Intercellular Tight Junctions Research Articles

High-Resolution Insights Into the in vitro Developing Blood-Brain Barrier: Novel Morphological Features of Endothelial Nanotube Function.

High-resolution electron microscopy (HREM) imaging of the in vitro blood-brain barrier (BBB), is a promising modality for investigating the dynamic morphological interplay underpinning BBB development. The successful establishment of BBB integrity is grounded in the brain endothelial cells (BEC’s) ability to occlude its paracellular spaces of brain capillaries through the expression of the intercellular tight junction (TJ) proteins. The impermeability of these paracellular spaces are crucial in the regulation of transcellular transport systems to achieve homeostasis of the central nervous system. To-date research describing morphologically, the dynamics by which TJ interaction is orchestrated to successfully construct a specialized barrier remains undescribed. In this study, the application of HREM illuminates the novel, dynamic and highly restrictive BEC paracellular pathway which is founded based on lateral membrane alignment which is the functional imperative for the mechanical juxtapositioning of TJ zones that underpin molecular bonding and sealing of the paracellular space. For the first time, we report on the secretion of a basement membrane in vitro, which allow BECs to orientate themselves into distinct basolateral and apicolateral domains and establish a 3-dimensional BEC construct. We report for the first time, on the expression of nanovesicles bound to the plasma membrane surfaces of the BECs. These membrane-bound vesicles are reported to possess an array of DNA/RNA constituents and chemotaxic properties affecting the formation of nanotubes that span the paracellular space between BECs, facilitating BBB construction, alluding to a functional role in mediating cell-to-cell communication. This study suggests that novel, ultrathin nanotubular (NT) structures are involved in functional roles in bringing into alignment the paracellular space of BECs. Immortalized mouse BECs (b.End3, b.End5) and primary rat cardiac microvascular ECs were used to further validate the in vitro BBB model by profiling variances in peripheral EC monolayer development. These cardiac capillary ECs presented with an opposite topographical profile: large fenestra and intercellular spaces, devoid of morphological ultrastructures. This comparative study alludes to the role of NT facilitation in TJ-induced hemifusion of apicolateral BEC membranes, as a structural event forming the basis for establishing a polarized BBB.

LncRSPH9-4 Facilitates Meningitic Escherichia coli-Caused Blood-Brain Barrier Disruption via miR-17-5p/MMP3 Axis.

Brain microvascular endothelial cells (BMECs) constitute the structural and functional basis for the blood–brain barrier (BBB) and play essential roles in bacterial meningitis. Although the BBB integrity regulation has been under extensive investigation, there is little knowledge regarding the roles of long non-coding RNAs (lncRNAs) in this event. The present study aimed to investigate the roles of one potential lncRNA, lncRSPH9-4, in meningitic E. coli infection of BMECs. LncRSPH9-4 was cytoplasm located and significantly up-regulated in meningitic E. coli-infected hBMECs. Electrical cell-substrate impedance sensing (ECIS) measurement and Western blot assay demonstrated lncRSPH9-4 overexpression in hBMECs mediated the BBB integrity disruption. By RNA-sequencing analysis, 639 mRNAs and 299 miRNAs were significantly differentiated in response to lncRSPH9-4 overexpression. We further found lncRSPH9-4 regulated the permeability in hBMECs by competitively sponging miR-17-5p, thereby increasing MMP3 expression, which targeted the intercellular tight junctions. Here we reported the infection-induced lncRSPH9-4 aggravated disruption of the tight junctions in hBMECs, probably through the miR-17-5p/MMP3 axis. This finding provides new insights into the function of lncRNAs in BBB integrity during meningitic E. coli infection and provides the novel nucleic acid targets for future treatment of bacterial meningitis.

The effect of feeding patterns on serum zonulin levels in infants at 3-4 months of age.

Zonulin so far is the only known endogenous modulator of intercellular tight junctions which regulate the intestinal permeability. Breastfeeding is considered to enhance the integrity of the gastrointestinal tract; however, limited data are available about the effect of feeding patterns on intestinal permeability. We aimed to investigate the potential association between the mode of feeding (breast versus formula milk) and the serum zonulin levels as a marker of intestinal permeability. One hundred fifty-seven full-term, healthy infants, born after an uncomplicated pregnancy, were enrolled within 72-96 h of life. Blood samples from 105 infants were obtained at 3 to 4 months of life. Serum zonulin levels were measured by ELISA. Out of 105 infants, 52.4% (55) were female, and 58.1% (61) were delivered by caesarian section at a mean gestational age of 38.9 (SD ± 1.0) weeks. At the time of blood sampling, median age was 3.4 (IQR 3.20-3.50) months, and mean weight was 6332 (SD ± 692) gr. Infants were divided in three groups according to the feeding patterns: exclusive breastfeeding (n = 42), mixed feeding (n = 41), and cow's milk formula (n = 22). The feeding pattern had no impact on infants' serum zonulin levels. Moreover, zonulin levels were not affected by infant's clinical and epidemiological characteristics such as body weight or family history of autoimmune disease.Conclusion: In our study, different feeding patterns were not associated with serum zonulin levels in healthy infants at 3-4 months of age. What is Known: • Serum zonulin is upregulated in conditions with increased intestinal permeability • Breast milk favors the physiological decline of the intestinal permeability after birth in the neonates What is New: • Serum zonulin levels were not affected by the feeding pattern (breast milk versus formula) in infants at 3-4 months of age • Clinical and epidemiological characteristics of infants had no impact on zonulin levels.

Assessment of brain-to-blood drug distribution using liquid chromatography.

Delivery of already existing and new drugs under development to the brain necessitates passage across the blood-brain barrier (BBB) with its tight intercellular junctions, molecular components and transporter systems. Consequently, it is critical to identify the extent of brain permeation and the partitioning across the BBB. The interpretation of brain-to-blood ratios is considered to be a significant and fundamental approach for estimating drug penetration through BBB, the brain-targeting ability and central nervous system (CNS) pharmacokinetics. Among the different bioanalytical techniques, liquid chromatography with various detectors has been widely used for determination of these ratios. This review defines the different approaches for sample preparation, extraction techniques and liquid chromatography procedures concerned with the determination of drugs in blood and brain tissues and the assessment of brain-to-blood levels. These approaches are expanded to cover the analysis of several drug classes such as CNS-acting drugs, chemotherapeutics, antidiabetics, herbal medicinal products, radiopharmaceuticals, antibiotics and antivirals. Accordingly, stability in biological matrices and matrix effects are investigated. The different administration/formulation effects and the possible deviations in these ratios are also disscussed.

Characteristics of Passive Solute Transport across Primary Rat Alveolar Epithelial Cell Monolayers.

Primary rat alveolar epithelial cell monolayers (RAECM) were grown without (type I cell-like phenotype, RAECM-I) or with (type II cell-like phenotype, RAECM-II) keratinocyte growth factor to assess passive transport of 11 hydrophilic solutes. We estimated apparent permeability (Papp) in the absence/presence of calcium chelator EGTA to determine the effects of perturbing tight junctions on “equivalent” pores. Papp across RAECM-I and -II in the absence of EGTA are similar and decrease as solute size increases. We modeled Papp of the hydrophilic solutes across RAECM-I/-II as taking place via heterogeneous populations of equivalent pores comprised of small (0.41/0.32 nm radius) and large (9.88/11.56 nm radius) pores, respectively. Total equivalent pore area is dominated by small equivalent pores (99.92–99.97%). The number of small and large equivalent pores in RAECM-I was 8.55 and 1.29 times greater, respectively, than those in RAECM-II. With EGTA, the large pore radius in RAECM-I/-II increased by 1.58/4.34 times and the small equivalent pore radius increased by 1.84/1.90 times, respectively. These results indicate that passive diffusion of hydrophilic solutes across an alveolar epithelium occurs via small and large equivalent pores, reflecting interactions of transmembrane proteins expressed in intercellular tight junctions of alveolar epithelial cells.

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.

Par3/integrin β1 regulates embryo adhesion via changing endometrial luminal epithelium polarity†.

The objective is to investigate the pathophysiological significance of Par3 and integrin β1 with regard to the functionality of the endometrial luminal epithelium (LE). Design: laboratory study; setting: university research laboratory. Analysis involved endometrial aspirates and endometrial adenocarcinoma cells (HEC-1A) and endometrial carcinoma cells (RL95-2). We first examined the expression and localization of Par3 and integrin β1 in HEC-1A cells and RL95-2 cells. Then we knocked down Par3 and integrin β1 in HEC-1A cells and RL95-2 cells, respectively, and found that Par3/integrin β1 affected embryo adhesion by regulating the intercellular tight junctions' (TJs') structure and thus the polarity of the endometrial LE. These findings were also confirmed in the endometrium specimens from human and mice. The main outcome measures were the expression and localization of Par3 and integrin β1 in the endometrial epithelial cell lines and endometrium specimens and the regulations of Par3 and integrin β1 on TJs, polarity, and embryo adhesion. Following the knockdown of Par3 in HEC-1A cells, there was a reduction in the complexity of the TJs and cell polarity, and the adhered blastocysts number was significantly increased. However, the reduction of integrin β1 in RL95-2 cells resulted in effects that directly opposed those following the knockdown of Par3 in HEC-1A cells. Estrogen and progesterone reduced the expression of Par3 and promoted the expression of integrin β1 in HEC-1A cells. Par3/integrin β1 regulates embryo adhesion by regulating intercellular TJs' structure and polarity of endometrial LE under the action of ovarian hormones.

Target specific tight junction modulators

Intercellular tight junctions represent a formidable barrier against paracellular drug absorption at epithelia (e.g., nasal, intestinal) and the endothelium (e.g., blood-brain barrier). In order to enhance paracellular transport of drugs and increase their bioavailability and organ deposition, active excipients modulating tight junctions have been applied. First-generation of permeation enhancers (PEs) acted by unspecific interactions, while recently developed PEs address specific physiological mechanisms. Such target specific tight junction modulators (TJMs) have the advantage of a defined specific mechanism of action. To date, merely a few of these novel active excipients has entered into clinical trials, as their lack in safety and efficiency in vivo often impedes their commercialisation. A stronger focus on the development of such active excipients would result in an economic and therapeutic improvement of current and future drugs.

α-Helical peptides on plasma-treated polymers promote ciliation of airway epithelial cells

Airway respiratory epithelium forms a physical barrier through intercellular tight junctions, which prevents debris from passing through to the internal environment while ciliated epithelial cells expel particulate-trapping mucus up the airway. Polymeric solutions to loss of airway structure and integrity have been unable to fully restore functional epithelium. We hypothesised that plasma treatment of polymers would permit adsorption of α-helical peptides and that this would promote functional differentiation of airway epithelial cells. Five candidate plasma compositions are compared; Air, N2, H2, H2:N2 and Air:N2. X-ray photoelectron spectroscopy shows changes in at% N and C 1s peaks after plasma treatment while electron microscopy indicates successful adsorption of hydrogelating self-assembling fibres (hSAF) on all samples. Subsequently, adsorbed hSAFs support human nasal epithelial cell attachment and proliferation and induce differentiation at an air-liquid interface. Transepithelial measurements show that the cells form tight junctions and produce cilia beating at the normal expected frequency of 10–11 Hz after 28 days in culture. The synthetic peptide system described in this study offers potential superiority as an epithelial regeneration substrate over present “gold-standard” materials, such as collagen, as they are controllable and can be chemically functionalised to support a variety of in vivo environments. Using the hSAF peptides described here in combination with plasma-treated polymeric surfaces could offer a way of improving the functionality and integration of implantable polymers for aerodigestive tract reconstruction and regeneration.

The Therapeutic use of the Zonulin Inhibitor AT-1001 (Larazotide) for a Variety of Acute and Chronic Inflammatory Diseases.

The involvement of intercellular tight junctions and, in particular, the modulation of their competency by the zonulin pathway with a subsequent increase in epithelial and endothelial permeability, has been described in several chronic and acute inflammatory diseases. In this scenario, Larazotide, a zonulin antagonist, could be employed as a viable therapeutic strategy. The present review aims to describe recent research and current observations about zonulin involvement in several diseases and the use of its inhibitor Larazotide for their treatment. A systematic search was conducted on PubMed and Google Scholar, resulting in 209 publications obtained with the following search query: "Larazotide," "Larazotide acetate," "AT-1001," "FZI/0" and "INN-202." After careful examination, some publications were removed from consideration because they were either not in English or were not directly related to Larazotide. The obtained publications were subdivided according to Larazotide's mechanism of action and different diseases: celiac disease, type 1 diabetes, other autoimmune diseases, inflammatory bowel disease, Kawasaki disease, respiratory (infective and/or non-infective) diseases, and other. A substantial role of zonulin in many chronic and acute inflammatory diseases has been demonstrated in both in vivo and in vitro, indicating the possible efficacy of a Larazotide treatment. Moreover, new possible molecular targets for this molecule have also been demonstrated.

CAN SUCRALFATE ENEMA PREVENT COLITIS IN COLONIC SEGMENTS WITHOUT FECAL TRANSIT?

ABSTRACT - BACKGROUND:Oxidative stress is one of the main mechanisms associated with the rupture of the defense mechanisms of the colonic epithelial barrier; it reduces the tissue content of the claudin-3 and occludin proteins, which are the main constituents of intercellular tight junctions. Sucralfate (SCF) has antioxidant activity and has been used to treat different forms of colitis.AIM: This study aimed to measure the tissue claudin-3 and occludin content of the colon mucosa without fecal transit, subjected to intervention with SCF.METHODS: Thirty-six rats were subjected to left colon colostomy and distal mucous fistula. They were divided into two groups according to euthanasia that was performed 2 or 4 weeks after the intervention. Each group was divided into three subgroups according to the enema applied daily: saline alone, SCF at 1 g/kg/day, or SCF at 2 g/kg/day. Colitis was diagnosed by the histological analysis adopting the previous validate scale. The tissue expression of both proteins was identified by immunohistochemical technique. The content of proteins was quantified by computer-assisted image analysis.RESULTS: The inflammatory score was high in colonic segments without fecal transit, and enemas with SCF reduced the inflammatory score in these segments, mainly in those animals submitted to intervention with SCF in greater concentration and for a longer period of intervention. There was an increase in tissue content of claudin-3 and occludin, related to SCF concentration. The tissue content of both proteins was not related to the intervention time.CONCLUSION: Enemas with SCF reduced the inflammation and increased the tissue content of claudin-3 and occludin in colonic mucosa without fecal stream.

Fabrication of cyclic olefin polymer and polydimethylsiloxane co-bonded microfluidic device and its appliactions in terahertz biological effects on intestinal cells

<sec>The current research on the terahertz biological effects at a cellular level is limited by the conventional petri dishes used for cell culture, which cannot be directly used for confocal laser microscopy. In this research, the cycloolefin polymer (COP), a material that possesses low terahertz absorption rate but excellent optical property for microscopy, is bonded to polydimethylsiloxane (PDMS), thereby developing a novel COP-PDMS microfluidic device by using the techniques of soft etching, photolithography, plasma cleaning, high-temperature and high-pressure incubation. The bonding strength of resulting device is tested by using a push-tension meter. The results indicate that the developed device shows a bonding strength as strong as the device fabricated by quartz and PDMS, which is thought as the tightest binding in the multiple types of microfluidic device. In addition, by perfusing the device chamber at a high flow rate (200 μL/s) and long-term time-course (2 weeks), which simulates the dynamic shearing stress occurring in <i>in-vivo</i> organs and tissues, this COP-PDMS microfluidic device can still maintain the original shape and sealing property, indicating that this device qualifies the requirements of the following dynamic cell culture.</sec><sec>The biological effects of terahertz on the cells are explored by using this COP-PDMS microfluidic device mentioned above. In this device, we develop the dynamic culture of intestinal epithelial cells Caco-2 with a perfusion rate of 0.05 μL/s, which meets the findings of the <i>in-vivo</i> gastrointestinal lumen shearing stress. The Caco-2 cells are then irradiated with 0.1 THz wave with the power of 15 mW/cm<sup>2</sup> for 3 days, and the irradiation duration is 10 min per day. The biological effects of terahertz irradiation on the intercellular tight junction protein ZO-1, the Paxillin relating to the cell adhesion and migration, and the cytoskeletal microfilament protein F-actin of Caco-2 cells are detected in the device directly using the technique of immunofluorescence staining.</sec><sec>The results show that the morphology of cell adhesion as well as the level and distribution of ZO-1 and Paxillin are changed. In brief, the protein expression of ZO-1 and Paxillin are induced more by the terahertz irradiation, while the F-actin is not influenced by the irradiation. As can be seen from the F-actin results, the cells without terahertz irradiation show a spread and outward shape with regular smooth cell edge while a contraction and burr shape of cell edge are shown after irradiation, suggesting that the cell adhesion is weakened after irradiation. Even though the expression level of F-actin is consistent, the changed morphology indicates that terahertz may regulate the interaction and aggregation among actin proteins in cells. Interestingly, the ZO-1 presents diffuse distribution in the cells and its location on the cell membrane is not obvious, that is, a large amount of ZO-1 expresses not only on the cell membrane but also in the intracellular matrix after the irradiation. The expression of Paxillin is enhanced after terahertz irradiation, and some cells show local aggregation and distribution of Paxillin. These indicate that the terahertz irradiation might affect the biomolecular mechanism of synthesis and distribution of protein. The COP-PDMS co-bonded microfluidic device developed in this study provides a convenient and effective platform for exploring the biological effects of terahertz irradiation on cells, and is expected to be further used for real-time research on the effects of terahertz on cells and molecules in the future.</sec>

SERUM ZONULIN LEVELS IN HYPOTHYROID AND EUTHYROID PATIENTS WITH HASHIMOTO'S THYROIDITIS - A PILOT STUDY.

Human zonulin is a protein that regulates the intercellular tight junctions in various tissues and organs of the human body. Hashimoto's thyroiditis (HT) is one of the most common endocrine autoimmune disorder, but the role of increased intestinal permeability in its pathogenesis is still being studied. This pilot cross-sectional study investigates serum zonulin concentration in adults with Hashimoto's thyroiditis and assesses the relationship between zonulin levels, clinical hormonal and immunological characteristics. A group of 62 adults with HT participated in this study and were divided into three groups: hypothyroid (n=33) euthyroid (n=25) and hyperthyroid (n=4). Serum zonulin was determined using an ELISA method. Age, gender and BMI were different between groups (hypothyroid and euthyroid ones). Serum zonulin values ranged from 2.6 to 198.0 ng/mL in participants. A direct positive correlation was found between serum zonulin levels and weight and BMI (r = 0.351, p = 0.008 and r = 0.236, p = 0.05, respectively). There is no correlation between zonulin and thyroid hormones or autoantibodies in Hashimoto thyroiditis patients. There is a difference in zonulin levels between the studied groups, but they are not statistically significant.

Non-invasive monitoring of the kinetic infiltration and therapeutic efficacy of nanoparticle-labeled chimeric antigen receptor T cells in glioblastoma via 7.0-Tesla magnetic resonance imaging

Background aimsChimeric antigen receptor (CAR) T-cell therapy is a promising treatment strategy in solid tumors. In vivo cell tracking techniques can help us better understand the infiltration, persistence and therapeutic efficacy of CAR T cells. In this field, magnetic resonance imaging (MRI) can achieve high-resolution images of cells by using cellular imaging probes. MRI can also provide various biological information on solid tumors. MethodsThe authors adopted the amino alcohol derivatives of glucose-coated nanoparticles, ultra-small superparamagnetic particles of iron oxide (USPIOs), to label CAR T cells for non-invasive monitoring of kinetic infiltration and persistence in glioblastoma (GBM). The specific targeting CARs included anti-human epidermal growth factor receptor variant III and IL13 receptor subunit alpha 2 CARs. ResultsWhen using an appropriate concentration, USPIO labeling exerted no negative effects on the biological characteristics and killing efficiency of CAR T cells. Increasing hypointensity signals could be detected in GBM models by susceptibility-weighted imaging MRI ranging from 3 days to 14 days following the injection of USPIO-labeled CAR T cells. In addition, nanoparticles and CAR T cells were found on consecutive histopathological sections. Moreover, diffusion and perfusion MRI revealed significantly increased water diffusion and decreased vascular permeability on day 3 after treatment, which was simultaneously accompanied by a significant decrease in tumor cell proliferation and increase in intercellular tight junction on immunostaining sections. ConclusionThese results establish an effective imaging technique that can track CAR T cells in GBM models and validate their early therapeutic effects, which may guide the evaluation of CAR T-cell therapies in solid tumors.

Association of adverse outcomes of intrahepatic cholestasis of pregnancy with zonulin levels

Our aim was to investigate serum zonulin levels in intrahepatic cholestasis of pregnancy (ICP) and to determine the usefulness of zonulin in ICP follow-up. A prospective case-control study was carried out which included 88 pregnant women (44 patients with ICP and 44 controls). Maternal serum samples obtained from all participants and zonulin levels were determined by enzyme-linked immunosorbent assay (ELISA). Compared with controls, women with ICP had significantly higher zonulin levels (mean 0.728 ± 0.520 ng/mL vs. 1.303 ± 0.63 ng/mL, p <.001). According to the receiver operating characteristic (ROC) analysis performed for the predictive value of zonulin levels for ICP, the area under the curve (AUC) was 0.761 (95% CI: 0.661–0.860). Multivariable logistic regression analysis revealed serum zonulin levels was independently associated with adverse perinatal outcomes (OR = 1.278, 95% CI: 0.232–7.041), severity ICP (OR: 7.535, 95% CI: 1.597–13.553) and also unresponsiveness to treatment in ICP (OR: 4.178, 95% CI: 0.929–8.784). IMPACT STATEMENT What is already known on this subject? Zonulin is a regulator protein that increases the intestinal permeability by modulating the intercellular tight junctions (TJ). It is the only physiological protein known to control intestinal permeability and damage of the intestinal barrier is one of the causes of absorption disorders, inflammation and autoimmunity. ICP is a relatively non-threatening condition to women but is linked with a higher risk of preterm delivery, foetal distress and foetal death. What do the results of this study add? This study showed that increased levels of zonulin are associated with adverse perinatal outcomes, severity of ICP and unresponsiveness to treatment in ICP. What are the implications of these findings for clinical practice and/or further research? Focussing on preservation of intestinal permeability may be an alternative preventive strategy to reduce the adverse perinatal outcomes and severity of ICP. Further longitudinal studies are needed to verify the relationships among zonulin levels and pregnancy-related diseases.

The esophageal mucosal barrier in health and disease: mucosal pathophysiology and protective mechanisms.

Diseases of the esophagus, such as gastroesophageal reflux (GER), can result in changes to mucosal integrity, neurological function, and the microbiome. Although poorly understood, both age and GER can lead to changes to the enteric nervous system. In addition, the esophagus has a distinct microbiome that can be altered in GER. Mucosal integrity is also at risk due to persistent damage from acid. Diagnostic tools, such as ambulatory pH/impedance testing and esophageal mucosal impedance, can assess short-term and longitudinal GER burden, which can also assess the risk for mucosal compromise. The quality of the mucosal barrier is determined by its intercellular spaces, tight junctions, and tight junction proteins, which are represented by claudins, occludins, and adhesion molecules. Fortunately, there are protective factors for mucosal integrity that are secreted by the esophageal submucosal mucous glands and within saliva that are augmented by mastication. These protective factors have potential as therapeutic targets for GER. In this article, we aim to review diagnostic tools used to predict mucosal integrity, aging, and microbiome changes to the esophagus and esophageal mucosal defense mechanisms.

Zanthoxylum nitidum extract attenuates BMP-2-induced inflammation and hyperpermeability.

Bone morphogenetic protein-2 (BMP-2) is commonly applied in spinal surgery to augment spinal fusion. Nevertheless, its pro-inflammatory potential could induce dangerous side effects such as vascular hyper-permeability, posing the need for manners against this condition. The present study aims to investigate the protective effect of Zanthoxylum nitidum (ZN) on BMP-2-related hyperpermeability and inflammation on the human umbilical vein endothelial cells (HUVECs). The results revealed that, in a concentration-dependent manner, BMP-2 enhanced the production of pro-inflammatory cytokines, including interleukin (IL)-1α, IL-1β, and tumor necrosis factor-α, which were, however, suppressed by ZN. ZN inhibited BMP-2-induced inflammatory response by suppressing the phosphorylation of NF-κBp65 and IκB, and the abnormal nuclear translocation of p65. Moreover, the inhibited expression intercellular tight junction protein VE-cadherin and Occludin caused by BMP-2 was blocked by ZN. The hyper-permeability of HUVECs induced by BMP-2, as expressed as the higher fluorescent intensity of dextran, was also reversed by ZN. Overall, these findings demonstrated that ZN antagonized BMP-2-induced inflammation and hyperpermeability. It could be a therapeutic candidate for the treatment of BMP-2-induced side effects during spinal fusion.

Passing across the blood-brain barrier in glioblastoma multiforme (GBM)

Introduction and purpose: Blood-brain barrier (BBB) consists of capillary endothelium, in which there are three types of intercellular junctions - adherent, tight and gap junctions.Efficient therapy involves delivering a therapeutic dose of drug into a specific site in the body, and maintaining this dose for adequate time afterwards. The aim of this study is to review current knowledge of new strategies in drug delivery to CNS and the effectiveness of these methods in glioblastoma multiforme (GBM) treatment. This review was performed using the PubMed database. A brief description of the state of knowledge: Methods for delivering drugs to the brain are divided into invasive and non-invasive. Invasive methods involve temporary disrupting tight intercellular junctions of the vascular endothelial cells and delivering drugs intracerebrally or intraventricularly during neurosurgical procedures. In recent years, there has been a growing interest in the use of nanoparticles as drug carriers to the central nervous system via blood-brain barrier. The usage of nanoparticles implies many advantages, such as non-invasive, low cost, good biodegradability, stability, ability to carry various types of agents, selectivity and ability to control drug release. Conclusions: Limited options in treating brain located tumors, including glioblastoma multiforme, due to difficulties in drug penetration through the BBB engages scientists to search for new treatments. Crossing the BBB using invasive methods based on interruption of cell junctions show promising results, but they are associated with i.a. a high risk of uncontrolled influx of toxins to the CNS or ion-electrolyte imbalance, which may lead to neuronal dysfunction. Invasive methods can be effective only in tumors, while treatment of diseases such as Alzheimer’s disease is impossible. Recent studies show that nanoparticles would be a great, non-invasive alternative, but they are difficult to use with relatively low permeability through undamaged BBB. In some studies using nanoparticles as nanocarriers (EDVDox) or SYMPHONY method (combining photothermal therapy with GNS and immunotherapy of checkpoints in a mouse model) against GBM shows positive results. More research is required to confirm the effectiveness and safety of these treatments.

Suppression of Connexin 43 Leads to Strial Vascular Hyper-Permeability, Decrease in Endocochlear Potential, and Mild Hearing Loss.

Objective: Connexin 43 (Cx43) is a protein constituent of gap junctions (GJs) in various barrier cells, especially astrocytes and microglia of the blood-brain-barrier (BBB), where it plays an important role in intercellular communication and regulation of the barrier. Despite the importance of Cx43 in other blood barriers, not much attention has been paid to expression and function of Cx43 in the blood-labyrinth-barrier (BLB) of the stria vascularis in the cochlea.Methods: We used multiple research approaches, including immunocytochemical staining, patch-clamp dye loading technique, real-time quantitative reverse transcription (RT)-PCR, western blot, measurement of endocochlear potential (EP) with an electrode through the scala media, and auditory brainstem response to test hearing function.Results: We found Cx43 expressed in vascular endothelial cells (ECs) and perivascular resident macrophages (PVMs) in the stria vascularis of adult C57BL/6 mouse cochleae. In particular, we found Cx43 expressed in foot processes of PVMs at points of contact with the endothelium. Consistent with Cx43 expression in vivo, we also found Cx43 expressed in EC-EC and EC-PVM interfaces in a co-cultured cell line model. Using a patch-clamp dye loading technique, we demonstrated that Alexa Fluor® 568 dye injected into PVMs diffuses to connected neighboring ECs. The functional coupling between the ECs and PVMs is blocked by 18α-Glycyrrhetinic acid (18α-GA), a GJ blocker. Suppression of Cx43 with small interfering RNA (siRNA) in vivo significantly elevated hearing threshold and caused the EP to drop and the blood barrier to become more permeable. In further study, using in vitro primary EC cell line models, we demonstrated that suppression of Cx43 disrupts intercellular tight junctions (TJs) in the EC monolayer and increases endothelial monolayer permeability.Conculsion: Taken together, these findings underscore the importance of Cx43 expression in the normal ear for maintaining BLB integrity, normal EP, and hearing function.

Development of a miniaturized 96-Transwell air\u2013liquid interface human small airway epithelial model

In order to overcome the challenges associated with a limited number of airway epithelial cells that can be obtained from clinical sampling and their restrained capacity to divide ex vivo, miniaturization of respiratory drug discovery assays is of pivotal importance. Thus, a 96-well microplate system was developed where primary human small airway epithelial (hSAE) cells were cultured at an air–liquid interface (ALI). After four weeks of ALI culture, a pseudostratified epithelium containing basal, club, goblet and ciliated cells was produced. The 96-well ALI cultures displayed a cellular composition, ciliary beating frequency, and intercellular tight junctions similar to 24-well conditions. A novel custom-made device for 96-parallelized transepithelial electric resistance (TEER) measurements, together with dextran permeability measurements, confirmed that the 96-well culture developed a tight barrier function during ALI differentiation. 96-well hSAE cultures were responsive to transforming growth factor β1 (TGF-β1) and tumor necrosis factor α (TNF-α) in a concentration dependent manner. Thus, the miniaturized cellular model system enables the recapitulation of a physiologically responsive, differentiated small airway epithelium, and a robotic integration provides a medium throughput approach towards pharmaceutical drug discovery, for instance, in respect of fibrotic distal airway/lung diseases.