Localization Of Claudin-1 Research Articles

VEGF-A165a and angiopoietin-2 differently affect the barrier formed by retinal endothelial cells

Exposure to VEGF-A165a over several days leads to a persistent dysfunction of the very tight barrier formed by immortalized endothelial cells of the bovine retina (iBREC). Elevated permeability of the barrier is indicated by low cell index values determined by electric cell-substrate impedance measurements, by lower amounts of claudin-1, and by disruption of the homogenous and continuous staining of vascular endothelial cadherin at the plasma membrane. Because of findings that suggest modulation of VEGF-A's detrimental effects on the inner blood-retina barrier by the angiogenic growth factor angiopoietin-2, we investigated in more detail in vitro whether this growth factor indeed changes the stability of the barrier formed by retinal endothelial cells or modulates effects of VEGF-A. In view of the clinical relevance of anti-VEGF therapy, we also studied whether blocking VEGF-A-driven signaling is sufficient to prevent barrier dysfunction induced by a combination of both growth factors. Although angiopoietin-2 stimulated proliferation of iBREC, the formed barrier was not weakened at a concentration of 3 nM: Cell index values remained high and expression or subcellular localization of claudin-1 and vascular endothelial cadherin, respectively, were not affected. Angiopoietin-2 enhanced the changes induced by VEGF-A165a and this was more pronounced at lower concentrations of VEGF-A165a. Specific inhibition of the VEGF receptors with tivozanib as well as interfering with binding of VEGF-A to its receptors with bevacizumab prevented the detrimental effects of the growth factors; dual binding of angiopoietin-2 and VEGF-A by faricimab was marginally more efficient. Uptake of extracellular angiopoietin-2 by iBREC can be efficiently prevented by addition of faricimab which is also internalized by the cells. Exposure of the cells to faricimab over several days stabilized their barrier, confirming that inhibition of VEGF-A signaling is not harmful to this cell type. Taken together, our results confirm the dominant role of VEGF-A165a in processes resulting in increased permeability of retinal endothelial cells in which angiopoietin-2 might play a minor modulating role.

Downregulation of CLDN1 impairs trophoblast invasion and endovascular trophoblast differentiation in early-onset preeclampsia

IntroductionTo investigate the role of claudin-1 (CLDN1) in trophoblast invasion and endovascular trophoblast (enEVT) differentiation in early-onset preeclampsia (EOPE). MethodsThe expression and localization of CLDN1 in normal (n = 18) and EOPE (n = 20) placental tissues were detected by immunohistochemical (IHC) staining, quantitative real-time polymerase chain reaction (qRT‒PCR) and Western blotting. Next, invasion, migration and tube formation assays were performed to explore the involvement of CLDN1 in trophoblast invasion and enEVT differentiation in trophoblast cell lines (HTR8/SVneo). Then, invasion and enEVT markers were analyzed via Western blotting and qRT‒PCR, respectively. Finally, we established an EOPE mouse model to detect the Cldn1 protein level. ResultsCLDN1 expression was significantly decreased in EOPE placental tissues. Knockdown of CLDN1 suppressed HTR8/SVneo cell invasion, migration and the ability to penetrate the endothelial tube. Conversely, overexpression of CLDN1 promoted trophoblast invasion and the ability to invade the endothelial tube. Inhibition of CLDN1 decreased the protein expression of VIM and SNAIL along with downregulating IL1B and PECAM1 mRNA levels, while overexpression of CLDN1 gave the opposite results. In the EOPE mouse model, we found a decrease in Cldn1 expression in EOPE mouse placentas. DiscussionThese results suggest that the downregulation of CLDN1 in trophoblast cells is involved in the pathogenesis of early-onset preeclampsia by affecting trophoblast invasion and enEVT differentiation.

Pharmacologic Comparison of High-Dose Hesperetin and Quercetin on MDCK II Cell Viability, Tight Junction Integrity, and Cell Shape.

The modulation of tight junction (TJ) integrity with small molecules is important for drug delivery. High-dose baicalin (BLI), baicalein (BLE), quercetin (QUE), and hesperetin (HST) have been shown to open TJs in Madin-Darby canine kidney (MDCK) II cells, but the mechanisms for HST and QUE remain unclear. In this study, we compared the effects of HST and QUE on cell proliferation, morphological changes, and TJ integrity. HST and QUE were found to have opposing effects on the MDCK II cell viability, promotion, and suppression, respectively. Only QUE, but not HST, induced a morphological change in MDCK II into a slenderer cell shape. Both HST and QUE downregulated the subcellular localization of claudin (CLD)-2. However, only QUE, but not HST, downregulated CLD-2 expression. Conversely, only HST was shown to directly bind to the first PDZ domain of ZO-1, a key molecule to promote TJ biogenesis. The TGFβ pathway partially contributed to the HST-induced cell proliferation, since SB431541 ameliorated the effect. In contrast, the MEK pathway was not involved by both the flavonoids, since U0126 did not revert their TJ-opening effect. The results offer insight for using HST or QUE as naturally occurring absorption enhancers through the paracellular route.

Formation and function of the meningeal arachnoid barrier around the developing mouse brain.

The arachnoid barrier, a component of the blood-cerebrospinal fluid barrier (B-CSFB) in the meninges, is composed of epithelial-like, tight-junction-expressing cells. Unlike other central nervous system (CNS) barriers, its' developmental mechanisms and timing are largely unknown. Here, we show that mouse arachnoid barrier cell specification requires the repression of Wnt-β-catenin signaling and that constitutively active β-catenin can prevent its formation. We also show that the arachnoid barrier is functional prenatally and, in its absence, a small molecular weight tracer and the bacterium group B Streptococcus can cross into the CNS following peripheral injection. Acquisition of barrier properties prenatally coincides with the junctional localization of Claudin 11, and increased E-cadherin and maturation continues after birth, where postnatal expansion is marked by proliferation and re-organization of junctional domains. This work identifies fundamental mechanisms that drive arachnoid barrier formation, highlights arachnoid barrier fetal functions, and provides novel tools for future studies on CNS barrier development.

Abstract 387: Blood brain barrier (BBB) disruption by tumor treating fields (TTFields) in a human 3D in vitro model

Abstract The clinical translatability of novel drug delivery systems begins with basic scientific breakthroughs. Our recent discovery of the ability of Tumor Treating Fields (TTFields) to potentially and transiently disrupt the blood brain barrier (BBB) using our murine in vitro and in vivo models, led us to validate our findings in a human 3D in vitro model established in our lab. The model consists of primary brain microvascular endothelial cells co-cultured with immortalized perciytes in a transwell system. TTFields are alternating electric fields of low intensity (1-3V/cm) and intermediate frequency (100-300kHz), which are effective and approved for the treatment of glioblastoma (GBM) using 200kHz frequency. Our murine data point out that TTFields could disrupt the BBB optimally at 100kHz. To investigate if TTFields exhibit similar effects in the human cell-based in vitro model, it was subjected to TTFields at various frequencies for 24-96h. Cells were afterwards made to recover for 24-96h. To assess BBB integrity and compromise, transendothelial electrical resistance (TEER) was measured before start of TTFields, immediately after end of TTFields, as well as 24-96h after TTFields. In addition, a permeability assay was performed. Finally, immunofluorescence (IF) staining visualized the effects of TTFields on tight junction protein claudin-5 localization. TTFields application of all investigated frequencies significantly decreased TEER. However, the strongest effects were observed with 100kHz after 72h. IF staining revealed delocalization of claudin-5 from the cell boundaries to the cytoplasm. Restoration of cell integrity was already evident as early as 24h, with complete recovery after 48h. Results using our human 3D in vitro model validated our previous observations from murine in vitro and in vivo models that TTFields could transiently disrupt the BBB. These findings provide fundamental pre-clinical data for translation from bench to bedside. Accordingly, TTFields demonstrate to be a promising novel approach in opening the BBB to facilitate drug delivery for improved treatment of central nervous system diseases including devastating brain tumors such as GBM. Citation Format: Ellaine Salvador, Almuth F. Kessler, Theresa Köppl, Sebastian Schönhärl, Malgorzata Burek, Catherine Tempel Brami, Tali Voloshin, Moshe Giladi, Ralf-Ingo Ernestus, Mario Löhr, Carola Y. Förster, Carsten Hagemann. Blood brain barrier (BBB) disruption by tumor treating fields (TTFields) in a human 3D in vitro model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 387.

Acidosis regulation of epithelial barrier function and immune signaling

A common but often underappreciated aspect of the inflammatory microenvironment is extracellular acidification. As with other chronic inflammatory diseases, tissue acidification has been observed in inflammatory bowel disease (IBD) patients. Colonic pH as low as 4 have been reported in IBD patients. In contrast, the pH of a healthy colon is between a pH of 7.0‐7.4. Recently, we identified an adaptive tissue response in intestinal epithelial cells (IEC) which buffered extracellular acidification during neutrophil transepithelial migration, suggesting pH homeostasis is an important aspect of acute inflammation. Extensions of these studies identified a previously undescribed CREB‐dependent pathway for sensing extracellular pH and a unique acidosis gene signature in IEC. The full impact of acidosis on IEC function remains unclear. To better understand the impact of chronic extracellular acidification on IEC function, we investigated changes in IEC immune signaling and barrier function in the setting of acute and chronic exposure to acidosis. We initially examined barrier function in T84 IEC cultured under acidic conditions and observed a significant decrease in barrier function, as determined by transepithelial electrical resistance (TER) and paracellular flux, in cells exposed to acidosis in a pH range of 6.0 – 7.4. Interestingly, following acute acidification, treatment with low pH for less than 8hr, barrier function recovered within 16hr. However, following chronic acidification, treatment with low pH beyond 16hr resulted in an appreciable barrier dysfunction for several days. We extended these studies to examine expression and localization of tight‐junction proteins in IEC exposed to acidosis and observed a time and dose‐dependent decrease in ZO‐1 and E‐cadherin expression, with almost complete loss of expression after 72hr at a pH of 6.0. Likewise, expression and localization of claudins 1, 2, and 4 decreased over time. In parallel to a loss of barrier function, IEC exposed to chronic acidosis revealed a prominent increase in the expression of inflammatory markers including several chemokines (IL‐8, CCL20, TNF) as well as anti‐microbial peptides (mucins and beta‐defensins).Thus, chronic extracellular acidosis induces the loss of IEC tight junction function, while inducing the expression of several pro‐inflammatory chemokines and the production of various anti‐microbial peptides. These results identify extracellular acidification as significant signaling mechanism which induces a loss of epithelial features and the acquisition of a more immune‐like phenotype. Furthering our understandings of the mechanisms involved in acidosis induced phenotype switching in IEC has the potential to uncover novel therapeutic drug targets.

Osteopontin-induced vascular hyperpermeability through tight junction disruption in diabetic retina

Diabetic retinopathy is a major cause of blindness in developed countries, and is characterized by deterioration of barrier function causing vascular hyperpermeability and retinal edema. Vascular endothelial growth factor (VEGF) is a major mediator of diabetic macular edema. Although anti-VEGF drugs are the first-line treatment for diabetic macular edema, some cases are refractory to anti-VEGF therapy. Osteopontin (OPN) is a phosphoglycoprotein with diverse functions and expressed in various cells and tissues. Elevated OPN level has been implicated in diabetic retinopathy, but whether OPN is involved in hyperpermeability remains unclear. Using streptozotocin-induced diabetic mice (STZ mice) and human retinal endothelial cells (HRECs), we tested the hypothesis that up-regulated OPN causes tight junction disruption, leading to vascular hyperpermeability. The serum and retinal OPN concentrations were elevated in STZ mice compared to controls. Intravitreal injection of anti-OPN neutralizing antibody (anti-OPN Ab) suppressed vascular hyperpermeability and prevented decreases in claudin-5 and ZO-1 gene expression levels in the retina of STZ mice. Immunohistochemical staining of retinal vessels in STZ mice revealed claudin-5 immunoreactivity with punctate distribution and attenuated ZO-1 immunoreactivity, and these changes were prevented by anti-OPN Ab. Intravitreal injection of anti-OPN Ab did not change VEGF gene expression or protein concentration in retina of STZ mice. In an in vitro study, HRECs were exposed to normal glucose or high glucose with or without OPN for 48 h, and barrier function was evaluated by transendothelial electrical resistance and Evans blue permeation. Barrier function deteriorated under high glucose condition, and was further exacerbated by the addition of OPN. Immunofluorescence localization of claudin-5 and ZO-1 demonstrated punctate appearance with discontinuous junction in HRECs exposed to high glucose and OPN. There were no changes in VEGF and VEGF receptor-2 expression levels in HRECs by exposure to OPN. Our results suggest that OPN induces tight junction disruption and vascular hyperpermeability under diabetic conditions. Targeting OPN may be an effective approach to manage diabetic retinopathy.

High-Dose Acetaminophen Alters the Integrity of the Blood-Brain Barrier and Leads to Increased CNS Uptake of Codeine in Rats.

The consumption of acetaminophen (APAP) can induce neurological changes in human subjects; however, effects of APAP on blood–brain barrier (BBB) integrity are unknown. BBB changes by APAP can have profound consequences for brain delivery of co-administered drugs. To study APAP effects, female Sprague–Dawley rats (12–16 weeks old) were administered vehicle (i.e., 100% dimethyl sulfoxide (DMSO), intraperitoneally (i.p.)) or APAP (80 mg/kg or 500 mg/kg in DMSO, i.p.; equivalent to a 900 mg or 5600 mg daily dose for a 70 kg human subject). BBB permeability was measured via in situ brain perfusion using [14C]sucrose and [3H]codeine, an opioid analgesic drug that is co-administered with APAP (i.e., Tylenol #3). Localization and protein expression of tight junction proteins (i.e., claudin-5, occludin, ZO-1) were studied in rat brain microvessels using Western blot analysis and confocal microscopy, respectively. Paracellular [14C]sucrose “leak” and brain [3H]codeine accumulation were significantly enhanced in rats treated with 500 mg/kg APAP only. Additionally, claudin-5 localization and protein expression were altered in brain microvessels isolated from rats administered 500 mg/kg APAP. Our novel and translational data show that BBB integrity is altered following a single high APAP dose, results that are relevant to patients abusing or misusing APAP and/or APAP/opioid combination products.

Blocking of VEGF-A is not sufficient to completely revert its long-term effects on the barrier formed by retinal endothelial cells

The VEGF-A-induced functional impairment of the barrier formed by retinal endothelial cells (REC) can be prevented and even – at least temporarily – reverted by trapping the growth factor in a complex with a VEGF-binding protein or by inhibiting the activity of the VEGF receptor 2 (VEGFR2). In an approach to emulate the clinically relevant situation of constant exposure to effectors, we investigated (1) whether prolonged exposure to VEGF-A165 for up to six days results in a different type of disturbance of the barrier formed by immortalized bovine REC (iBREC) and (2) whether alterations of the barrier induced by VEGF-A165 can indeed be sustainably reverted by subsequent treatment with the VEGF-A-binding proteins ranibizumab or brolucizumab. As a measure of barrier integrity, the cell index (CI) of iBREC cultivated on gold electrodes was monitored continuously. CI values declined shortly after addition of the growth factor and then remained low for more than six days over which considerable amounts of both extra- and intracellular VEGF-A were measured. Interestingly, the specific VEGFR2 inhibitor nintedanib normalized the lowered CI when added to iBREC pre-treated with VEGF-A165 for one day, but failed to do so when cells had been exposed to the growth factor for six days. Expression of the tight junction (TJ) protein claudin-5 was unchanged early after addition of VEGF-A165 but higher after prolonged treatment, whereas decreased amounts of the TJ-protein claudin-1 remained low, and increased expression of the plasmalemma vesicle-associated protein (PLVAP) remained high during further exposure. After two days, the characteristic even plasma membrane stainings of claudin-1 or claudin-5 appeared weaker or disordered, respectively. After six days the subcellular localization of claudin-5 was similar to that of control cells again, but claudin-1 remained relocated from the plasma membrane. To counteract these effects of VEGF-A165, brolucizumab or ranibizumab was added after one day, resulting in recovery of the then lowered CI to normal values within a few hours. However, despite the VEGF antagonist being present, the CI declined again two days later to values that were just slightly higher than without VEGF inhibition during further assessment for several days. At this stage, neither the supernatants nor whole cell extracts from iBREC treated with VEGF-A165 and its antagonists contained significant amounts of free VEGF-A. Treatment of VEGF-A165-challenged iBREC with ranibizumab or brolucizumab normalized expression of claudin-1 and claudin-5, but not completely that of PLVAP. Interestingly, the characteristic VEGF-A165-induced relocalization of claudin-1 from the plasma membrane was reverted within one day by any of the VEGF antagonists, but reappeared despite their presence after further exposure for several days. Taken together, barrier dysfunction induced by VEGF-A165 results from deregulated para- and transcellular flow but the precise nature or magnitude of underlying changes on a molecular level clearly depend on the time of exposure, evolving into a stage of VEGF-A165-independent barrier impairment. These findings also provide a plausible explanation for resistance to treatment with VEGF-A antagonists frequently observed in clinical practice.

Mlc1-Expressing Perivascular Astrocytes Promote Blood-Brain Barrier Integrity.

In the mammalian brain, perivascular astrocytes (PAs) closely juxtapose blood vessels and are postulated to have important roles in the control of vascular physiology, including regulation of the blood-brain barrier (BBB). Deciphering specific functions for PAs in BBB biology, however, has been limited by the ability to distinguish these cells from other astrocyte populations. In order to characterize selective roles for PAs in vivo, a new mouse model has been generated in which the endogenous megalencephalic leukoencephalopathy with subcortical cysts 1 (Mlc1) gene drives expression of Cre fused to a mutated estrogen ligand-binding domain (Mlc1-T2A-CreERT2). This knock-in mouse model, which we term MLCT, allows for selective identification and tracking of PAs in the postnatal brain. We also demonstrate that MLCT-mediated ablation of PAs causes severe defects in BBB integrity, resulting in premature death. PA loss results in aberrant localization of Claudin 5 and -VE-Cadherin in endothelial cell junctions as well as robust microgliosis. Collectively, these data reveal essential functions for Mlc1-expressing PAs in regulating endothelial barrier integrity in mice and indicate that primary defects in astrocytes that cause BBB breakdown may contribute to human neurologic disorders.SIGNIFICANCE STATEMENT Interlaced among the billions of neurons and glia in the mammalian brain is an elaborate network of blood vessels. Signals from the brain parenchyma control the unique permeability properties of cerebral blood vessels known as the blood-brain barrier (BBB). However, we understand very little about the relative contributions of different neural cell types in the regulation of BBB functions. Here, we show that a specific subpopulation of astrocyte is essential for control of BBB integrity, with ablation of these cells leading to defects in endothelial cell junctions, BBB breakdown, and resulting neurologic deficits.

Angulin-1 seals tricellular contacts independently of tricellulin and claudins.

Tricellular tight junctions (tTJs) are specialized tight junctions (TJs) that seal the intercellular space at tricellular contacts (TCs), where the vertices of three epithelial cells meet. Tricellulin and angulin family membrane proteins are known constituents of tTJs, but the molecular mechanism of tTJ formation remains elusive. Here, we investigated the roles of angulin-1 and tricellulin in tTJ formation in MDCK II cells by genome editing. Angulin-1-deficient cells lost the plasma membrane contact at TCs with impaired epithelial barrier function. The C terminus of angulin-1 bound to the TJ scaffold protein ZO-1, and disruption of their interaction influenced the localization of claudins at TCs, but not the tricellular sealing. Strikingly, the plasma membrane contact at TCs was formed in tricellulin- or claudin-deficient cells. These findings demonstrate that angulin-1 is responsible for the plasma membrane seal at TCs independently of tricellulin and claudins.

NMR-Guided Repositioning of Non-Steroidal Anti-Inflammatory Drugs into Tight Junction Modulators.

Bioavailability is a major bottleneck in the clinical application of medium molecular weight therapeutics, including protein and peptide drugs. Paracellular transport of these molecules is hampered by intercellular tight junction (TJ) complexes. Therefore, safe chemical regulators for TJ loosening are desired. Here, we showed a potential application of select non-steroidal anti-inflammatory drugs (NSAIDs) as TJ modulators. Based on our previous observation that diclofenac and flufenamic acid directly bound various PDZ domains with a broad specificity, we applied solution nuclear magnetic resonance techniques to examine the interaction of other NSAIDs and the first PDZ domain (PDZ1) of zonula occludens (ZO)-1, ZO-1(PDZ1). Inhibition of ZO-1(PDZ1) is expected to provide loosening of the epithelial barrier function because the domain plays a crucial role in maintaining TJ integrity. Accordingly, diclofenac and indomethacin were found to decrease the subcellular localization of claudin (CLD)-2 but not occludin and ZO-1 at the apicolateral intercellular compartment of Madin–Darby canine kidney (MDCK) II cells. These NSAIDs exhibited 125–155% improved paracellular efflux of fluorescein isothiocyanate insulin for the Caco-2 cell monolayer. We propose that these NSAIDs can be repurposed as drug absorption enhancers for peptide drugs.

Protective effect of the sphingosine-1 phosphate receptor agonist siponimod on disrupted blood brain barrier function

Sphingosine 1 phosphate (S1P) is a bioactive sphingolipid that exerts several functions in physiological and pathological conditions. The modulation of one of its receptors, S1P1, plays an important role in the egress of lymphocytes from lymph nodes and is a useful target in multiple sclerosis (MS) treatment. A new drug, siponimod (BAF-312) has been recently approved for the treatment of secondary progressive MS and has affinity for two S1P receptors, S1P1 and S1P5. The two receptors are expressed by endothelial cells that, as components of the blood–brain barrier (BBB), prevent the access of solutes and lymphocytes into the central nervous system, function often compromised in MS. Using an in vitro BBB model exposed to inflammatory cytokines (TNFα and IFNγ, 5 UI and 10 UI respectively), we evaluated the effects of BAF-312 (100 nM) on expression and function of endothelial tight junctional proteins (Zo-1 and claudin-5), regulation of transendothelial electrical resistance (TEER) and permeability to FITC-conjugated dextran. Zo-1 expression, as well as TEER values, were promptly recovered (24 h) when both S1P1 and S1P5 were activated by BAF-312. In contrast, at this time point, activation of S1P5 with the selective agonist UC-42-WP04 (300 nM) or with BAF-312, under blockade of S1P1 with the selective antagonist NIBR-0213 (1 μM), resulted in recovery of expression and localization of claudin-5 and reduction of TNFα/INFγ-induced expression of metalloproteinase 9. Only after a prolonged BAF-312 exposure (48 h), S1P1 was involved through activation of the PI3K/Akt pathway. The PI3K inhibitor LY294002 (10 µM) prevented in fact the effects of BAF-312 on all the parameters examined. In conclusion, BAF-312, by modulating both S1P1 and S1P5, may strengthen BBB properties, thus providing additional effects in the treatment of MS.

Towards the development of a human in vitro model of the blood-brain barrier for virus-associated acute encephalopathy: assessment of the time- and concentration-dependent effects of TNF-α on paracellular tightness.

The pathogenesis of virus-associated acute encephalopathy (VAE) involves brain edema caused by disruption of the blood-brain barrier (BBB). We aimed to develop an in vitro VAE model using an in vitro BBB model, to evaluate the dynamics of vascular dysfunction caused by tumor necrosis factor (TNF)-α. A co-culture model, consisting of Transwell®-grown human brain microvascular endothelial cells and pericytes, was treated with serially diluted TNF-α. Transendothelial electrical resistance (TER) was measured using cellZscope®. A permeability assay, using fluorescein isothiocyanate-conjugated sodium or dextran, was performed. Changes in claudin-5 localization and expression after TNF-α treatment were observed using immunofluorescence staining and western blot analysis. The TER decreased and permeability increased after TNF-α treatment; recovery time was dependent on TNF-α concentration. Claudin-5 was delocalized after TNF-α treatment and recovered in a TNF-α concentration-dependent manner. The expression of claudin-5 decreased 24h after the TNF-α treatment and completely recovered 48h after TNF-α treatment. Claudin-5 delocalization was likely associated with vascular hyperpermeability. To conclude, we evaluated vascular endothelial cell permeability and injury in VAE using an in vitro BBB model treated with TNF-α. This system can be useful for developing novel therapeutic strategies for VAE and designing treatments that target vascular permeability.

A head-to-head Caco-2 assay comparison of the mechanisms of action of the intestinal permeation enhancers: SNAC and sodium caprate (C10)

Salcaprozate sodium (SNAC) and sodium caprate (C10) are the two leading intestinal permeation enhancers (PEs) in oral peptide formulations in clinical trials. There is debate over their mechanism of action on intestinal epithelia. The aims were: (i) to compare their effects on the barrier function by measuring transepithelial electrical resistance (TEER), permeability of FITC-4000 (FD4) across Caco-2 monolayers, and on immunohistochemistry of tight junction (TJ)-associated proteins; and (ii) to compare cellular parameters using conventional end-point cytotoxicity assays and quantitative high content analysis (HCA) of multiple sub-lethal parameters in Caco-2 cells. C10 (8.5 mM) reversibly reduced TEER and increased FD4 permeability across monolayers, whereas SNAC had no effects on either parameter except at cytotoxic concentrations. C10 exposure induced reorganization of three TJ proteins, whereas SNAC only affected claudin-5 localization. High concentrations of C10 and SNAC were required to cause end-point toxicology changes in vitro. SNAC was less potent than C10 at inducing lysosomal and nuclear changes and plasma membrane perturbation. In parallel, HCA revealed that both agents displayed detergent-like features that reflect initial membrane fluidization followed by changes in intracellular parameters. In conclusion, FD4 permeability increases in monolayers in response to C10 were in the range of concentrations that altered end-point cytotoxicity and HCA parameters. For SNAC, while HCA parameters were also altered in a similar overall pattern as C10, they did not lead to increased paracellular flux. These assays show that both agents are primarily surfactants, but C10 has additional TJ-opening effects. While these in vitro assays illucidate their epithelial mechanism of action, clinical experience suggests that they over-estimate their toxicology in the dynamic intestinal environment.

Serum-derived factors of breast cancer patients with brain metastases alter permeability of a human blood\u2013brain barrier model

BackgroundThe most threatening metastases in breast cancer are brain metastases, which correlate with a very poor overall survival, but also a limited quality of life. A key event for the metastatic progression of breast cancer into the brain is the migration of cancer cells across the blood–brain barrier (BBB).MethodsWe adapted and validated the CD34+ cells-derived human in vitro BBB model (brain-like endothelial cells, BLECs) to analyse the effects of patient serum on BBB properties. We collected serum samples from healthy donors, breast cancer patients with primary cancer, and breast cancer patients with, bone, visceral or cerebral metastases. We analysed cytokine levels in these sera utilizing immunoassays and correlated them with clinical data. We used paracellular permeability measurements, immunofluorescence staining, Western blot and mRNA analysis to examine the effects of patient sera on the properties of BBB in vitro.ResultsThe BLECs cultured together with brain pericytes in transwells developed a tight monolayer with a correct localization of claudin-5 at the tight junctions (TJ). Several BBB marker proteins such as the TJ proteins claudin-5 and occludin, the glucose transporter GLUT-1 or the efflux pumps PG-P and BCRP were upregulated in these cultures. This was accompanied by a reduced paracellular permeability for fluorescein (400 Da). We then used this model for the treatment with the patient sera. Only the sera of breast cancer patients with cerebral metastases had significantly increased levels of the cytokines fractalkine (CX3CL1) and BCA-1 (CXCL13). The increased levels of fractalkine were associated with the estrogen/progesterone receptor status of the tumour. The treatment of BLECs with these sera selectively increased the expression of CXCL13 and TJ protein occludin. In addition, the permeability of fluorescein was increased after serum treatment.ConclusionWe demonstrate that the CD34+ cell-derived human in vitro BBB model can be used as a tool to study the molecular mechanisms underlying cerebrovascular pathologies. We showed that serum from patients with cerebral metastases may affect the integrity of the BBB in vitro, associated with elevated concentrations of specific cytokines such as CX3CL1 and CXCL13.

Norrin restores blood-retinal barrier properties after vascular endothelial growth factor–induced permeability

Vascular endothelial growth factor (VEGF) contributes to blood-retinal barrier (BRB) dysfunction in several blinding eye diseases, including diabetic retinopathy. Signaling via the secreted protein norrin through the frizzled class receptor 4 (FZD4)/LDL receptor-related protein 5-6 (LRP5-6)/tetraspanin 12 (TSPAN12) receptor complex is required for developmental vascularization and BRB formation. Here, we tested the hypothesis that norrin restores BRB properties after VEGF-induced vascular permeability in diabetic rats or in animals intravitreally injected with cytokines. Intravitreal co-injection of norrin with VEGF completely ablated VEGF-induced BRB permeability to Evans Blue-albumin. Likewise, 5-month diabetic rats exhibited increased permeability of FITC-albumin, and a single norrin injection restored BRB properties. These results were corroborated in vitro, where co-stimulation of norrin with VEGF or stimulation of norrin after VEGF exposure restored barrier properties, indicated by electrical resistance or 70-kDa RITC-dextran permeability in primary endothelial cell culture. Interestingly, VEGF promoted norrin signaling by increasing the FZD4 co-receptor TSPAN12 at cell membranes in an MAPK/ERK kinase (MEK)/ERK-dependent manner. Norrin signaling through β-catenin was required for BRB restoration, but glycogen synthase kinase 3 α/β (GSK-3α/β) inhibition did not restore BRB properties. Moreover, levels of the tight junction protein claudin-5 were increased with norrin and VEGF or with VEGF alone, but both norrin and VEGF were required for enriched claudin-5 localization at the tight junction. These results suggest that VEGF simultaneously induces vascular permeability and promotes responsiveness to norrin. Norrin, in turn, restores tight junction complex organization and BRB properties in a β-catenin-dependent manner.

Intracellular claudin-1 at the invasive front of tongue squamous cell carcinoma is associated with lymph node metastasis.

Claudins are the major component of tight junctions, which form a primary barrier to paracellular diffusion and maintain cell polarity in normal epithelia and endothelia. In cancer cells, claudins play additional roles besides serving as components of the tight junctions, and participate in anoikis or invasion. Among the claudin family proteins, claudin‐1 has the most promising potential, both diagnostically and prognostically, in many types of cancers, including oral, gastric, liver, and colon cancers. However, conflicting results have been reported in relation to the degree of claudin‐1 expression and the prognosis, suggesting that the expression level of claudin‐1 alone is not sufficient to analyze the relationship between claudin‐1 and cancer progression. As endocytic trafficking of claudin‐1 has been reported in several epithelial cell types in vitro, we aimed to determine whether intracellular localization of claudin‐1 is the missing aspect between claudin‐1 and cancer. We investigated the expression of claudin‐1 in 83 tongue squamous cell carcinoma (TSCC) pathological specimens. Although the expression level of claudin‐1 based on immunohistochemistry was not associated with TSCC progression, within the high claudin‐1 expression group, the incidence of intracellular localization of claudin‐1 was correlated with cervical lymph node metastasis. In an in vitro experiment, claudin‐1 was constitutively internalized in TSCC‐derived cells. Motility of TSCC‐derived cells was increased by deficiency of claudin‐1, suggesting that the decrease in cell‐surface claudin‐1 promoted the cell migration. Therefore, intracellular localization of claudin‐1 at the invasion front may represent a promising diagnostic marker of TSCC.

Brazilian Green Propolis Rescues Oxidative Stress-Induced Mislocalization of Claudin-1 in Human Keratinocyte-Derived HaCaT Cells.

Claudin-1 (CLDN1) is expressed in the tight junction (TJ) of the skin granular layer and acts as a physiological barrier for the paracellular transport of ions and nonionic molecules. Ultraviolet (UV) and oxidative stress may disrupt the TJ barrier, but the mechanism of and protective agents against this effect have not been clarified. We found that UVB and hydrogen peroxide (H2O2) caused the internalization of CLDN1 and increased the paracellular permeability of lucifer yellow, a fluorescent marker, in human keratinocyte-derived HaCaT cells. Therefore, the mechanism of mislocalization of CLDN1 and the protective effect of an ethanol extract of Brazilian green propolis (EBGP) were investigated. The UVB- and H2O2-induced decreases in CLDN1 localization were rescued by EBGP. H2O2 decreased the phosphorylation level of CLDN1, which was also rescued by EBGP. Wild-type CLDN1 was distributed in the cytosol after treatment with H2O2, whereas T191E, its H2O2-insensitive phosphorylation-mimicking mutant, was localized at the TJ. Both protein kinase C activator and protein phosphatase 2A inhibitor rescued the H2O2-induced decrease in CLDN1 localization. The tight junctional localization of CLDN1 and paracellular permeability showed a negative correlation. Our results indicate that UVB and H2O2 could induce the elevation of paracellular permeability mediated by the dephosphorylation and mislocalization of CLDN1 in HaCaT cells, which was rescued by EBGP. EBGP and its components may be useful in preventing the destruction of the TJ barrier through UV and oxidative stress.

Role of lactadherin in intestinal barrier integrity in experimental neonatal necrotizing enterocolitis

Necrotizing enterocolitis (NEC) is one of the most widespread and devastating gastrointestinal diseases in neonates. Destruction of the intestinal barrier is the main underlying cause of NEC. The aim of this study was to determine the role of lactadherin in preventing NEC in a neonatal rat model and investigate the molecular mechanism of lactadherin-mediated protection of the intestinal barrier. Neonatal rats were divided into three groups: dam feeding (DF), NEC (NEC), and NEC supplemented with 10 μg/(g·day) recombinant human lactadherin (NEC+L). Intestinal permeability, tissue damage, and cell junction protein expression and localization were evaluated. We found that lactadherin reduced weight loss caused by NEC, reduced the incidence of NEC from 100% to 46.7%, and reduced the mean histological score for tissue damage to 1.40 compared with 2.53 in the NEC group. Intestinal permeability of lactadherin-treated rats was significantly reduced when compared with that of the NEC group. In addition, the expression levels of JAM-A, claudin 3, and E-calcium in the ileum of NEC group animals increased compared with those in the ileum of DF group animals, and these levels decreased in the NEC+L group. Lactadherin changed the localization of claudin 3, occludin, and E-cadherin in epithelial cells. The mechanism underlying lactadherin-mediated protection of the intestinal barrier might be restoring the correct expression levels and localization of tight junction and adherent junction proteins. These findings suggest a new candidate agent for the prevention of NEC in newborns.