysregulation in intestinal or blood-brain barrier permeability. The causal relationship between these peptides and ADHD requires further ... (AU)

Blood-brain barrier genetic disruption leads to protective barrier formation at the Glia Limitans.

Inflammation of the central nervous system (CNS) induces endothelial blood-brain barrier (BBB) opening as well as the formation of a tight junction barrier between reactive astrocytes at the Glia Limitans. We hypothesized that the CNS parenchyma may acquire protection from the reactive astrocytic Glia Limitans not only during neuroinflammation but also when BBB integrity is compromised in the resting state. Previous studies found that astrocyte-derived Sonic hedgehog (SHH) stabilizes the BBB during CNS inflammatory disease, while endothelial-derived desert hedgehog (DHH) is expressed at the BBB under resting conditions. Here, we investigated the effects of endothelial Dhh on the integrity of the BBB and Glia Limitans. We first characterized DHH expression within endothelial cells at the BBB, then demonstrated that DHH is down-regulated during experimental autoimmune encephalomyelitis (EAE). Using a mouse model in which endothelial Dhh is inducibly deleted, we found that endothelial Dhh both opens the BBB via the modulation of forkhead box O1 (FoxO1) transcriptional activity and induces a tight junctional barrier at the Glia Limitans. We confirmed the relevance of this glial barrier system in human multiple sclerosis active lesions. These results provide evidence for the novel concept of "chronic neuroinflammatory tolerance" in which BBB opening in the resting state is sufficient to stimulate a protective barrier at the Glia Limitans that limits the severity of subsequent neuroinflammatory disease. In summary, genetic disruption of the BBB generates endothelial signals that drive the formation under resting conditions of a secondary barrier at the Glia Limitans with protective effects against subsequent CNS inflammation. The concept of a reciprocally regulated CNS double barrier system has implications for treatment strategies in both the acute and chronic phases of multiple sclerosis pathophysiology.

Involvement of claudin-5 in H2S-induced acute lung injury.

Acute exposure to hydrogen sulfide (H2S) can cause fatal acute lung injury (ALI). However, the mechanisms of H2S-induced ALI are still not fully understood. This study aims to investigate the role of the tight junction protein claudin-5 in H2S-induced ALI. In our study, Sprague-Dawley (SD) rats were exposed to H2S to establish the ALI model, and in parallel, human pulmonary microvascular endothelial cells (HPMECs) were incubated with NaHS (a H2S donor) to establish a cell model. Lung immunohistochemistry and electron microscopy assays were used to identify H2S-induced ALI, and the expression of claudin-5, p-AKT/t-AKT and p-FoxO1/t-FoxO1 was detected. Our results show that H2S promoted the formation of ALI by morphological investigation and decreased claudin-5 expression. Dexamethasone (Dex) could partly attenuate NaHS-mediated claudin-5 downregulation, and the protective effects of Dex could be partially blocked by LY294002, a PI3K/AKT/FoxO1 pathway antagonist. Moreover, as a consequence of the altered phosphorylation of AKT and FoxO1, a change in claudin-5 with the same trend was observed. Therefore, the tight junction protein claudin-5 might be considered a therapeutic target for the treatment of ALI induced by H2S and other hazardous gases.

Inner Blood-Retinal Barrier Regulation in Retinopathies.

The neural retina is protected from the blood circulation by the presence of a highly selective inner blood-retinal barrier (iBRB). The presence of sophisticated tight junctions (TJs) between the endothelial cells (ECs) of the iBRB helps mediate the very low passive permeability of the tissue, permitting entry of nutrients into the retina but excluding harmful toxic material and inflammatory cells. The most highly enriched TJ protein is claudin-5, which is critical in mediating the passive paracellular diffusion barrier properties of the iBRB. In numerous retinal degeneration pathologies, TJ disruption is observed, and a more refined understanding of this disruption could be used for therapeutic benefit.

Oxygen-Glucose Deprivation/Reoxygenation-Induced Barrier Disruption at the Human Blood-Brain Barrier is Partially Mediated Through the HIF-1 Pathway.

The blood-brain barrier (BBB) plays an important role in brain homeostasis. Hypoxia/ischemia constitutes an important stress factor involved in several neurological disorders by inducing the disruption of the BBB, ultimately leading to cerebral edema formation. Yet, our current understanding of the cellular and molecular mechanisms underlying the BBB disruption following cerebral hypoxia/ischemia remains limited. Stem cell-based models of the human BBB present some potentials to address such issues. Yet, such models have not been validated in regard of its ability to respond to hypoxia/ischemia as existing models. In this study, we investigated the cellular response of two iPSC-derived brain microvascular endothelial cell (BMEC) monolayers to respond to oxygen-glucose deprivation (OGD) stress, using two induced pluripotent stem cells (iPSC) lines. iPSC-derived BMECs responded to prolonged (24 h) and acute (6 h) OGD by showing a decrease in the barrier function and a decrease in tight junction complexes. Such iPSC-derived BMECs responded to OGD stress via a partial activation of the HIF-1 pathway, whereas treatment with anti-angiogenic pharmacological inhibitors (sorafenib, sunitinib) during reoxygenation worsened the barrier function. Taken together, our results suggest such models can respond to hypoxia/ischemia similarly to existing in vitro models and support the possible use of this model as a screening platform for identifying novel drug candidates capable to restore the barrier function following hypoxic/ischemic injury.

Hypoxic glioblastoma release exosomal VEGF-A induce the permeability of blood-brain barrier.

Exosomes are nano-vesicles released by tumor cells to modulate extracellular environment. Accumulating evidence revealed that glioblastoma derived exosomes contain multiple pro-angiogenic factors to induce the proliferation of endothelial cells. Here, we investigated the role of GBM-derived exosomes in inducing the permeability of the blood-brain barrier. We found that VEGF-A was over-expressed in hypoxic GBM-derived exosomes, which enhance the permeability of a BBB in vitro model by interrupting the expression of claudin-5 and occludin. In vivo permeability assay showed hypoxic GBM-derived exosomes remained functional in the blood circulation and induced the permeability of BBB.

Dose-dependent expression of claudin-5 is a modifying factor in schizophrenia.

Schizophrenia is a neurodevelopmental disorder that affects up to 1% of the general population. Various genes show associations with schizophrenia and a very weak nominal association with the tight junction protein, claudin-5, has previously been identified. Claudin-5 is expressed in endothelial cells forming part of the blood-brain barrier (BBB). Furthermore, schizophrenia occurs in 30% of individuals with 22q11 deletion syndrome (22q11DS), a population who are haploinsufficient for the claudin-5 gene. Here, we show that a variant in the claudin-5 gene is weakly associated with schizophrenia in 22q11DS, leading to 75% less claudin-5 being expressed in endothelial cells. We also show that targeted adeno-associated virus-mediated suppression of claudin-5 in the mouse brain results in localized BBB disruption and behavioural changes. Using an inducible 'knockdown' mouse model, we further link claudin-5 suppression with psychosis through a distinct behavioural phenotype showing impairments in learning and memory, anxiety-like behaviour and sensorimotor gating. In addition, these animals develop seizures and die after 3-4 weeks of claudin-5 suppression, reinforcing the crucial role of claudin-5 in normal neurological function. Finally, we show that anti-psychotic medications dose-dependently increase claudin-5 expression in vitro and in vivo while aberrant, discontinuous expression of claudin-5 in the brains of schizophrenic patients post mortem was observed compared to age-matched controls. Together, these data suggest that BBB disruption may be a modifying factor in the development of schizophrenia and that drugs directly targeting the BBB may offer new therapeutic opportunities for treating this disorder.

The Endothelin ETB Receptor Antagonist BQ788 Protects against Brain Edema after Fluid Percussion Injury by Decreasing Vascular Endothelial Growth Factor-A Expression in Mice.

Brain edema is a severe morbid complication of brain injury, characterized by excessive fluid accumulation and an elevation of intracranial pressure. However, effective anti-brain edema drugs are lacking. One of the causes of brain edema is disruption of blood-brain barrier (BBB) function, which results in extravasation of intravascular fluid. After brain damage, astrocytes are activated, and astrocyte-derived vascular endothelial growth factor-A (VEGF-A) is known to induce BBB dysfunction. Therefore maintaining BBB integrity by regulating astrocyte function is a potentially effective strategy for treating brain edema. In this review, we focus on the endothelin ETB receptor and its role in regulation of astrocyte functions. In mice, brain damage was induced by fluid percussion injury (FPI), and the resulting BBB disruption and brain edema were observed in the mouse cerebrum. BQ788, a selective ETB receptor antagonist, attenuated the FPI-induced BBB disruption and brain edema. Levels of brain VEGF-A increased after FPI, mainly in reactive astrocytes. BQ788 suppressed the FPI-induced increase in VEGF-A expression in reactive astrocytes. Moreover, intraventricular administration of VEGF neutralizing antibody also attenuated FPI-induced BBB disruption and brain edema. Claudin-5 is an endothelial tight junction protein essential for normal BBB structure and function. Levels of claudin-5 protein were reduced by FPI. Furthermore, VEGF neutralizing antibody blocked FPI-induced decrease in claudin-5. These results suggest that the ETB receptor antagonist BQ788 protects against brain edema by inhibiting VEGF-A-mediated decrease in claudin-5.

Claudin-5, -7, and -18 suppress proliferation mediated by inhibition of phosphorylation of Akt in human lung squamous cell carcinoma.

Abnormal expression of claudin (CLDN) subtypes has been reported in various solid cancers. However, it is unknown which subtype plays a key role in the regulation of proliferation in cancer cells. The expression of CLDN3-5, 7, and 18 in human lung squamous carcinoma tissues was lower than that in normal tissue. Here, we examined which combination of exogenous CLDNs expression inhibits proliferation and the molecular mechanism using human lung squamous RERF-LC-AI cells. Real-time polymerase chain reaction and western blotting showed that CLDN3-5, 7, and 18 are little expressed in RERF-LC-AI cells. In the exogenously transfected cells, CLDN5, 7, and 18 were distributed in the cell-cell contact areas concomitant with ZO-1, a tight junctional scaffolding protein, whereas CLDN3 and 4 were not. Cell proliferation was individually and additively suppressed by CLDN5, 7, and 18. The expression of these CLDNs showed no cytotoxicity compared with mock cells. CLDN5, 7, and 18 increased p21 and decreased cyclin D1, resulting in the suppression of cell cycle G1-S transition. The expression of these CLDNs inhibited phosphorylation of Akt without affecting phosphorylated ERK1/2. Furthermore, these CLDNs inhibited the nuclear localization of Akt and its association with 3-phosphoinositide-dependent protein kinase-1 (PDK1). The suppression of G1-S transition caused by CLDN5, 7, and 18 was rescued by the expression of constitutively active-Akt. We suggest that the reduction of CLDN5, 7, and 18 expression loses the suppressive ability of interaction between PDK1 and Akt and causes sustained phosphorylation of Akt, resulting in the disordered proliferation in lung squamous carcinoma cells.

Claudin-5 is associated with elevated TATI and CA125 levels in mucinous ovarian borderline tumors.

BACKGROUND/AIM: Claudin proteins represent a large family of integral membrane proteins crucial for tight junction (TJ) formation and function and are abnormally regulated in several human cancers. The aim of the present study was to study the expression levels of claudin-5 in pre-malignant disease as borderline mucinous ovarian tumors. Previous reports have suggested that claudin-5 over-expression correlates with aggressive behaviour in serous ovarian adenocarcinoma, breast cancer and in pancreatic andenocarcinoma. PATIENTS AND METHODS: We investigated the expression of claudin-5 in mucinous ovarian borderline tumors and its correlation with clinico-pathological parameters and the expression of serum markers cancer antigen (CA) 125 and tumor-associated trypsin inhibitor (TATI). RESULTS: A total of 29 mucinous borderline tumor tissue samples were analyzed using immunohistochemical staining for claudin-5. An association between strong claudin-5 expression and higher serum levels of TATI (p=0.04) and CA125 (p=0.008) were found. There was also an association between claudin-5 expression and the presence of ascites (p=0.02). CONCLUSION: Changes in claudin-5 expression may play a role in malignant transformation.

Strong claudin 5 expression is a poor prognostic sign in pancreatic adenocarcinoma.

We investigated the expression of claudin 5 in 88 ductal adenocarcinomas of the pancreas. The results were correlated with patient prognosis, with claudin 5 expression in blood vessels, with the expression level of bcl2 and bax and with apoptosis. Claudin 5 expression was detected in 24 (38%) cases. It was not associated with tumour size or spread, but strong claudin 5 expression correlated with a worse survival (p = 0.005). Claudin 5 also associated with a higher extent of apoptosis and greater expression of bax protein. In the tumour vasculature, some vessels displayed a loss of claudin 5 expression. The presence of this loss was associated with tumour grade and the presence of nodal metastases (p = 0.02, p = 0.022, respectively). These results indicate that claudin 5 is upregulated in a proportion of pancreatic ductal adenocarcinomas. The association of strong claudin 5 expression with a worse survival is in line with some earlier reports indicating that this protein is involved with increased locomotion and more aggressive spread of carcinomas. The association of claudin 5 with apoptosis and bax might be due to stronger cellular kinetics found in such tumours. The loss of claudin 5 expression in the tumour vasculature points to a leaky vessel type; this might also ease the access of tumours to vessels and be reflected in its association with the presence of nodal metastases.

Chymase-mediated intestinal epithelial permeability is regulated by a protease-activating receptor/matrix metalloproteinase-2-dependent mechanism.

Mast cells regulate intestinal barrier function during disease and homeostasis. Secretion of the mast cell-specific serine protease chymase regulates homeostasis. In the present study, we employ in vitro model systems to delineate the molecular pathways involved in chymase-mediated intestinal epithelial barrier dysfunction. Chymase stimulation of intestinal epithelial (Caco-2 BBe) cell monolayers induced a significant reduction in transepithelial resistance, indicating decreased intestinal epithelial barrier function. The chymase-induced intestinal epithelial barrier dysfunction was characterized by chymase-induced protease-activated receptor (PAR)-2 activation and matrix metalloproteinase (MMP)-2 expression and activation. Consistent with this observation, in vitro analysis revealed chymase-induced PAR-2 activation and increased MAPK activity and MMP-2 expression. Pharmacological and small interfering RNA-mediated antagonism of PAR-2 and MMP-2 significantly attenuated chymase-stimulated barrier dysfunction. Additionally, the chymase/MMP-2-mediated intestinal epithelial dysfunction was associated with a significant reduction in the tight junction protein claudin-5, which was partially restored by MMP-2 inhibition. Finally, incubation of Caco-2 BBe cells with chymase-sufficient, but not chymase-deficient, bone marrow-derived mast cells decreased barrier function, which was attenuated by the chymase inhibitor chymostatin. Collectively, these results suggest that mast cell/chymase-mediated intestinal epithelial barrier function is mediated by PAR-2/MMP-2-dependent pathways.

Influenza infects lung microvascular endothelium leading to microvascular leak: role of apoptosis and claudin-5.

Severe influenza infections are complicated by acute lung injury, a syndrome of pulmonary microvascular leak. The pathogenesis of this complication is unclear. We hypothesized that human influenza could directly infect the lung microvascular endothelium, leading to loss of endothelial barrier function. We infected human lung microvascular endothelium with both clinical and laboratory strains of human influenza. Permeability of endothelial monolayers was assessed by spectrofluorimetry and by measurement of the transendothelial electrical resistance. We determined the molecular mechanisms of flu-induced endothelial permeability and developed a mouse model of severe influenza. We found that both clinical and laboratory strains of human influenza can infect and replicate in human pulmonary microvascular endothelium, leading to a marked increase in permeability. This was caused by apoptosis of the lung endothelium, since inhibition of caspases greatly attenuated influenza-induced endothelial leak. Remarkably, replication-deficient virus also caused a significant degree of endothelial permeability, despite displaying no cytotoxic effects to the endothelium. Instead, replication-deficient virus induced degradation of the tight junction protein claudin-5; the adherens junction protein VE-cadherin and the actin cytoskeleton were unaffected. Over-expression of claudin-5 was sufficient to prevent replication-deficient virus-induced permeability. The barrier-protective agent formoterol was able to markedly attenuate flu-induced leak in association with dose-dependent induction of claudin-5. Finally, mice infected with human influenza developed pulmonary edema that was abrogated by parenteral treatment with formoterol. Thus, we describe two distinct mechanisms by which human influenza can induce pulmonary microvascular leak. Our findings have implications for the pathogenesis and treatment of acute lung injury from severe influenza.