Search Results (61)

Current in vitro models fail to recapitulate specific physiological properties of the human blood–brain barrier (BBB); hence the need for a reliable platform to study central nervous system diseases and drug permeability. To mimic the normally tight blood–brain interface, primary human endothelial cells (HAECs) and primary human astrocytes (A) were grown in a confined space of the physical scaffold created by gelatin methacrylate (GelMA) hydrogel to allow optimal astrocyte–endothelial cell direct/indirect interaction. Evidence for a physiologically relevant BBB was established by assessing the expression of tight junction markers conferring the barrier function, and by measuring biophysical attributes using the trans-endothelial electrical resistance (TEER) and the Evans blue albumin (EBA) permeability assay. An HAEC+A three-dimensional (3D) co-culture was associated with 12-fold higher claudin-5 (CLDN5) and cadherin-1 (CDH1 or Epithelial [E]-cadherin) transcriptional levels than two-dimensional (2D) models. This model conferred the highest TEER (45 Ω·cm²) in 3D HAEC+A, which value was 30 Ω·cm² in 2D (p < 0.01) and 25 Ω·cm² in 3D HAEC cultures (p < 0.001). Functionally, in 3D HAEC+A co-cultures, higher TEER resulted in 10-fold and 7-fold lower EBA permeability at 120 min, in HAECs alone or in to 2D co-cultures (p < 0.01). The established human primary cell model has acquired features mimicking the human BBB in vitro, and is now poised to be tested for the permeability of the BBB to pharmacological agents, parasites, cells (such as brain-tropic cancer cell metastasis) and any mechanisms that might involve traversing the BBB. Full article

We previously observed elevated plasma levels of citrulline and arginine in diabetic retinopathy patients compared to diabetic controls. We tested our hypothesis that citrulline plus arginine induces angiogenesis and increases permeability in retinal endothelial cells. Human retinal microvascular endothelial cells (HRMECs) were treated with citrulline, arginine, or citrulline + arginine, and angiogenesis was measured with cell proliferation, migration, and tube formation assays. Permeability was measured in HRMEC monolayers via trans-endothelial electrical resistance (TEER) and FITC-labeled dextran. We also measured arginase activity, arginase-1 and arginase-2 expression, protein expression and phosphorylation of endothelial nitric oxide synthase (eNOS), and nitric oxide (NO) production. Citrulline + arginine induced endothelial cell proliferation (p = 0.018), migration (p = 0.011), and tube formation (p = 0.0042). Citrulline + arginine also increased FITC-dextran flow-through (p = 1.5 × 10⁻⁵) and decreased TEER (p = 0.010). Citrulline + arginine had no effect on arginase activity, but it increased eNOS (p = 6.3 × 10⁻⁴) and phosphorylated eNOS (p = 0.029), as well as NO production (p = 0.025). Inhibiting eNOS prevented the increase in NO (p = 0.0092), inhibited citrulline + arginine-induced cell migration (p = 0.0080) and tube formation (p = 0.0092), and blocked citrulline + arginine-related alterations in FITC-dextran flow-through (p = 3.6 × 10⁻⁴) and TEER (p = 3.9 × 10⁻⁴). These data suggest that citrulline + arginine treatment induces angiogenesis and increases permeability in retinal endothelial cells by activating eNOS and increasing NO production. Full article

Methamphetamine (METH) is a powerful addictive psychostimulant that gives rise to severe abusers worldwide. While many studies have reported on the neurotoxicity of METH, blood–brain barrier (BBB) dysfunction has recently attracted attention as an essential target in METH-induced pathological changes in the brain. However, its mechanism has not been fully understood. We found that METH increased paracellular permeability and decreased vascular integrity through FITC–dextran and trans-endothelial electrical resistance (TEER) assay in primary human brain endothelial cells (HBMECs). Also, redistribution of tight junction proteins (zonula occluden-1 and claudin-5) and reorganization of F-actin cytoskeleton were observed in METH-exposed HBMECs. To determine the mechanism of METH-induced BBB disruption, the RhoA/ROCK signaling pathway was examined in METH-treated HBMECs. METH-activated RhoA, followed by an increase in the phosphorylation of downstream effectors, myosin light chain (MLC) and cofilin, occurs in HBMECs. Pretreatment with ROCK inhibitors Y-27632 and fasudil reduced the METH-induced increase in phosphorylation of MLC and cofilin, preventing METH-induced redistribution of junction proteins and F-actin cytoskeletal reorganization. Moreover, METH-induced BBB leakage was alleviated by ROCK inhibitors in vitro and in vivo. Taken together, these results suggest that METH induces BBB dysfunction by activating the RhoA/ROCK signaling pathway, which results in the redistribution of junction proteins via F-actin cytoskeletal reorganization. Full article

Polychlorinated biphenyls (PCBs) are heterogeneous, synthetic, and widespread organochlorine compounds, and are one of the persistent organic pollutants present in improperly dumped waste and electronic equipment (e-waste), with a high bioaccumulation potential. In this study, the toxicity of Aroclor 1254 (a mixture of commercial PCBs) in human corneal epithelial cells (HCEpiCs), in an in vitro model of an ocular barrier, was evaluated. Aroclor 1254 (0.1–10 μg/mL) reduced cell viability, trans-endothelial electric resistance (TEER) and cell migration. Moreover, it induced an inflammatory response, as indicated by the increase in cPLA₂ activity, PGE2 production, phosphorylation of ERK 1/2 and p-38, and release of inflammatory cytokines. Aroclor 1254 can damage corneal cells, compromising the integrity of the eye’s outermost barrier. This damage may facilitate the occurrence of infectious processes that are physiologically prevented by the corneal barrier. Full article

The placenta plays a critical role in nutrient and oxygen exchange during pregnancy, yet the effects of medicinal drugs on this selective barrier remain poorly understood. To overcome this, this study presents a cost-effective bioimpedance spectroscopy (BIS) system to assess tight junction integrity and monolayer formation in BeWo b30 cells, a widely used model of the multinucleated maternal–fetal exchange surface of the placental barrier. Cells were cultured on collagen-coated porous membranes and treated with forskolin to induce controlled syncytialization. Electrical impedance was measured using an entry level impedance analyzer, while immunofluorescence staining was used to confirm monolayer formation and syncytialization. The measurements and staining confirmed the formation of a confluent monolayer on day 4. In fact, the electrical resistance tripled for treated samples indicating a more electrically restrictive barrier. This resistance remained constant for treated samples reflecting the intact barrier’s integrity over the next 3 days. The measurements show that, on day 4, the electrical capacitance of the cells decreased for the treated samples as opposed to the untreated samples. This reflects that the surface area of the BeWo b30 cells decreased when the samples were treated with forskolin. Finally, a COMSOL model was developed to explore the effects of electrode positioning, depth, and distance on TEER measurements, explaining discrepancies in the literature. In fact, there was a substantial 97% and 39.4% difference in the obtained TEER values. This study demonstrates the AD2 device’s feasibility for monitoring placental barrier integrity and emphasizes the need for standardized setups for comparable results. The system can hence be used to analyze drug effects and nutrient transfer across the placental barrier. Full article

This study explores the potential of cold atmospheric plasma (CAP) to facilitate the delivery of large-molecule drugs to the brain. The blood–brain barrier (BBB) restricts the passage of most drugs, hindering treatment for neurological disorders. CAP generates reactive oxygen and nitrogen species (RONS) that may disrupt the BBB’s tight junctions, potentially increasing drug permeability. An in vitro BBB model and an immortalized cell line (bEND.3) were used in this experiment. Fluorescein isothiocyanate dextran (FD-4), a model drug, was added to the cells to determine drug permeability. Custom microplasma was used to produce reactive oxygen species (ROS). Trans-endothelial electrical resistance (TEER) measurements assessed the integrity of the BBB after the CAP treatment. A decrease in TEER was observed in the CAP-treated group compared to the controls, suggesting increased permeability. Additionally, fluorescence intensity measurements from the basal side of the trans-well plate indicated higher drug passage in the CAP-treated group. Moreover, the higher presence of ROS in the plasma-treated cells confirmed the potential of CAP in drug delivery. These findings suggest that CAP may be a promising approach for enhancing brain drug delivery. Full article

Tight junctional complexes (TJCs) between cerebral microvascular endothelial cells (CMECs) are essential parts of the blood–brain barrier (BBB), whose regulation closely correlates to the BBB’s integrity and function. hCMEC/D3 is the typical cell line used to imitate and investigate the barrier function of the BBB via the construction of an in vitro model. This study aims to investigate the protective effect of the deep-sea-derived fibrinolytic compound FGFC1 against H₂O₂-induced dysfunction of TJCs and to elucidate the underlying mechanism. The barrier function was shown to decline following exposure to 1 mM H₂O₂ in an in vitro model of hCMEC/D3 cells, with a decreasing temperature-corrected transendothelial electrical resistance (tcTEER) value. The decrease in the tcTEER value was significantly inhibited by 80 or 100 µM FGFC1, which suggested it efficiently protected the barrier integrity, allowing it to maintain its function against the H₂O₂-induced dysfunction. According to immunofluorescence microscopy (IFM) and quantitative real-time polymerase chain reaction (qRT-PCR), compared to the H₂O₂-treated group, 80~100 µM FGFC1 enhanced the expression of claudin-5 (CLDN-5) and VE-cadherin (VE-cad). And this enhancement was indicated to be mainly achieved by both up-regulation of CLDN-5 and inhibition of the down-regulation by H₂O₂ of VE-cad at the transcriptional level. Supported by FGFC1’s molecular docking to these proteins with reasonable binding energy, FGFC1 was proved to exert a positive effect on TJCs’ barrier function in hCMEC/D3 cells via targeting CLDN-5 and VE-cad. This is the first report on the protection against H₂O₂-induced barrier dysfunction by FGFC1 in addition to its thrombolytic effect. With CLDN-5 and VE-cad as the potential target proteins of FGFC1, this study provides evidence at the cellular and molecular levels for FGFC1’s reducing the risk of bleeding transformation following its application in thrombolytic therapy for cerebral thrombosis. Full article

Blood–brain barrier (BBB) dysfunction is a key feature in neuroimmunological and neurodegenerative diseases. In this study, we developed a microfluidic human BBB-on-a-chip to model barrier dysfunction and immune cell migration using immortalized TY10 brain endothelial cells, pericytes, and astrocytes. It was found that immortalized TY10 brain endothelial cells developed a microvascular structure under flow. Pericytes were localized on the basal side surrounding the TY10 microvascular structure, showing an in vivo-like structure. Barrier integrity increased under co-culture with pericytes. In addition, both ethylenediaminetetraacetic acid (EDTA) and anti-Claudin-5 (CLDN5) neutralizing antibody caused a decrease in the transendothelial electrical resistance (TEER). EDTA caused the leakage of 20 kDa dextran, suggesting different effects on the BBB based on the mechanism of action, whereas anti-CLDN5 antibody did not cause leakage. In the tri-culture model, human T cells migrated through endothelial vessels towards basal C-X-C motif chemokine ligand 12 (CXCL12). The live-imaging analysis confirmed the extravasation of fluorescence-labelled T cells in a CXCL12-concentration- and time-dependent manner. Our BBB model had an in vivo-like structure and successfully represented barrier dysfunction and transendothelial T cell migration. In addition, our study suggests that the inhibition of CLDN5 attenuates the BBB in humans. This platform has various potential uses in relation to the BBB in both drug discovery research and in elucidating the mechanisms of central nervous system diseases. Full article

The blood–brain barrier (BBB) is the specialised microvasculature system that shields the central nervous system (CNS) from potentially toxic agents. Attempts to develop therapeutic agents targeting the CNS have been hindered by the lack of predictive models of BBB crossing. In vitro models mimicking the human BBB are of great interest, and advances in induced pluripotent stem cell (iPSC) technologies and the availability of reproducible differentiation protocols have facilitated progress. In this study, we present the efficient differentiation of three different wild-type iPSC lines into brain microvascular endothelial cells (BMECs). Once differentiated, cells displayed several features of BMECs and exhibited significant barrier tightness as measured by trans-endothelial electrical resistance (TEER), ranging from 1500 to >6000 Ωcm². To assess the functionality of our BBB models, we analysed the crossing efficiency of adeno-associated virus (AAV) vectors and peptide-conjugated antisense oligonucleotides, both currently used in genetic approaches for the treatment of rare diseases. We demonstrated superior barrier crossing by AAV serotype 9 compared to serotype 8, and no crossing by a cell-penetrating peptide-conjugated antisense oligonucleotide. In conclusion, our study shows that iPSC-based models of the human BBB display robust phenotypes and could be used to screen drugs for CNS penetration in culture. Full article

Previous studies have revealed that norrin can reverse vascular endothelial-growth-factor (VEGF)-induced permeability in a β-catenin-dependent pathway. Here, we have explored the contribution of disheveled-1 (DVL1) in norrin-induced blood-retinal barrier (BRB) restoration. We provide evidence that in addition to canonical signaling, DVL1 promotes tight junction (TJ) stabilization through a novel, non-canonical signaling pathway involving direct claudin-5 (CLDN5) binding. Immunofluorescence staining of rat retinal cross-sections showed enriched expression of DVL1 and 3 at endothelial capillaries and co-localization with CLDN5 and ZO-1 at the TJ complex in primary bovine retinal endothelial cells (BRECs). Barrier properties of BRECs were determined via measurements of trans-endothelial electrical resistance (TEER) or permeability to 70 kDa RITC-dextran. These studies demonstrated that norrin restoration of barrier properties after VEGF treatment required DVL1 as an siRNA knockdown of Dvl1 but not Dvl2 or Dvl3, reduced basal barrier properties and ablated norrin-induced barrier restoration. However, loss of Dvl1 did not decrease β-catenin signaling activity as measured by Axin2 mRNA expression, suggesting the contribution of a non-canonical pathway. DVL and TJ protein interactions were analyzed via co-immunoprecipitation of endogenous protein in BRECs, which demonstrated that DVL1 interacts with both CLDN5 and ZO-1, while DVL3 interacts only with ZO-1. These interactions were most abundant after inducing BRB restoration by treating BRECs with VEGF and norrin. DVL has previously been shown to form intramolecular bindings between the C-terminal PDZ-binding motif (PDZ-BM) with an internal PDZ domain. Co-transfection of HEK293 cells with DVL1 and CLDN5 or relevant mutants revealed that DVL1 interacts with CLDN5 through the DVL PDZ domain binding, CLDN5 PDZ-BM, in competition with DVL1 PDZ-BM, since DVL/CLDN5 interaction increases with deletion of the DVL1 PDZ-BM and decreases by co-expressing the C-terminal fragment of DVL1 containing the PDZ-BM or through deletion of CLDN5 PDZ-BM. In BREC cells, transfection of the C-terminal fragment of DVL1 downregulates the expression of CLDN5 but does not affect the expression of other proteins of the TJs, including ZO-1, occludin, CLDN1 or VE-cadherin. Blocking DVL1/CLDN5 interaction increased basal permeability and prevented norrin induction of barrier properties after VEGF. Combined with previous data, these results demonstrate that norrin signals through both a canonical β-catenin pathway and a non-canonical signaling pathway by which DVL1 directly binds to CLDN5 to promote barrier properties. Full article

To examine the epithelial–mesenchymal transition (EMT) that is induced on the human corneal stroma, two- and three-dimensional (2D and 3D) cultures of human corneal stroma fibroblasts (HCSFs) were used. In this study, HCSF 2D monolayers and 3D spheroids were characterized by (1) scanning electron microscopy (SEM), (2) trans-endothelial electrical resistance (TEER) measurements and fluorescein isothiocyanate (FITC)-dextran permeability, (3) cellular metabolic measurements, (4) the physical properties of 3D HCSF spheroids, and (5) the extracellular matrix (ECM) molecule gene expressions, including collagen (COL) 1, 4 and 6, and fibronectin (FN), a tissue inhibitor of metalloproteinase (TIMP) 1–4, matrix metalloproteinase (MMP) 2, 3, 9 and 14, and several endoplasmic reticulum (ER) stress-related factors. In the 2D HCSFs, TGF-β2 concentration-dependently generated (1) a considerable increase in ECM deposits revealed by SEM, (2) an increase in TEER values and a decrease in FITC-dextran permeability, (3) increases in both mitochondrial and glycolytic functions, and a substantial upregulation of COL1, COL4, FN, αSMA, TIMP1, TIMP, and most ER stress-related genes and the downregulation of COL6 and MMP3. In the case of 3D spheroids, TGF-β2 induced the downsizing and stiffening of 3D spheroids and the upregulation of COL6, MMP14, and most ER stress-related genes. These findings suggest that TGF-β2 significantly induced a number of EMT-associated biological events including planar proliferation, cellular metabolic functions, and the production of ECM molecules in the 2D cultured HCSF cells, but these effects were significantly less pronounced in the case of 3D HCSF spheroids. Full article

The presence of the blood–brain barrier (BBB) limits the delivery of therapies into the brain. There has been significant interest in overcoming the BBB for the effective delivery of therapies to the brain. Inorganic nanomaterials, especially silica nanoparticles with varying surface chemistry and surface topology, have been recently used as permeation enhancers for oral protein delivery. In this context, nanoparticles with varying sizes and surface chemistries have been employed to overcome this barrier; however, there is no report examining the effect of nanoscale roughness on BBB permeability. This paper reports the influence of nanoscale surface roughness on the integrity and permeability of the BBB in vitro, using smooth surface Stöber silica nanoparticles (60 nm) compared to rough surface virus-like silica nanoparticles (VSNP, 60 nm). Our findings reveal that VSNP (1 mg/mL) with virus-mimicking-topology spiky surface have a greater effect on transiently opening endothelial tight junctions of the BBB than the same dose of Stöber silica nanoparticles (1 mg/mL) by increasing the FITC-Dextran (70 kDa) permeability 1.9-fold and by decreasing the trans-endothelial electrical resistance (TEER) by 2.7-fold. This proof-of-concept research paves the way for future studies to develop next-generation tailored surface-modified silica nanoparticles, enabling safe and efficient macromolecule transport across the BBB. Full article

Three highly homologous isoforms of TGF-β, TGF-β-1~3, are involved in the regulation of various pathophysiological conditions such as wound healing processes in different manners, despite the fact that they bind to the same receptors during their activation. The purpose of the current investigation was to elucidate the contributions of TGF-β-1 ~3 to the pathology associated with conjunctiva. For this purpose, the biological effects of these TGF-β isoforms on the structural and functional properties of two-dimensional (2D) and three-dimensional (3D) cultured human conjunctival fibroblasts (HconF) were subjected to the following analyses: 1) transendothelial electrical resistance (TEER), a Seahorse cellular metabolic measurement (2D), size and stiffness measurements of the 3D HTM spheroids, and the qPCR gene expression analyses of extracellular matrix (ECM) components (2D and 3D). The TGF-β isoforms caused different effects on the proliferation of the HconF cell monolayer evaluated by TEER measurements. The differences included a significant increase in the presence of 5 ng/mL TGF-β-1 and -2 and a substantial decrease in the presence of 5 ng/mL TGF-β-3, although there were no significant differences in the response to the TGF-β isoforms for cellular metabolism among the three groups. Similar to planar proliferation, the TGF-β isoforms also induced diverse effects toward the mechanical aspects of 3D HconF spheroids, where TGF-β-1 increased stiffness, TGF-β-2 caused no significant effects, and TGF-β-3 caused the downsizing of the spheroids and stiffness enhancement. The mRNA expression of the ECMs were also modulated in diverse manners by the TGF-β isoforms as well as the culture conditions for the 2D vs. 3D isoforms. Many of these TGF-β-3 inducible effects were markedly different from those caused by TGF-β1 and TGF-β-2. The findings presented herein suggest that the three TGF-β isoforms induce diverse and distinctly different effects on cellular properties and the expressions of ECM molecules in HconF and that these changes are independent of cellular metabolism, thereby inducing different effects on the epithelial and subepithelial proliferation of human conjunctiva. Full article

The “leaky gut” syndrome describes a damaged (leaky) intestinal mucosa and is considered a serious contributor to numerous chronic diseases. Chronic inflammatory bowel diseases (IBD) are particularly associated with the “leaky gut” syndrome, but also allergies, autoimmune diseases or neurological disorders. We developed a complex in vitro inflammation-triggered triple-culture model using 21-day-differentiated human intestinal Caco-2 epithelial cells and HT29-MTX-E12 mucus-producing goblet cells (90:10 ratio) in close contact with differentiated human macrophage-like THP-1 cells or primary monocyte-derived macrophages from human peripheral blood. Upon an inflammatory stimulus, the characteristics of a “leaky gut” became evident: a significant loss of intestinal cell integrity in terms of decreased transepithelial/transendothelial electrical resistance (TEER), as well as a loss of tight junction proteins. The cell permeability for FITC-dextran 4 kDa was then increased, and key pro-inflammatory cytokines, including TNF-alpha and IL-6, were substantially released. Whereas in the M1 macrophage-like THP-1 co-culture model, we could not detect the release of IL-23, which plays a crucial regulatory role in IBD, this cytokine was clearly detected when using primary human M1 macrophages instead. In conclusion, we provide an advanced human in vitro model that could be useful for screening and evaluating therapeutic drugs for IBD treatment, including potential IL-23 inhibitors. Full article

The blood–brain barrier (BBB) is a functional interface that provides selective permeability, protection from toxic substances, transport of nutrients, and clearance of brain metabolites. Additionally, BBB disruption has been shown to play a role in many neurodegenerative conditions and diseases. Therefore, the aim of this study was to establish a functional, convenient, and efficient in vitro co-cultured BBB model that can be used for several physiological conditions related to BBB disruption. Mouse brain-derived endothelial (bEnd.3) and astrocyte (C8-D1A) cells were co-cultured on transwell membranes to establish an intact and functional in vitro model. The co-cultured model and its effects on different neurological diseases and stress conditions, including Alzheimer’s disease (AD), neuroinflammation, and obesity, have been examined by transendothelial electrical resistance (TEER), fluorescein isothiocyanate (FITC) dextran, and tight junction protein analyses. Scanning electron microscope images showed evidence of astrocyte end-feet processes passing through the membrane of the transwell. Moreover, the co-cultured model showed effective barrier properties in the TEER, FITC, and solvent persistence and leakage tests when compared to the mono-cultured model. Additionally, the immunoblot results showed that the expression of tight junction proteins such as zonula occludens-1 (ZO-1), claudin-5, and occludin-1 was enhanced in the co-culture. Lastly, under disease conditions, the BBB structural and functional integrity was decreased. The present study demonstrated that the co-cultured in vitro model mimicked the BBB’s structural and functional integrity and, under disease conditions, the co-cultured model showed similar BBB damages. Therefore, the present in vitro BBB model can be used as a convenient and efficient experimental tool to investigate a wide range of BBB-related pathological and physiological studies. Full article