Transendothelial Electrical Resistance Research Articles

Brain microvascular endothelial cells and blood-brain barrier dysfunction in psychotic disorders.

Although there is convergent evidence for blood-brain barrier (BBB) dysfunction and peripheral inflammation in schizophrenia (SZ) and bipolar disorder (BD), it is unknown whether BBB deficits are intrinsic to brain microvascular endothelial cells (BMECs) or arise via effects of peripheral inflammatory cytokines. We examined BMEC function using stem cell-based models to identify cellular and molecular deficits associated with BBB dysfunction in SZ and BD. Induced pluripotent stem cells (iPSCs) from 4 SZ, 4 psychotic BD and 4 healthy control (HC) subjects were differentiated into BMEC-"like" cells. Gene expression and protein levels of tight junction proteins were assessed. Transendothelial electrical resistance (TEER) and permeability were assayed to evaluate BBB function. Cytokine levels were measured from conditioned media. BMECs derived from human iPSCs in SZ and BD did not show differences in BBB integrity or permeability compared to HC BMECs. Outlier analysis using TEER revealed a BBB-deficit (n = 3) and non-deficit (n = 5) group in SZ and BD lines. Stratification based on BBB function in SZ and BD patients identified a BBB-deficit subtype with reduced barrier function, tendency for increased permeability to smaller molecules, and decreased claudin-5 (CLDN5) levels. BMECs from the BBB-deficit group show increased matrix metallopeptidase 1 (MMP1) activity, which correlated with reduced CLDN5 and worse BBB function, and was improved by tumor necrosis factor α (TNFα) and MMP1 inhibition. These results show potential deficits in BMEC-like cells in psychotic disorders that result in BBB disruption and further identify TNFα and MMP1 as promising targets for ameliorating BBB deficits.

TIMP-1 Protects Tight Junctions of Brain Endothelial Cells From MMP-Mediated Degradation.

The blood-brain barrier (BBB) plays a critical role in central nervous system homeostasis, and the integrity ofBBB is disrupted in many neurodegenerative diseases. Matrix metalloproteinases (MMPs) degrade the tight junctions (TJs) of endothelial cells and basement membrane components essential to BBB integrity, which leads to increased BBB permeability and allows inflammatory cells and neurotoxic substances to enter the brain. Tissue inhibitors of metalloproteinases (TIMPs), endogenous inhibitors of MMPs, regulate MMP activity, thereby maintaining BBB integrity. The disruptive impacts of MMP-3 and MMP-9 on BBB and protective effect of TIMP-1 were investigated in a simplified in vitro model of the BBB, which was generated using rat brain microvascular endothelial cells (RBMEC). The main features of BBB formation, including permeability and the trans-endothelial electrical resistance (TEER), were monitored over time after the addition of MMP-3 and MMP-9 and their complexes with TIMP-1 inhibitor. Our results indicated that MMP-3 and MMP-9 caused a dose-dependent disruption of the BBB, with 1.5µM MMPs resulting in an over threefold increase in permeability, while TIMP-1 inhibition protected the integrity of the BBB model and recovered TEER and permeability of RBMECs. The disruption and recovery of tight junction proteins of RBMECs after MMP and TIMP treatment were also detected using fluorescent microscopy. MMP-9 and MMP-3 disrupt the BBB bydegrading tight junctions in endothelial cells, and TIMP-1 could inhibit the disruptive effect of MMP-3 and MMP-9 by showing potential as therapeutic proteinagainst MMP-related diseases where BBB disruption plays a role.

Virus-like Silica Nanoparticles Improve Permeability of Macromolecules across the Blood-Brain Barrier In Vitro.

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.

Curcumin reduces blood-nerve barrier abnormalities and cytotoxicity to endothelial cells and pericytes induced by cisplatin.

Cisplatin is a platinum-based antineoplastic agent used to treat cancers of solid organs. Neuropathy is one of its major side effects, necessitating dose reduction or cessation. Previous studies suggested that cisplatin causes microvascular toxicity, including pericyte detachment. This study aimed to clarify whether these alterations occurred in the blood-nerve barrier (BNB) of capillaries after cisplatin treatment. Electron microscopic analysis of rat sciatic nerves with cisplatin neuropathy showed increased frequency and severity of pericyte detachment. Moreover, the vascular basement membrane did not tightly encircle around the endothelial cells and pericytes. Cultured human umbilical vein endothelial cells and human brain vascular pericytes showed reduced viability, increased caspase-3 activity and enhanced oxidative stress following cisplatin treatment. In addition, cisplatin decreased transendothelial electrical resistance (TEER) and the expression of the tight junction proteins occludin and zonula occludens-1. Curcumin, a polyphenol found in the root of Curcuma longa, had favourable effects on cisplatin neuropathy in previous work. Therefore, curcumin was tested to determine whether it had any effect on these abnormalities. Curcumin alleviated pericyte detachment, cytotoxicity, oxidative stress, TEER reduction and tight junction protein expression. These data indicate that cisplatin causes BNB disruption in the nerves and might result in neuropathy. Curcumin might improve neuropathy via the restoration of BNB. Whether alterations in the BNB occur and curcumin is effective in patients with cisplatin neuropathy remain to be investigated.

Reactive astrocytes associated with prion disease impair the blood brain barrier

BackgroundImpairment of the blood-brain barrier (BBB) is considered to be a common feature among neurodegenerative diseases, including Alzheimer's, Parkinson's and prion diseases. In prion disease, increased BBB permeability was reported 40 years ago, yet the mechanisms behind the loss of BBB integrity have never been explored. Recently, we showed that reactive astrocytes associated with prion diseases are neurotoxic. The current work examines the potential link between astrocyte reactivity and BBB breakdown. ResultsIn prion-infected mice, the loss of BBB integrity and aberrant localization of aquaporin 4 (AQP4), a sign of retraction of astrocytic endfeet from blood vessels, were noticeable prior to disease onset. Gaps in cell-to-cell junctions along blood vessels, together with downregulation of Occludin, Claudin-5 and VE-cadherin, which constitute tight and adherens junctions, suggested that loss of BBB integrity is linked with degeneration of vascular endothelial cells. In contrast to cells isolated from non-infected adult mice, endothelial cells originating from prion-infected mice displayed disease-associated changes, including lower levels of Occludin, Claudin-5 and VE-cadherin expression, impaired tight and adherens junctions, and reduced trans-endothelial electrical resistance (TEER). Endothelial cells isolated from non-infected mice, when co-cultured with reactive astrocytes isolated from prion-infected animals or treated with media conditioned by the reactive astrocytes, developed the disease-associated phenotype observed in the endothelial cells from prion-infected mice. Reactive astrocytes were found to produce high levels of secreted IL-6, and treatment of endothelial monolayers originating from non-infected animals with recombinant IL-6 alone reduced their TEER. Remarkably, treatment with extracellular vesicles produced by normal astrocytes partially reversed the disease phenotype of endothelial cells isolated from prion-infected animals. ConclusionsTo our knowledge, the current work is the first to illustrate early BBB breakdown in prion disease and to document that reactive astrocytes associated with prion disease are detrimental to BBB integrity. Moreover, our findings suggest that the harmful effects are linked to proinflammatory factors secreted by reactive astrocytes.

Propofol Suppresses LPS-induced BBB Damage by Regulating miR-130a-5p/ZO-1 Axis

The blood-brain barrier (BBB) is a highly selective semi-permeable barrier that separates circulating blood from the extracellular fluid of the brain and central nervous system, which is crucial for maintaining brain homeostasis. This study aimed to explore the role of propofol in BBB damage and further evaluate the underlying molecular mechanism. Lipopolysaccharide (LPS) was administered to mice to create an in vivo BBB damage mice model. Additionally, hCMEC/D3 cells as brain microvascular endothelial cells (BMECs) were treated with LPS to establish the in vitro BBB damage cell model. Subsequently, propofol was used for the BBB damage model. Evans blue staining and fluorescein sodium were utilized in the in vivo experiments to demonstrate BBB leakage and BBB permeability. Cell counting kit-8 (CCK-8) assay was used to assess cell viability and the trans-endothelial electrical resistance (TEER) value was measured using an epithelial voltmeter. Furthermore, enzyme-linked immunosorbent assay was performed to measure the levels of the inflammatory cytokines such as interleukin-1β (IL-1β) and tumor necrosis factor-alpha (TNF-α). The levels of miR-130a-5p and zonula occludens-1 (ZO-1) in brain tissues and cells were detected using reverse transcription-quantitative polymerase chain reaction, western blot, or immunofluorescence staining. Furthermore, a dual-luciferase reporter assay was used to demonstrate the association between miR-130a-5p and ZO-1. Propofol treatment suppressed BBB leakage, the amount of fluorescein sodium, and the levels of IL-1β and TNF-α in the LPS-induced BBB damage mice model. Meanwhile, propofol treatment increased the TEER value in the LPS-induced hCMEC/D3 cells. Additionally, propofol treatment significantly down-regulated miR-130a-5p and up-regulated ZO-1. More importantly, miR-130a-5p directly targeted ZO-1 and negatively regulated ZO-1 expression in hCMEC/D3 cells. Furthermore, miR-130a-5p mimic partially reversed the effect of propofol on the TEER value and the levels of inflammatory cytokines such as IL-1β and TNF-α in the LPS-induced hCMEC/D3 cells. Propofol suppressed LPS-induced BBB damage by regulating miR-130a-5p/ZO-1 axis. These findings suggested a potentially effective treatment approach for BBB damage.

Infusible Extracellular Matrix Biomaterial Promotes Vascular Integrity and Modulates the Inflammatory Response in Acute Traumatic Brain Injury.

Traumatic brain injury (TBI) affects millions of people each year and, in many cases, results in long-term disabilities. Once a TBI has occurred, there is a significant breakdown of the blood-brain barrierresulting in increased vascular permeability and progression of the injury. In this study, the use of an infusible extracellular matrix-derived biomaterial (iECM) for its ability to reduce vascular permeability and modulate gene expression in the injured brain is investigated. First, the pharmacokinetics of iECM administration in a mouse model of TBI is characterized, and the robust accumulation of iECM at the site of injury is demonstrated. Next, it is shown that iECM administration after injury can reduce the extravasation of molecules into the brain, and in vitro, iECM increases trans-endothelial electrical resistance across a monolayer of TNFα-stimulated endothelial cells. In gene expression analysis of brain tissue, iECM induces changes that are indicative of downregulation of the proinflammatory response 1-day post-injury/treatment and neuroprotection at 5 days post-injury/treatment. Therefore, iECM shows potential as a treatment for TBI.

TGF-β Isoforms Affect the Planar and Subepithelial Fibrogenesis of Human Conjunctival Fibroblasts in Different Manners.

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.

Transendothelial electrical resistance measurement by a microfluidic device for functional study of endothelial barriers in inflammatory bowel disease.

Introduction: Inflammatory bowel disease (IBD) is a chronic relapsing and remitting disease with a rising incidence globally. Circulating exosomes play great roles in IBD pathogenesis through exosomal cargoes, especially impacting the function of endothelial barriers. Transendothelial electrical resistance (TEER) measurement is a widely used non-invasive and label-free strategy to monitor endothelial barrier function in vitro. This study established a well-designed microfluidic device to monitor the TEER changes of endothelial cellular barrier on-chip after treated with exosome derived from IBD serum. Methods: The chip comprised two layers of microfluidic chambers with top layer for the perfusion of medium to maintain the nutrition and pressure during cell culture, and bottom layer for the extracellular matrix mimic using hydrogel, which are separated by a semipermeable membrane that permitted the formation of endothelial cell barrier. Four electrodes independent from the outlets were integrated to the chip for TEER detection. In vivo mouse models mouse models and proteome profiling were performed to finding relevant regulators. Results: With this platform, significant decrease of TEER was detected, indicating that IBD serum exosome impact the endothelial cellular barrier on-chip. In vivo mouse models, IBD serum exosome treated group showed great higher DAI scores, shorter colons, more severe histological features, and higher levers of S100A8 expression, promoting the disease progress. Proteome profiling showed that TFRC and ANXA5 have great potentials as novel regulators in IBD. Discussion: This in-house customized microfluidic chip emulates the endothelial barrier microenvironment and enables the TEER monitoring, and can be used to investigate endothelial barrier function in vitro. IBD serum exosome promote the severity of disease.

TNF-α evokes blood-brain barrier dysfunction through activation of Rho-kinase and neurokinin 1 receptor

Ischaemic stroke, accompanied by neuroinflammation, impairs blood–brain barrier (BBB) integrity through a complex mechanism involving activation of both RhoA/Rho kinase/myosin light chain-2 and neurokinin 1 receptor (NK1R). Using an in vitro model of human BBB composed of brain microvascular endothelial cells (BMEC), astrocytes and pericytes, this study examined the potential contributions of these elements to BBB damage induced by elevated availability of pro-inflammatory cytokine, TNF-α. Treatment of human BMECs with TNF-α significantly enhanced RhoA activity and the protein expressions of Rho kinase and phosphorylated Ser19MLC-2 while decreasing that of NK1R. Pharmacological inhibition of Rho kinase by Y-27632 and NK1R by CP96345 neutralised the disruptive effects of TNF-α on BBB integrity and function as ascertained by reversal of decreases in transendothelial electrical resistance and increases in paracellular flux of low molecular weight permeability marker, sodium fluorescein, respectively. Suppression of RhoA activation, mitigation of actin stress fibre formation and restoration of plasma membrane localisation of tight junction protein zonula occludens-1 appeared to contribute to the barrier-protective effects of both Y-27632 and CP96345. Attenuation of TNF-α-mediated increases in NK1R protein expression in BMEC by Y-27632 suggests that RhoA/Rho kinase pathway acts upstream to NK1R. In conclusion, specific inhibition of Rho kinase in cerebrovascular conditions, accompanied by excessive release of pro-inflammatory cytokine TNF-α, helps preserve endothelial cell morphology and inter-endothelial cell barrier formation and may serve as an important therapeutic target.

Alcohol and vascular endothelial function: Biphasic effect highlights the importance of dose.

Alcohol (ethanol) consumption has different influences on arterial disease, being protective or harmful depending on the amount and pattern of consumption. The mechanisms mediating these biphasic effects are unknown. Whereas endothelial cells play a critical role in maintaining arterial health, this study compared the effects of moderate and high alcohol concentrations on endothelial cell function. Human coronary artery endothelial cells (HCAEC) were treated with levels of ethanol associated with either low-risk/moderate drinking (i.e., 25 mM) or high-risk/heavy drinking (i.e., 50 mM) after which endothelial function was assessed. The effect of ethanol's primary metabolite acetaldehyde (10 and 25 μM) was also determined. Moderate ethanol exposure (25 mM) improved HCAEC barrier integrity as determined by increased transendothelial electrical resistance (TEER), inhibited cell adhesion molecule (CAM) mRNA expression, decreased inflammatory cytokine (interferon-γ and interleukin 6) production, inhibited monocyte chemotactic protein-1 (MCP-1) expression and monocyte adhesion, and increased homeostatic Notch signaling. In contrast, exposure to high-level ethanol (50 mM) decreased TEER, increased CAM expression and inflammatory cytokine production, and stimulated MCP-1 and monocyte adhesion, with no effect on Notch signaling. Reactive oxygen species (ROS) generation and endothelial nitric oxide synthase activity were increased by both alcohol treatments, and to a greater extent in the 50 mM ethanol group. Acetaldehyde-elicited responses were generally the same as those of the high-level ethanol group. Ethanol has biphasic effects on several endothelial functions such that a moderate level maintains the endothelium in a nonactivated state, whereas high-level ethanol causes endothelial dysfunction, as does acetaldehyde. These data show the importance of dose when considering ethanol's effects on arterial endothelium, and could explain, in part, the J-shaped relationship between alcohol concentration and atherosclerosis reported in some epidemiologic studies.

Truncated oxidized phospholipids exacerbate endothelial dysfunction and lung injury caused by bacterial pathogens

Oxidized phospholipids (OxPLs) are present at basal levels in circulation of healthy individuals, but a substantial increase and changes in composition of OxPLs may rapidly occur during microbial infections, sepsis, and trauma. Specifically, truncated oxidized phospholipids (Tr-OxPLs) exhibit detrimental effects on pulmonary endothelium, yet their role on modulation of lung injury caused by bacterial pathogens remains to be elucidated. This study investigated the effects of Tr-OxPL species: KOdiA-PC, POV-PC, PON-PC, PAz-PC, PGPC, and Lyso-PC on endothelial permeability and inflammatory responses to gram-positive bacterial particles. Results showed that all six tested Tr-OxPLs augmented endothelial barrier disruption caused by heat-killed Staphylococcus aureus (HKSA) as determined by VE-cadherin immunostaining and monitoring transendothelial electrical resistance. In parallel, even moderate elevation of Tr-OxPLs augmented HKSA-induced activation of NF-κB, secretion of IL-6 and IL-8, and protein expression of ICAM-1 and VCAM-1. In the mouse model of acute lung injury caused by intranasal injection of HKSA, intravenous Tr-OxPLs administration augmented HKSA-induced increase in BAL protein content and cell counts, tissue expression of TNFα, KC, IL1β, and CCL2, and promoted vascular leak monitored by lung infiltration of Evans Blue. These results suggest that elevated Tr-OxPLs act as critical risk factor worsening bacterial pathogen-induced endothelial dysfunction and lung injury.

Synergistic Effect of Treatment with Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus and Lipopolysaccharide on the Inflammatory Response of Porcine Pulmonary Microvascular Endothelial Cells.

The highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) often causes secondary bacterial infection in piglets, resulting in inflammatory lung injury and leading to high mortality rates and significant economic losses in the pig industry. Microvascular endothelial cells (MVECs) play a crucial role in the inflammatory response. Previous studies have shown that HP-PRRSV can infect porcine pulmonary MVECs and damage the endothelial glycocalyx. To further understand the role of pulmonary MVECs in the pathogenesis of HP-PRRSV and its secondary bacterial infection, in this study, cultured porcine pulmonary MVECs were stimulated with a HP-PRRSV HN strain and lipopolysaccharide (LPS). The changes in gene expression profiles were analyzed through transcriptome sequencing, and the differentially expressed genes were verified using qRT-PCR, Western blot, and ELISA. Furthermore, the effects on endothelial barrier function and regulation of neutrophil trans-endothelial migration were detected using the Transwell model. HP-PRRSV primarily induced differential expression of numerous genes associated with immune response, including IFIT2, IFIT3, VCAM1, ITGB4, and CCL5, whereas LPS triggered an inflammatory response involving IL6, IL16, CXCL8, CXCL14, and ITGA7. Compared to the individual effect of LPS, when given after HN-induced stimulation, it caused a greater number of changes in inflammatory molecules, such as VCAM1, IL1A, IL6, IL16, IL17D, CCL5, ITGAV, IGTB8, and TNFAIP3A, a more significant reduction in transendothelial electrical resistance, and higher increase in neutrophil transendothelial migration. In summary, these results suggest a synergistic effect of HP-PRRSV and LPS on the inflammatory response of porcine pulmonary MVECs. This study provides insights into the mechanism of severe lung injury caused by secondary bacterial infection following HP-PRRSV infection from the perspective of MVECs, emphasizing the vital role of pulmonary MVECs in HP-PRRSV infection.

Substance P reversibly compromises the integrity and function of blood-brain barrier

BackgroundSubstance P (SP) plays a role in vasodilatation and tissue integrity through its receptor, neurokinin 1 (NK1R). However, its specific effect on blood-brain barrier (BBB) remains unknown. MethodsThe impact of SP on the integrity/function of human BBB model in vitro, composed of brain microvascular endothelial cells (BMECs), astrocytes and pericytes, was assessed by measurements of transendothelial electrical resistance and paracellular flux of sodium fluorescein (NaF), respectively in the absence/presence of specific inhibitors targeting NK1R (CP96345), Rho-associated protein kinase (ROCK; Y27632) and nitric oxide synthase (NOS; N(G)-nitro-L-arginine methyl ester). Sodium nitroprusside (SNP), a NO donor, was employed as a positive control. The levels of tight junction proteins, zonula occludens-1, occludin and claudin-5 alongside RhoA/ROCK/myosin regulatory light chain-2 (MLC2) and extracellular signal‑regulated protein kinase (Erk1/2) proteins were detected by western analyses. Subcellular localisations of F-actin and tight junction proteins were visualized by immunocytochemistry. Flow cytometry was used to detect transient calcium release. ResultsExposure to SP increased RhoA, ROCK2 and phosphorylated serine-19 MLC2 protein levels and Erk1/2 phosphorylation in BMECs which were abolished by CP96345. These increases were independent of the changes in intracellular calcium availability. SP perturbed BBB in a time-dependent fashion through induction of stress fibres. Changes in tight junction protein dissolution or relocalisation were not involved in SP-mediated BBB breakdown. Inhibition of NOS, ROCK and NK1R mitigated the effect of SP on BBB characteristics and stress fibre formation. ConclusionSP promoted a reversible decline in BBB integrity independent of tight junction proteins expression or localisation.

A ROCK inhibitor suppresses the transforming growth factor-beta-2-induced endothelial–mesenchymal transition in Schlemm’s canal endothelial cells

In the normal eye, most of the aqueous humor drains through the trabecular meshwork (TM) and Schlemm’s canal (SC). The concentration of transforming growth factor beta 2 (TGF-β2) is increased in the aqueous humor of primary open angle glaucoma patients. TGF-β2 increases outflow resistance by affecting the TM and SC, and endothelial–mesenchymal transition (EndMT) of SC cells is involved in these changes. Here, we investigated the effect of a ROCK inhibitor on TGF-β2-induced EndMT in SC cells. The ROCK inhibitor Y-27632 suppressed the TGF-β2-induced increase in the trans-endothelial electrical resistance (TER) and proliferation of SC cells. Y-27632 suppressed the expression of α-SMA, N-cadherin, and Snail, which are upregulated by TGF-β2. Moreover, TGF-β2 decreased mRNA levels of bone morphogenetic protein (BMP) 4 and increased those of the BMP antagonist gremlin (GREM1), but Y-27632 significantly suppressed these changes. Y-27632 also inhibited TGF-β2-induced phosphorylation of p-38 mitogen-activated protein kinase (MAPK). BMP4 and the p-38 MAPK inhibitor SB203580 suppressed the TGF-β2-induced TER elevation in SC cells. Moreover, SB203580 suppressed TGF-β2-induced upregulation of fibronectin, Snail, and GREM1. These results indicate that a ROCK inhibitor inhibited the TGF-β2-induced EndMT in SC cells, implying the involvement of p38 MAPK and BMP4 signaling.

Induced pluripotent stem cell model revealed impaired neurovascular interaction in genetic small vessel disease Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy.

Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) is the most common genetic small vessel disease caused by variants in the NOTCH3 gene. Patients with CADASIL experience recurrent strokes, developing into cognitive defect and vascular dementia. CADASIL is a late-onset vascular condition, but migraine and brain MRI lesions appear in CADASIL patients as early as their teens and twenties, suggesting an abnormal neurovascular interaction at the neurovascular unit (NVU) where microvessels meet the brain parenchyma. To understand the molecular mechanisms of CADASIL, we established induced pluripotent stem cell (iPSC) models from CADASIL patients and differentiated the iPSCs into the major NVU cell types including brain microvascular endothelial-like cells (BMECs), vascular mural cells (MCs), astrocytes and cortical projection neurons. We then built an in vitro NVU model by co-culturing different neurovascular cell types in Transwells and evaluated the blood brain barrier (BBB) function by measuring transendothelial electrical resistance (TEER). Results showed that, while the wild-type MCs, astrocytes and neurons could all independently and significantly enhance TEER of the iPSC-BMECs, such capability of MCs from iPSCs of CADASIL patients was significantly impaired. Additionally, the barrier function of the BMECs from CADASIL iPSCs was significantly decreased, accompanied with disorganized tight junctions in iPSC-BMECs, which could not be rescued by the wild-type MCs or sufficiently rescued by the wild-type astrocytes and neurons. Our findings provide new insight into early disease pathologies on the neurovascular interaction and BBB function at the molecular and cellular levels for CADASIL, which helps inform future therapeutic development.

Isolation and Cultivation of Porcine Endothelial Cells, Pericytes and Astrocytes to Develop an In Vitro Blood-Brain Barrier Model for Drug Permeation Testing.

The blood-brain barrier (BBB) is the bottleneck in the development of new drugs to reach the brain. Due to the BBB, toxic substances cannot enter the brain, but promising drug candidates also pass the BBB poorly. Suitable in vitro BBB models are therefore of particular importance during the preclinical development process, as they can not only reduce animal testing but also enable new drugs to be developed more quickly. The aim of this study was to isolate cerebral endothelial cells, pericytes, and astrocytes from the porcine brain to produce a primary model of the BBB. Additionally, as primary cells are well suited by their properties but the isolation is complex and better reproducibility with immortalized cells must be ensured, there is a high demand for immortalized cells with suitable properties for use as a BBB model. Thus, isolated primary cells can also serve as the basis for a suitable immortalization technique to generate new cell lines. In this work, cerebral endothelial cells, pericytes, and astrocytes were successfully isolated and expanded using a mechanical/enzymatic method. Furthermore, in a triple coculture model, the cells showed a significant increase in barrier integrity compared with endothelial cell monoculture, as determined by transendothelial electrical resistance measurement and permeation studies using sodium fluorescein. The results demonstrate the opportunity to obtain all three cell types significantly involved in BBB formation from one species, thus providing a suitable tool for testing the permeation properties of new drug candidates. In addition, the protocols are a promising starting point to generate new cell lines of BBB-forming cells as a novel approach for BBB in vitro models.

Annexin A5 Inhibits Endothelial Inflammation Induced by Lipopolysaccharide-Activated Platelets and Microvesicles via Phosphatidylserine Binding.

Sepsis is caused by a dysregulated immune response to infection and is a leading cause of mortality globally. To date, no specific therapeutics are available to treat the underlying septic response. We and others have shown that recombinant human annexin A5 (Anx5) treatment inhibits pro-inflammatory cytokine production and improves survival in rodent sepsis models. During sepsis, activated platelets release microvesicles (MVs) with externalization of phosphatidylserine to which Anx5 binds with high affinity. We hypothesized that recombinant human Anx5 blocks the pro-inflammatory response induced by activated platelets and MVs in vascular endothelial cells under septic conditions via phosphatidylserine binding. Our data show that treatment with wildtype Anx5 reduced the expression of inflammatory cytokines and adhesion molecules induced by lipopolysaccharide (LPS)-activated platelets or MVs in endothelial cells (p < 0.01), which was not observed with Anx5 mutant deficient in phosphatidylserine binding. In addition, wildtype Anx5 treatment, but not Anx5 mutant, improved trans-endothelial electrical resistance (p < 0.05) and reduced monocyte (p < 0.001) and platelet (p < 0.001) adhesion to vascular endothelial cells in septic conditions. In conclusion, recombinant human Anx5 inhibits endothelial inflammation induced by activated platelets and MVs in septic conditions via phosphatidylserine binding, which may contribute to its anti-inflammatory effects in the treatment of sepsis.

Mouse embryonic stem cell-derived blood–brain barrier model: applicability to studying antibody triggered receptor mediated transcytosis

Blood brain barrier (BBB) models in vitro are an important tool to aid in the pre-clinical evaluation and selection of BBB-crossing therapeutics. Stem cell derived BBB models have recently demonstrated a substantial advantage over primary and immortalized brain endothelial cells (BECs) for BBB modeling. Coupled with recent discoveries highlighting significant species differences in the expression and function of key BBB transporters, the field is in need of robust, species-specific BBB models for improved translational predictability. We have developed a mouse BBB model, composed of mouse embryonic stem cell (mESC-D3)-derived brain endothelial-like cells (mBECs), employing a directed monolayer differentiation strategy. Although the mBECs showed a mixed endothelial-epithelial phenotype, they exhibited high transendothelial electrical resistance, inducible by retinoic acid treatment up to 400 Ω cm2. This tight cell barrier resulted in restricted sodium fluorescein permeability (1.7 × 10–5 cm/min), significantly lower than that of bEnd.3 cells (1.02 × 10–3 cm/min) and comparable to human induced pluripotent stem cell (iPSC)-derived BECs (2.0 × 10–5 cm/min). The mBECs expressed tight junction proteins, polarized and functional P-gp efflux transporter and receptor mediated transcytosis (RMT) receptors; collectively important criteria for studying barrier regulation and drug delivery applications in the CNS. In this study, we compared transport of a panel of antibodies binding species selective or cross-reactive epitopes on BBB RMT receptors in both the mBEC and human iPSC-derived BEC model, to demonstrate discrimination of species-specific BBB transport mechanisms.

An inhibitor of claudin-5 interactions, M01, alleviates neuroinflammation and vasogenic edema after blood-spinal cord barrier dysfunction.

Molecular docking modeling has confirmed that M01 (C30H28N4O5) acts as a potent inhibitor of claudin-5. Our prior data indicated that claudin-5 is essential to the structural integrity of the blood-spinal cord barrier (BSCB). The aim of this study was to investigate the effect of M01 on the integrity of the BSCB and its effect on neuroinflammation and vasogenic edema after blood-spinal cord barrier dysfunction in in-vitro and in-vivo models. Transwell chambers were used to construct an in-vitro model of the BSCB. Fluorescein isothiocyanate (FITC)-dextran permeability and leakage assays were performed to validate the reliability of the BSCB model. Semiquantitative analysis of inflammatory factor expression and nuclear factor-κB signaling pathway protein levels was performed using western blotting. The transendothelial electrical resistance of each group was measured, and the expression of a tight junction protein ZO-1 was determined via immunofluorescence confocal microscopy. Rat models of spinal cord injury were established by the modified Allen's weight-drop method. Histological analysis was carried out by hematoxylin and eosin staining. Locomotor activity was evaluated with Footprint analysis and the Basso-Beattie-Bresnahan scoring system. The M01 (10 μM) reduced the release of inflammatory factors and degradation of ZO-1 and improved the integrity of the BSCB by reversing vasogenic edema and leakage. M01 may represent a new strategy for the treatment of diseases related to BSCB destruction.