Changes In Transendothelial Electrical Resistance Research Articles

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.

Chlorpyrifos, permethrin and cyfluthrin effect on cell survival, permeability, and tight junction in an in-vitro model of the human blood-brain barrier (BBB)

The blood-brain barrier (BBB) is a structural and functional interface between the plasma and the human brain. Predictive BBB in-vitro models like immortalized human capillary microvascular endothelial cells (HCMEC/D3) can be used to explore the BBB disruption potential of daily exposed chemicals. The present study was focused on investigating the human BBB permeation potential of one organophosphate pesticide, chlorpyrifos (CPF), and two pyrethroids, permethrin (PMT) and cyfluthrin (CFT). HCMEC/D3 cells were exposed to the chemical and the time-dependent pass across BBB along with permeation coefficient (Papp) was calculated. Transendothelial electrical resistance (TEER) was measured for the cells to check the monolayer formation and later to check the reduction in integrity after chemical exposure. Real time PCR was conducted to investigate the effect of chemicals on the expression BBB´s tight and adherens junction proteins. Calculated Papp value for three chemicals was in the following order: CPF>CFT>PMT, where CPF showed the highest permeation coefficient. TEER calculation showed that the integrity decreased after CPF exposure which was in concordance with Papp value whereas for other chemicals, no change in TEER after exposure was observed. In addition, the transwell study showed a higher efflux ratio (ER) (>2) of CFT indicating that CFT could be a substrate for active transport. For CPF and PMT, ER was less than 2, so no active transport seems to be involved. The evaluation of the mRNA expression analysis revealed a statistically significant decrease in Occludin (OCLN) gene expression for CPF, VE-Cadherin (CDH5) for PMT and Zonula Occludens (ZO1) expression for CFT. Our study showed that CPF has the highest potential for inducing cell death, higher permeation, and capability to induce BBB dysfunction than among the above-mentioned chemicals. Additionally, the results of the permeation study could be useful to build a human PBPK model using in vitro-to-in vivo extrapolation approach.

Effects of fasudil on blood–brain barrier integrity

BackgroundCerebral infarction accounts for 85% of all stroke cases. Even in an era of rapid and effective recanalization using an intravascular approach, the majority of patients have poor functional outcomes. Thus, there is an urgent need for the development of therapeutic agents to treat acute ischemic stroke. We evaluated the effect of fasudil, a Rho kinase inhibitor, on blood brain barrier (BBB) functions under normoxia or oxygen–glucose deprivation (OGD) conditions using a primary cell-based in vitro BBB model.MethodsBBB models from rat primary cultures (brain capillary endothelial cells, astrocytes, and pericytes) were subjected to either normoxia or 6 h OGD/24 h reoxygenation. To assess the effects of fasudil on BBB functions, we evaluated real time impedance, transendothelial electrical resistance (TEER), sodium fluorescein permeability, and tight junction protein expression using western blotting. Lastly, to understand the observed protective mechanism on BBB functions by fasudil we examined the role of cyclooxygenase-2 and thromboxane A2 receptor agonist U-46619 in BBB-forming cells.ResultsWe found that treatment with 0.3–30 µM of fasudil increased cellular impedance. Fasudil enhanced barrier properties in a concentration-dependent manner, as measured by an increased (TEER) and decreased permeability. Fasudil also increased the expression of tight junction protein claudin-5. Reductions in TEER and increased permeability were observed after OGD/reoxygenation exposure in mono- and co-culture models. The improvement in BBB integrity by fasudil was confirmed in both of the models, but was significantly higher in the co-culture than in the monoculture model. Treatment with U-46619 did not show significant changes in TEER in the monoculture model, whereas it showed a significant reduction in TEER in the co-culture model. Fasudil significantly improved the U-46619-induced TEER reduction in the co-culture models. Pericytes and astrocytes have opposite effects on endothelial cells and may contribute to endothelial injury in hyperacute ischemic stroke. Overall, fasudil protects the integrity of BBB both by a direct protective effect on endothelial cells and by a pathway mediated via pericytes and astrocytes.ConclusionsOur findings suggest that fasudil is a BBB-protective agent against acute ischemic stroke.

Change of reception and lactate transport by cerebral endothelium under the influence of viral and bacterial inflammation in vitro

Aim. To study the effect of neuroinflammation inducers of viral and bacterial origin on the reception and transport of lactate in endothelial cells of the brain in vitro. Materials and Methods. In the study, we incubated endothelial cells with bacterial inducers (lipopolysaccharide, E. coli) of various concentrations and inducer of viral inflammation (Poly I:C). After the exposure, we evaluated the proliferative activity of endothelial cells, change in transendothelial electrical resistance, and the concentration of extracellular lactate. We then performed an im-munocytochemistry investigation to record changes in the number of endothelial cells expressing the target molecules GPR81, MCT-1, IL-1 β , TLR3 and ZO1. Conclusions. Disturbance of the transport and reception of lactate can contribute to the changes in structural and functional integrity of the blood-brain barrier caused by bacterial and viral inflammation.

Development of a highly sensitive in vitro endothelial cell toxicity assay for evaluating ricin toxin A chain-based vaccines or therapeutics

The ricin toxin A chain (RTA) is responsible for ricin intoxication due to inhibition of protein synthesis. RTA is also known to cause endothelial toxicity [via a 3 amino acid sequence (x)D(y) motif that acts as a natural disintegrin] resulting in vascular leak syndrome (VLS) in humans. An in vitro endothelial cell toxicity (ECT) assay was developed to evaluate if the ricin vaccine candidate (RVEc) exhibited endothelial toxicity, determined by altered transendothelial electrical resistance (TEER) across human umbilical vein endothelial cell (HUVEC) monolayers. Timepoints at 2 and 4 h were included to evaluate HUVEC monolayers before the effects of RTA ribotoxic activity are observed. Both the 3 μM and 6 μM RTA positive controls consistently demonstrated significantly reduced TEER values, compared to their corresponding vehicle control, in a time- and concentration-dependent manner at 2, 4, and 24 h. Fluorescent imaging of HUVECs exposed to 3 μM RTA showed cell rounding at 2 and 4 h and gap formation at 24 h. No changes in TEER or fluorescent imaging were observed after exposure to endothelial cell growth medium-2 (EGM-2) exchange (mock control). The negative controls, which included 2 mutant RTA vaccine derivatives [RVEc with an (x)D(y) VLS sequence modification to V76M or D75N] and bovine serum albumin (BSA), demonstrated no evidence of HUVEC toxicity at 3 μM and 6 μM concentrations. Overall, the performance of the ECT assay was consistent, allowing for the development of acceptance criteria that were related to time- and concentration-dependent decreases in TEER between 2 and 24 h.

Toll-like receptor 3-mediated inflammation by p38 is enhanced by endothelial nitric oxide synthase knockdown

BackgroundVascular dysfunction is commonly seen during severe viral infections. Endothelial nitric oxide synthase (eNOS), has been postulated to play an important role in regulating vascular homeostasis as well as propagation of the inflammatory reaction. We hypothesized that the loss of eNOS would negatively impact toll-like receptor 3 (TLR3) signaling and worsen vascular function to viral challenge.MethodsHuman microvascular endothelial cells (HMVECs) were exposed to either control or eNOS siRNA and then treated with Poly I:C, a TLR3 agonist and mimicker of dsRNA viruses. Cells were assessed for protein-protein associations, cytokine and chemokine analysis as well as transendothelial electrical resistance (TEER) as a surrogate of permeability.ResultsHMVECs that had reduced eNOS expression had a significantly elevated increase in IL-6, IL-8 and IP-10 production after Poly I:C. In addition, the knockdown of eNOS enhanced the change in TEER after Poly I:C stimulation. Western blot analysis showed enhanced phosphorylation of p38 in sieNOS treated cells with Poly I:C compared to siControl cells. Proximity ligation assays further demonstrated direct eNOS-p38 protein-protein interactions. The addition of the p38 inhibitor, SB203580, in eNOS knockdown cells reduced both cytokine production after Poly I:C, and as well as mitigated the reduction in TEER, suggesting a direct link between eNOS and p38 in TLR3 signaling.ConclusionsThese results suggest that reduction of eNOS increases TLR3-mediated inflammation in human endothelial cells in a p38-dependent manner. This finding has important implications for understanding the pathogenesis of severe viral infections and the associated vascular dysfunction.

Role of Fatty Acid Oxidation and Glucose Oxidation in Endothelial Cell Inflammation and Permeability Associated with Acute Lung Injury

Acute lung injury (ALI) is a common cause of respiratory failure in critically ill patients with a mortality rate of 38.5%. ALI can be precipitated by either direct insults such as pneumonia, aspiration or via indirect insults such as sepsis and multiple trauma, to the lungs. The vascular endothelium forming the innermost lining of all pulmonary blood vessels is the major barrier that protects air spaces against vascular fluid entry. Because of their localization, endothelial cells (EC) are constantly exposed to oxygen and nutrients that fuel cell metabolism. However, very little is known about the role of EC metabolism in the regulation of EC inflammation and permeability, the two major pathogenic features of ALI. Recent studies have highlighted that fatty acid oxidation (FAO) is central to the tight regulation of EC permeability required to maintain vessel functionality. The purpose of the present study is to understand the mechanistic link between cell metabolism and thrombin‐induced EC inflammation and permeability and to understand the relevance of this linkage in the pathophysiology of ALI. Human Pulmonary Artery Endothelial cells (HPAECs) were stimulated with proinflammatory mediator thrombin in the presence and absence of Etomoxir, an inhibitor of FAO or dichloroacetate (DCA), an inhibitor of pyruvate hydrogenase kinase (PHK). Our results indicate that both Etomoxir and DCA inhibited thrombin‐induced inflammatory responses as observed by decrease in the secretion of cytokines IL‐6 and IL‐8. In addition real time changes in transendothelial electrical resistance (TER) were measured to monitor endothelial permeability after exposure of HPAECs to Etomoxir or DCA, followed by thrombin treatment. Our data indicate that Etomoxir had no effect on thrombin‐induced decrease in TER however it delayed the recovery after 1 hour of thrombin challenge. DCA in contrast showed no effect on thrombin‐induced EC permeability. Together our data suggest that metabolic pathways are critical to EC inflammation and permeability induced by thrombin.Support or Funding InformationSupported by: NIH (HL130870)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Sera From Children After Cardiopulmonary Bypass Reduces Permeability of Capillary Endothelial Cell Barriers.

Children undergoing cardiopulmonary bypass develop clinically impactful capillary leak of unclear etiology. A widely held hypothesis that exposure of circulating cells to the cardiopulmonary bypass circuit induces the release of inflammatory mediators that act to disrupt intercellular junctions of capillary endothelial cells inducing paracellular capillary leak either directly or through new gene expression. Cohort study. Tertiary pediatric hospital. Twenty children undergoing surgery with cardiopulmonary bypass for congenital heart disease. Serum was collected before cardiopulmonary bypass, 2 hours after cardiopulmonary bypass, and 18 hours after cardiopulmonary bypass. None. We analyzed the effects of 10% patient sera on the "function, structure, and gene expression" of cultured human dermal and pulmonary microvascular endothelial cells. Changes in barrier "function" were measured using transendothelial electrical resistance. Associations between changes in transendothelial electrical resistance and subject characteristics were analyzed using linear mixed effects model with area under the resistance curve as outcome. Changes in junctional "structure" were assessed by analyzing the organization of the endothelial cell junctional proteins claudin-5 and VE-cadherin using immunofluorescence microscopy. Changes in inflammatory "gene expression" were measured using real-time quantitative reverse transcription-polymerase chain reaction. All serum samples induced a transient, 120-minute increase in transendothelial electrical resistance followed by persistent loss of barrier function. Unexpectedly, sera collected postcardiopulmonary bypass-induced significantly less loss of barrier function in both dermal and pulmonary capillary endothelial cell compared with precardiopulmonary bypass sera. Consistent with the transendothelial electrical resistance results, claudin-5 and vascular endothelial-cadherin junctional staining showed less disruption in cultures treated with postcardiopulmonary bypass sera. Expression of genes commonly associated with inflammation was largely unaffected by patient sera. Contrary to the hypothesis, sera taken from children after cardiopulmonary bypass induces less capillary barrier disruption relative to sera taken from children before cardiopulmonary bypass, and none of the sera induced significant changes in expression of inflammatory genes.

Α-Melanocyte-Stimulating Hormone Protects Early Diabetic Retina from Blood-Retinal Barrier Breakdown and Vascular Leakage via MC4R

Background/Aims: Blood-retinal barrier (BRB) breakdown and vascular leakage is the leading cause of blindness of diabetic retinopathy (DR). Hyperglycemia-induced oxidative stress and inflammation are primary pathogenic factors of this severe DR complication. An effective interventional modality against the pathogenic factors during early DR is needed to curb BRB breakdown and vascular leakage. This study sought to examine the protective effects of α-Melanocyte-stimulating hormone (α-MSH) on early diabetic retina against vascular hyperpermeability, electrophysiological dysfunction, and morphological deterioration in a rat model of diabetes and probe the mechanisms underlying the α-MSH’s anti-hyperpermeability in both rodent retinas and simian retinal vascular endothelial cells (RF6A). Methods: Sprague Dawley rats were injected through tail vein with streptozotocin to induce diabetes. The rats were intravitreally injected with α-MSH or saline at Week 1 and 3 after hyperglycemia. In another 2 weeks, Evans blue assay, transmission electron microscopy, electroretinogram (ERG), and hematoxylin and eosin (H&E) staining were performed to examine the protective effects of α-MSH in diabetic retinas. The expression of pro-inflammatory factors and tight junction at mRNA and protein levels in retinas was analyzed. Finally, the α-MSH’s anti-hyperpermeability was confirmed in a high glucose (HG)-treated RF6A cell monolayer transwell culture by transendothelial electrical resistance (TEER) measurement and a fluorescein isothiocyanate-Dextran assay. Universal or specific melanocortin receptor (MCR) blockers were also employed to elucidate the MCR subtype mediating α-MSH’s protection. Results: Evans blue assay showed that BRB breakdown and vascular leakage was detected, and rescued by α-MSH both qualitatively and quantitatively in early diabetic retinas; electron microscopy revealed substantially improved retinal and choroidal vessel ultrastructures in α-MSH-treated diabetic retinas; scotopic ERG suggested partial rescue of functional defects by α-MSH in diabetic retinas; and H&E staining revealed significantly increased thickness of all layers in α-MSH-treated diabetic retinas. Mechanistically, α-MSH corrected aberrant transcript and protein expression of pro-inflammatory factor and tight junction genes in the diseased retinas; moreover, it prevented abnormal changes in TEER and permeability in HG-stimulated RF6A cells, and this anti-hyperpermeability was abolished by a universal MCR blocker or an antagonist specific to MC4R. Conclusions: This study showed previously undescribed protective effects of α-MSH on inhibiting BRB breakdown and vascular leakage, improving electrophysiological functions and morphology in early diabetic retinas, which may be due to its down-regulating pro-inflammatory factors and augmenting tight junctions. α-MSH acts predominantly on MC4R to antagonize hyperpermeability in retinal microvessel endothelial cells.

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction.

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). This process is characterized by the transmigration of activated T cells across brain endothelial cells (ECs), the main constituents of the BBB. However, the consequences on brain EC function upon interaction with such T cells are largely unknown. Here we describe an assay that allows for the evaluation of primary mouse brain microvascular EC (MBMEC) function and barrier integrity during the interaction with T cells over time. The assay makes use of impedance cell spectroscopy, a powerful tool for studying EC monolayer integrity and permeability, by measuring changes in transendothelial electrical resistance (TEER) and cell layer capacitance (Ccl). In direct contact with ECs, stimulated but not naïve T cells are capable of inducing EC monolayer dysfunction, as visualized by a decrease in TEER and an increase in Ccl. The assay records changes in EC monolayer integrity in a continuous and automated fashion. It is sensitive enough to distinguish between different strengths of stimuli and levels of T cell activation and it enables the investigation of the consequences of a targeted modulation of T cell-EC interaction using a wide range of substances such as antibodies, pharmacological reagents and cytokines. The technique can also be used as a quality control for EC integrity in in vitro T-cell transmigration assays. These applications make it a versatile tool for studying BBB properties under physiological and pathophysiological conditions.

Modulating the endotheliopathy of trauma: Factor concentrate versus fresh frozen plasma.

Transfusion of balanced ratios of plasma to platelets and red blood cells has been shown to reduce early death from exsanguination in trauma patients. Aside from hemostasis, recent work has shown that plasma reduces vascular endothelial permeability, inflammation, and organ edema after hemorrhagic shock (HS), all components of the endotheliopathy of trauma. We hypothesized that Kcentra could have protective effects on the endotheliopathy of trauma comparable with fresh frozen plasma (FFP). In vitro, endothelial cell (EC) barrier function was assessed by measuring changes in transendothelial electrical resistance for Kcentra, FFP, and albumin. In vivo, a modified Miles assay was used on mice to study the effects of Kcentra, FFP, and albumin on vascular permeability induced by VEGF-A. The same groups were studied in a second in vivo model of pulmonary vascular leak induced by HS and laparotomy. The identification of proteins in Kcentra was assessed by liquid chromatography/mass spectrometry. We found that FFP and Kcentra inhibit EC permeability. We also found that Kcentra and FFP have equivalent capacity to restore EC adherens junction breakdown induced by VEGF-A. In vivo, we found that Kcentra and FFP, but not albumin, significantly inhibited vascular permeability induced by VEGF-A and HS-induced vascular permeability in mice. Investigation of the protein content of Kcentra by mass spectroscopy revealed that there are a number of proteins in Kcentra, derived from plasma that may have contributory roles in the noted effects of Kcentra on vascular leak. Taken together, we have demonstrated that FFP and Kcentra inhibit vascular permeability in vivo and in vitro. These beneficial effects of Kcentra may be due in part to the modulation of vascular function by soluble factors present in Kcentra aside from the known clotting factors II, VII, IX, and X. The clinical implications of these findings are unknown and warrant further investigation.

C5a alters blood-brain barrier integrity in a human in vitro model of systemic lupus erythematosus.

The blood-brain barrier (BBB) plays a crucial role in brain homeostasis, thereby maintaining the brain environment precise for optimal neuronal function. Its dysfunction is an intriguing complication of systemic lupus erythematosus (SLE). SLE is a systemic autoimmune disorder where neurological complications occur in 5-50% of cases and is associated with impaired BBB integrity. Complement activation occurs in SLE and is an important part of the clinical profile. Our earlier studies demonstrated that C5a generated by complement activation caused the loss of brain endothelial layer integrity in rodents. The goal of the current study was to determine the translational potential of these studies to a human system. To assess this, we used a two dimensional in vitro BBB model constructed using primary human brain microvascular endothelial cells and astroglial cells, which closely emulates the in vivo BBB allowing the assessment of BBB integrity. Increased permeability monitored by changes in transendothelial electrical resistance and cytoskeletal remodelling caused by actin fiber rearrangement were observed when the cells were exposed to lupus serum and C5a, similar to the observations in mice. In addition, our data show that C5a/C5aR1 signalling alters nuclear factor-κB translocation into nucleus and regulates the expression of the tight junction proteins, claudin-5 and zonula occludens 1 in this setting. Our results demonstrate for the first time that C5a regulates BBB integrity in a neuroinflammatory setting where it affects both endothelial and astroglial cells. In addition, we also demonstrate that our previous findings in a mouse model, were emulated in human cells in vitro, bringing the studies one step closer to understanding the translational potential of C5a/C5aR1 blockade as a promising therapeutic strategy in SLE and other neurodegenerative diseases.

Involvement of local lamellipodia in endothelial barrier function.

Recently we observed that endothelial cells cultured in tightly confluent monolayers display frequent local lamellipodia, and that thrombin, an agent that increases endothelial permeability, reduces lamellipodia protrusions. This led us to test the hypothesis that local lamellipodia contribute to endothelial barrier function. Movements of subcellular structures containing GFP-actin or VE-cadherin-GFP expressed in endothelial cells were recorded using time-lapse microscopy. Transendothelial electrical resistance (TER) served as an index of endothelial barrier function. Changes in both lamellipodia dynamics and TER were assessed during baseline and after cells were treated with either the barrier-disrupting agent thrombin, or the barrier-stabilizing agent sphingosine-1-phosphate (S1P). The myosin II inhibitor blebbistatin was used to selectively block lamellipodia formation, and was used to test their role in the barrier function of endothelial cell monolayers and isolated, perfused rat mesenteric venules. Myosin light chain (MLC) phosphorylation was assessed by immunofluorescence microscopy. Rac1 and RhoA activation were evaluated using G-LISA assays. The role of Rac1 was tested with the specific inhibitor NSC23766 or by expressing wild-type or dominant negative GFP-Rac1. The results show that thrombin rapidly decreased both TER and the lamellipodia protrusion frequency. S1P rapidly increased TER in association with increased protrusion frequency. Blebbistatin nearly abolished local lamellipodia protrusions while cortical actin fibers and stress fibers remained intact. Blebbistatin also significantly decreased TER of cultured endothelial cells and increased permeability of isolated rat mesenteric venules. Both thrombin and S1P increased MLC phosphorylation and activation of RhoA. However, thrombin and S1P had differential impacts on Rac1, correlating with the changes in TER and lamellipodia protrusion frequency. Overexpression of Rac1 elevated, while NSC23766 and dominant negative Rac1 reduced barrier function and lamellipodia activity. Combined, these data suggest that local lamellipodia, driven by myosin II and Rac1, are important for dynamic changes in endothelial barrier integrity.

AN ALL HUMAN 3D IN VITRO MODEL OF THE BLOOD BRAIN BARRIER IN NANOPARTICLE DELIVERY AND CANCER METASTASIS STUDIES

BACKGROUND: The blood-brain barrier (B-BB) is a dynamic functional unit consisting of endothelial cells, astrocytic endfeet and pericytes embedded in a specialized basement membrane. Most in vitro studies examine the ability of therapeutics to cross the B-BB using monolayer cultures or mixed species co-cultures and rely on Trans-Endothelial Electrical Resistance (TEER) measurements without verification of tight-junction (TJ) formation. More reliable high throughput in vitro models are therefore essential in brain tumour drug delivery and metastatic tumour behavioural studies. We are developing and testing a 3-dimensional in vitro model incorporating aspects of known B-BB in vivo characteristics. METHODS: A 3D model of the B-BB was constructed using human brain endothelial cells (hCMEC/D3), human astrocytes (SC-1800/UP010) and human brain vascular pericytes (HBVP) in mono-, co-, tri- cultures. TEER values were measured using a cell monitoring system- cellZscope®. TJ proteins (ZO-1 and occludin) were examined by Western blotting and immunocytochemistry. Effects of extracellular matrix (ECM) molecules (laminin, fibronectin, collagen type IV, agrin, perlecan) were assessed on endothelial cell proliferation, adhesion and TEER changes using an Electric Cell-Substrate Impedance Sensing (ECIS) system. Modified chitosyme nanoparticles and metastatic lung cancer cells were tested for effects on B-BB integrity. RESULTS: Co-cultures and tri-cultures resulted in significantly higher TEER values compared to endothelial cells alone (p < 0.05). TEER values for co-culture of ECs and astrocytes were 4620 Ω/cm2, and for tri-cultivation including pericytes, 4141.50 Ω/cm2 compared to single EC cultures (2244 Ω/cm2). Western blot revealed higher expression of ZO-1 and occludin in co- and tri-cultures. The individual optimal ECM concentrations for highest and longest TEER values differed depending on ECM molecule and concentration. Cell adhesion (%) correlated with TEER values. Recovery time of B-BB integrity following chitosyme nanoparticle/metastatic cell treatment, differed in mono-, co-, tri-cultures. CONCLUSIONS: Astrocytes contribute to B-BB formation in this in vitro model. Motile pericytes may positively or negatively influence barrier formation or TJ tightness. ECM has varied and profound effects on TEER values. Multiple measures are needed to confirm TJ formation in addition to TEER values as these may not reflect ‘true’ B-BB integrity. Using compounds with known low B-BB penetration and analysing TJ protein expression in combination with novel therapeutics will aid in providing meaningful data to bridge the gap between drug discovery and pre-clinical studies. In addition, our brain metastatic research group utilizes this model to test effect of CD15 knockdown in lung metastatic cells on endothelial cell adhesion and B-BB passage. SECONDARY CATEGORY: Tumor Biology.

African Trypanosome-Induced Blood-Brain Barrier Dysfunction under Shear Stress May Not Require ERK Activation.

African trypanosomes are tsetse fly transmitted protozoan parasites responsible for human African trypanosomiasis, a disease characterized by a plethora of neurological symptoms and death. How the parasites under microvascular shear stress (SS) flow conditions in the brain cross the blood-brain barrier (BBB) is not known. In vitro studies using static models comprised of human brain microvascular endothelial cells (BMEC) show that BBB activation and crossing by trypanosomes requires the orchestration of parasite cysteine proteases and host calcium-mediated cell signaling. Here, we examine BMEC barrier function and the activation of extracellular signal-regulated kinase (ERK)1/2 and ERK5, mitogen-activated protein kinase family regulators of microvascular permeability, under static and laminar SS flow and in the context of trypanosome infection. Confluent human BMEC were cultured in electric cell-substrate impedance sensing (ECIS) and parallel-plate glass slide chambers. The human BMEC were exposed to 2 or 14 dyn/cm(2) SS in the presence or absence of trypanosomes. Real-time changes in transendothelial electrical resistance (TEER) were monitored and phosphorylation of ERK1/2 and ERK5 analyzed by immunoblot assay. After reaching confluence under static conditions human BMEC TEER was found to rapidly increase when exposed to 2 dyn/cm(2) SS, a condition that mimics SS in brain postcapillary venules. Addition of African trypanosomes caused a rapid drop in human BMEC TEER. Increasing SS to 14 dyn/cm(2), a condition mimicking SS in brain capillaries, led to a transient increase in TEER in both control and infected human BMEC. However, no differences in ERK1/2 and ERK5 activation were found under any condition tested. African trypanosomiasis alters BBB permeability under low shear conditions through an ERK1/2 and ERK5 independent pathway.

Acute effects of ionizing radiation on human endothelial barrier function

The human vasculature is critical to healthy functioning of the tissues of the body and a major factor in maintaining homeostasis is the endothelial barrier. In the brain, the blood–brain barrier (BBB) is highly specialized in order to sustain the neural tissue.Here, we have examined the effects of radiation on BBB models using a unique variety of endpoints to assess barrier function. These include trans-endothelial electrical resistance (TEER), morphological effects, localization of adhesion and cell junction proteins (in two-dimensional monolayers and in three-dimensional tissue models) and permeability of molecules through the endothelial barrier. Two culture conditions were used to represent conditions on the inside or the lumen of vessels and conditions on the outside or ablumenal side of vessels. For the lumen, cells were cultured in serum and growth factor containing media, and for the ablumenal side of vessels, cells were cultured in serum-free defined media.Initial experiments with gamma rays in serum-free conditions revealed a previously unknown acute effect involving cell detachment and the loss of the clinically relevant cell adhesion molecule—cell platelet endothelial adhesion molecule (PECAM)-1 [ 1]. Gamma radiation (5 Gy) induced a rapid and transient decrease in TEER at 3 h, with effects also seen at the lower radiotherapy dose of 2 Gy. This dip in resistance correlated with the transient loss PECAM-1 in discrete areas where cells often detached from the monolayer leaving gaps. Loss in PECAM-1 occurred at least in part as detached microparticles. Redistribution of PECAM-1 microparticles was also seen in three-dimensional human tissue models. By 6 h, the remaining cells had migrated to reseal the barrier, coincident with TEER returning to control levels. Resealed monolayers contained fewer cells per unit area and their barrier function was weakened as corroborated by an increased permeability over 24 h. Because PECAM-1 is involved in barrier function and platelet aggregation, this effect is likely highly relevant to cancer radiotherapy using gamma rays.These studies were extended to include low linear energy transfer (LET) photons of X-rays and ion particles present in the space environment—low LET ion particles including high energy (1 GeV) protons and helium ions. X-rays under serum-free conditions also showed an acute response involving a dip in TEER at 3 h and the loss of PECAM-1 between cells as microparticles. Ion particles, however, did not show these effects of photons under serum-free conditions. Both protons and helium ions at doses up to 5 Gy did not produce this transient change in TEER or PECAM-1, although some longer term effects in TEER were noted.In the presence of serum and growth factors, however, all radiations tested showed short-term effects in TEER, that of a series of symmetrical peaks which diminished in size over several hours. For 1 GeV protons and helium ions, this effect could be fitted to an equation for under-damped oscillation, a pattern typical of a mechanism for timing of events in periodic processes. For gamma and X-rays, the underdamped oscillation was present but superimposed on a drop in resistance at 3 h similar to that seen in serum-free conditions.In conclusion, we have shown two acute effects of low LET radiation on the human endothelial barrier. First, a short-term effect of photons but not ion particles involving a single dip in TEER and the loss of PECAM-1 at 3 h after irradiation, and second an underdamped oscillation of TEER induced by both photons and ion particles that to date does not appear to be associated with the loss of PECAM-1 or any other junction molecules.

Monocytic adhesion molecule expression and monocyte–endothelial cell dysfunction are increased in patients with peripheral vascular disease versus patients with abdominal aortic aneurysms

BackgroundStatin therapy is used in the medical management of patients with peripheral vascular disease (PVD) and abdominal aortic aneurysm (AAA) for the pleiotropic and anti-inflammatory benefits. We hypothesize that the inflammatory mechanisms of monocyte–endothelial cell interactions in endothelial barrier dysfunction are more significant in patients with PVD compared with those with AAA. The purpose of this study was to assess patient peripheral blood monocyte adhesion molecules by flow cytometry and monocyte-induced endothelial barrier dysfunction by using an in vitro endothelial cell layer and electric cell-substrate impedance sensing (ECIS) system. MethodsPeripheral blood was collected from patients with either PVD (ankle-brachial index <0.9, toe–arm index <0.8, or required lower extremity vascular intervention) or AAA (aortic diameter >3.0cm). Monocytes were isolated from fresh whole blood using an accuspin-histopaque technique. The separated monocytes underwent flow cytometry analysis to evaluate the expression levels of the cell membrane adhesion molecules: CD18, CD11a/b/c, and very late antigen-4. Endothelial cell function was assessed by adding monocytes to an endothelial monolayer on ECIS arrays and coculturing overnight. Peak changes in transendothelial electrical resistance were measured and compared between patient groups. ResultsTwenty-eight monocyte samples were analyzed for adhesion molecules (PVD, 19 and AAA, 9) via flow cytometry, and 11 patients were evaluated for endothelial dysfunction (PVD, 7 and AAA, 4) via ECIS. There was no significant difference between risk factors among PVD and AAA patients except for age, where AAA patients were significantly older than PVD patients in both flow cytometry and ECIS groups (P=0.02 and 0.01, respectively). There were significantly higher levels of adhesion molecules CD11a, CD18, and CD11c (averaged mean fluorescent intensity P values: 0.047, 0.038, and 0.014, respectively) in PVD patients compared with AAA patients. No significant difference was found for CD11b and very late antigen-4 expression (P=0.21 and 0.15, respectively). There was significantly more monocyte–endothelial cell dysfunction in patients with PVD versus patients with AAA, with a maximal effect seen at 15h after monocyte addition (P=0.032). ConclusionsPatients with PVD have increased expression levels of certain monocyte adhesion molecules and greater monocyte-induced endothelial layer dysfunction compared with those with AAA. This may lead to other methods of targeted therapy to improve outcomes of these vascular patients.

Hsp90 inhibition prevents bacterial lipopolysaccharide‐induced and RhoA‐mediated signaling leading to paracellular hyper‐permeability in human lung microvascular endothelial cells

We have previously shown that hsp90 inhibitors protect and restore bacterial lipopolysaccharide (LPS) ‐ mediated barrier dysfunction of human lung microvascular endothelial cells (HLMVEC). Since RhoA is a well‐known mediator of endothelial barrier dysfunction, we investigated the role of hsp90 in LPS‐induced and RhoA mediated HLMVEC hyper‐permeability. LPS (1 EU/ml) challenge of HLMVEC significantly increased RhoA tyrosine nitration and RhoA activity. Pre‐treatment (1 hr) with the hsp90 inhibitor, 17‐AAG (2 uM) suppressed both RhoA nitration and RhoA activity. Pre‐treatment with GSK429286 (10 uM), an inhibitor of the RhoA effector, Rho kinase (ROCK) partially rescued the LPS‐mediated HLMVEC hyper‐permeability, as reflected in changes in transendothelial electrical resistance (TER) of HLMVEC monolayers. Furthermore, GSK429286 pretreatment abolished the LPS‐induced phosphorylation of both CREB (Ser133) and STAT3 (Tyr705), two pro‐inflammatory transcription factors regulated by hsp90. These data suggest that the barrier protective effects of Hsp90 inhibition in HLMVEC involve suppression of RhoA‐ROCK signaling and inhibition of CREB and STAT3. (Supported by HL093460 and HL101902)

A Role for VEGFR2 Activation in Endothelial Responses Caused by Barrier Disruptive OxPAPC Concentrations

IntroductionOxidation products of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphatidylcholine (OxPAPC) differentially modulate endothelial cell (EC) barrier function in a dose-dependent fashion. Vascular endothelial growth factor receptor-2 (VEGFR2) is involved in the OxPAPC-induced EC inflammatory activation. This study examined a role of VEGFR2 in barrier dysfunction caused by high concentrations of OxPAPC and evaluated downstream signaling mechanisms resulting from the effect of OxPAPC in EC from pulmonary and systemic circulation.MethodsEC monolayer permeability in human pulmonary artery endothelial cells (HPAEC) and human aortic endothelial cells (HAEC) was monitored by changes in transendothelial electrical resistance (TER) across EC monolayers. Actin cytoskeleton was examined by immunostaining with Texas Red labeled phalloidin. Phosphorylation of myosin light chains (MLC) and VE-Cadherin was examined by Western blot and immunofluorescence techniques. The role of VEGFR2 in OxPAPC-induced permeability and cytoskeletal arrangement were determined using siRNA-induced VEGFR2 knockdown.ResultsLow OxPAPC concentrations (5–20 µg/ml) induced a barrier protective response in both HPAEC and HAEC, while high OxPAPC concentrations (50–100 µg/ml) caused a rapid increase in permeability ; actin stress fiber formation and increased MLC phosphorylation were observed as early as 30 min after treatment. VEGFR2 knockdown dramatically decreased the amount of MLC phosphorylation and stress fiber formation caused by high OxPAPC concentrations with modest effects on the amount of VE-cadherin phosphorylation at Y731. We present evidence that activation of Rho is involved in the OxPAPC/VEGFR2 mechanism of EC permeability induced by high OxPAPC concentrations. Knockdown of VEGFR2 did not rescue the early drop in TER but prevented further development of OxPAPC-induced barrier dysfunction.ConclusionsThis study shows that VEGFR2 is involved in the delayed phase of EC barrier dysfunction caused by high OxPAPC concentrations and contributes to stress fiber formation and increased MLC phosphorylation.

Free Insulin-like Growth Factor Binding Protein-3 (IGFBP-3) Reduces Retinal Vascular Permeability in Association with a Reduction of Acid Sphingomyelinase (ASMase)

To examine the effect of free insulin-like growth factor (IGF) binding protein-3 (IGFBP-3), independent of the effect of insulin-like growth factors, in modulating retinal vascular permeability. We assessed the ability of a form of IGFBP-3 that does not bind IGF-1 (IGFBP-3NB), to regulate the blood retinal barrier (BRB) using two distinct experimental mouse models, laser-induced retinal vessel injury and vascular endothelial growth factor (VEGF)-induced retinal vascular permeability. Additionally, in vitro studies were conducted. In the animal models, BRB permeability was quantified by intravenous injection of fluorescein labeled serum albumin followed by digital confocal image analysis of retinal flat-mounts. Claudin-5 and vascular endothelial-cadherin (VE-cadherin) localization at interendothelial junctions was studied by immunofluorescence. In vitro changes in transendothelial electrical resistance (TEER) and flux of fluorescent dextran in bovine retinal endothelial monolayers (BREC) were measured after IGFBP-3NB treatment. Acid (ASMase) and neutral (NSMase) sphingomyelinase mRNA levels and activity were measured in mouse retinas. Four days postinjury, laser-injured mouse retinas injected with IGFBP-3NB plasmid demonstrated reduced vascular permeability compared with retinas of laser-injured mouse retinas injected with control plasmid. IGFBP-3NB administration resulted in a significant decrease in laser injury-associated increases in ASMase and NSMase mRNA and activity when compared with laser alone treated mice. In vivo, intravitreal injection of IGFBP-3NB reduced vascular leakage associated with intravitreal VEGF injection. IGFBP-3NB partially restored VEGF-induced in vivo permeability and dissociation of claudin-5 and VE-cadherin at junctional complexes. When IGFBP-3NB was applied basally to bovine retinal endothelial cells (BREC) in vitro, TEER increased and macromolecular flux decreased. Intravitreal administration of IGFBP-3NB preserves junctional integrity in the presence of VEGF or laser injury by reducing BRB permeability in part by modulating sphingomyelinase levels.