Decrease In Transendothelial Electrical Resistance Research Articles

Hyperglycaemia perturbs blood-brain barrier integrity through its effects on endothelial cell characteristics and function

ABSTRACT Breakdown of blood-brain barrier (BBB) represents a key pathology in hyperglycemia-mediated cerebrovascular damage after an ischemic stroke. As changes in the level and nature of vasoactive agents released by endothelial cells (ECs) may contribute to BBB dysfunction, this study first explored the specific impact of hyperglycemia on EC characteristics and secretome. It then assessed whether secretome obtained from ECs subjected to normoglycaemia or hyperglycemia might regulate pericytic cytokine profile differently. Using a triple cell culture model of human BBB, composed of brain microvascular EC (BMEC), astrocytes and pericytes, this study showed that exposure to hyperglycemia (25 mM D-glucose) for 72 h impaired the BBB integrity and function as evidenced by decreases in transendothelial electrical resistance and increases in paracellular flux of sodium fluorescein. Dissolution of zonula occludens-1, a tight junction protein, and appearance of stress fibers appeared to play a key role in this pathology. Despite elevations in angiogenin, endothelin-1, interleukin-8 and basic fibroblast growth factor levels and a decrease in placental growth factor levels in BMEC subjected to hyperglycemia vs normoglycaemia (5.5 mM D-glucose), tubulogenic capacity of BMECs remained similar in both settings. Similarly, pericytes subjected to secretome obtained from hyperglycemic BMEC released higher quantities of macrophage migration inhibitory factor and serpin and lower quantities of monocyte chemoattractant protein-1, intercellular adhesion molecule, interleukin-6 and interleukin-8. Taken together these findings indicate the complexity of the mechanisms leading to BBB disruption in hyperglycemic settings and emphasize the importance of endothelial cell-pericyte axis in the development of novel therapeutic strategies.

Abstract 12294: Poldip2 Mediates Brain Vascular Permeability by Regulating ZO-1 Phosphorylation and Localization at the Interendothelial Border

Introduction: Poldip2 is a novel regulator of cerebral vascular permeability involved in aggravating blood-brain barrier disruption following ischemic stroke; however, the underlying mechanisms are incompletely understood. Tight junctions are crucial for the formation of endothelial barriers and are composed of transmembrane proteins and adaptor proteins such as ZO-1. Cerebral ischemia and inflammation lead to ZO-1 redistribution resulting in vascular permeability. Hypothesis: We hypothesized that Poldip2 may mediate ZO-1 phosphorylation and promote brain endothelial monolayer permeability following cerebral ischemia. Methods: Cerebral ischemia was induced in endothelial-specific Poldip2 knockout and control mice. Vascular permeability was assessed by Evans blue dye extravasation. For in vitro analysis, primary rat brain microvascular endothelial cells were used to investigate the effect of Poldip2 depletion by siRNA on TNF-α-induced permeability and trans-endothelial electrical resistance (TEER). Immunocytochemistry was carried out to investigate the effects of Poldip2 depletion or overexpression on ZO-1 localization. Results: Endothelial-specific Poldip2 deletion abolished Evans blue dye extravasation after cerebral ischemia induction (p<0.05). Poldip2 knockdown in vitro suppressed TNF-α-induced endothelial cell (EC) monolayer permeability (FITC-dextran and biotin-avidin assays, p<0.05 and p<0.001) and prevented decreases in TEER (p<0.01). Poldip2 depletion prevented TNF-α-induced ZO-1 disruption at interendothelial junctions (p<0.05). Poldip2 overexpression increased endothelial permeability (p<0.05), decreased TEER (p<0.05), reduced ZO-1 localization at cell-cell junctions (p<0.0001) and enhanced reactive oxygen species (ROS) production (p<0.05). Treatment with the antioxidant N-acetyl cysteine (NAC) abrogated Poldip2-induced ZO-1 disruption (p<0.0001). Immunoprecipitation studies demonstrated that Poldip2 overexpression induced tyrosine phosphorylation of ZO-1 (p<0.0001), an effect prevented by NAC treatment (p<0.01). Conclusions: Poldip2 induces ROS-mediated tyrosine phosphorylation of the TJ protein ZO-1 and promotes EC permeability following cerebral ischemia

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.

Understanding the mechanisms of ultrasound-targeted microbubble cavitation-mediated blood brain barrier opening

Ultrasound-targeted microbubble cavitation (UTMC) transiently opens the blood brain barrier (BBB). We previously determined that UTMC induces BBB hyperpermeability through an influx of calcium. As activation of RhoA is a calcium-dependent pathway that causes cytoskeletal reorganization, leading to the breakdown of tight junctions, we tested the hypothesis that UTMC-induced activation of RhoA leads to BBB hyperpermeability. We utilized a transwell model with brain endothelial cells and astrocytes on opposite sides of a support membrane. Ultrasound (1 MHz, 250 kPa, 10 μs pulse duration, 10 ms pulse interval) was applied in the presence of lipid microbubbles for 20 s. BBB permeability was assessed using dextran flux and transendothelial electrical resistance (TEER) measured across the membrane. Integrity of tight junctions was evaluated by staining for ZO-1. UTMC reduced TEER (p < 0.05), confirming reduced barrier integrity. One hour after UTMC, there was a significant decrease in ZO-1 mean pixel intensity (p < 0.05). Treatment of cells with Rho inhibitor II (Y16) significantly reduced UTMC-induced dextran flux across the BBB (p < 0.05) and UTMC-induced decrease in TEER. In our co-culture model of the BBB, UTMC induces hyperpermeability through modulation of tight junctions, at least in part through a RhoA-dependent mechanism.

Toxoplasma Gondii Induces Blood-Brain Barrier Dysfunction in a Human Stem Cell- Derived Model


 
 
 Background:
 Toxoplasma gondii is a single-celled obligate intracellular parasite commonly found in cat feces and animal issues and is known to cause cerebral infections in immune- compromised individuals and neonates. Once ingested, T. gondii reaches the host's blood supply and reaches the brain, where infection ensues. However, it remains unclear how T. gondii crosses the blood-brain barrier to reach the brain. In this study, we utilize a human stem cell-derived BBB model with near in vivo properties to investigate the effects of T. gondii on critical barrier properties that permit entry into the brain parenchyma.
 Methods:
 Human-induced pluripotent stem cells (iPSCs) were used to derive brain microvascular endothelial cells (BMECs). BMECs were exposed to tachyzoites, the infectious rapid replicating stage of T. gondii, at a multiplicity of infection (MOI) of 0.1 and 1.0 for 24 hours. Following exposure, several critical barrier properties were monitored, including trans- endothelial electrical resistance (TEER) and tight junction analysis.
 Results:
 BMECs treated with both MOI of 0.1 and 1.0 showed a significant reduction in all three tight junction proteins (Claudin-5, Occludin, and ZO-1), as indicated by an increase in discontinuous junctions and a decrease in area fraction index. Barrier integrity is directly associated with tight junctional continuity and localization. Furthermore, tachyzoites at an MOI of 0.1 and 1.0 demonstrated a reduction in barrier tightness, as observed by a decrease in TEER.
 Conclusion and Potential Impact:
 These results indicate that T. gondii may, in part, have access to the brain by inducing disruption in tight junction proteins, thus increasing barrier leakiness via a para-cellular route. These data may help guide future investigations on T. gondii's ability to evoke blood-brain barrier dysfunction and reach the vulnerable brain tissue.
 
 

Critical role of Rho proteins in myosin light chain di-phosphorylation during early phase of endothelial barrier disruption

We previously reported the Rho-associated coiled-coil containing protein kinase (ROCK)-mediated di-phosphorylation of myosin light chain (MLC) and actin bundle formation at the cell periphery as early events of the endothelial barrier disruption. We herein examined the role of RhoA during early events of barrier disruption. Treatment of cultured porcine aortic endothelial cells with simvastatin prevented the decrease in trans-endothelial electrical resistance, MLC di-phosphorylation and peripheral actin bundle formation seen 3 min after thrombin stimulation. Co-treatment with geranylgeranyl pyrophosphate rescued the thrombin-induced events. Thrombin increased a GTP-bound form of RhoA and phosphorylation of myosin phosphatase target subunit 1 (MYPT1) at the ROCK site. The intracellular introduction of the inhibitory protein of RhoA inhibited the thrombin-induced di-phosphorylation of MLC. However, knockdown of either one of RhoA, RhoB or RhoC failed to inhibit thrombin-induced MLC di-phosphorylation. The findings suggest that Rho proteins play a critical role during early events of thrombin-induced barrier disruption.

Delay of endothelial cell senescence protects cerebral barrier against age-related dysfunction: role of senolytics and senomorphics

ABSTRACT Accumulation of senescent cells in cerebrovasculature is thought to play an important role in age-related disruption of blood–brain barrier (BBB). Using an in vitro model of human BBB, composed of brain microvascular endothelial cells (BMECs), astrocytes and pericytes, this study explored the so-called correlative link between BMEC senescence and the BBB dysfunction in the absence or presence of functionally distinct senotherapeutics. Replicative senescence was deemed present at passage ≥19 where BMECs displayed shortened telomere length, reduced proliferative and tubulogenic potentials and increased NADPH oxidase activity, superoxide anion production (markers of oxidative stress), S-β-galactosidase activity and γ-H2AX staining. Significant impairments observed in integrity and function of a model of BBB established with senescent BMECs, ascertained successively by decreases in transendothelial electrical resistance and increases in paracellular flux, revealed a close correlation between endothelial cell senescence and BBB dysfunction. Disruptions in the localization or expression of tight junction proteins, zonula occludens-1, occludin, and claudin-5 in senescent BMECs somewhat explained this dysfunction. Indeed, treatment of relatively old BMEC (passage 16) with a cocktail of senolytics (dasatinib and quercetin) or senomorphics targeting transcription factor NF-κB (QNZ), p38MAPK signaling pathway (BIRB-796) or pro-oxidant enzyme NADPH oxidase (VAS2870) until passage 20 rendered these cells more resistant to senescence and totally preserved BBB characteristics by restoring subcellular localization and expression of tight junction proteins. In conclusion, attempts that effectively mitigate accumulation of senescent endothelial cells in cerebrovasculature may prevent age-related BBB dysfunction and may be of prophylactic or therapeutic value to extend lifelong health and wellbeing.

Application of blood brain barrier models in pre-clinical assessment of glioblastoma-targeting CAR-T based immunotherapies

Human blood brain barrier (BBB) models derived from induced pluripotent stem cells (iPSCs) have become an important tool for the discovery and preclinical evaluation of central nervous system (CNS) targeting cell and gene-based therapies. Chimeric antigen receptor (CAR)-T cell therapy is a revolutionary form of gene-modified cell-based immunotherapy with potential for targeting solid tumors, such as glioblastomas. Crossing the BBB is an important step in the systemic application of CAR-T therapy for the treatment of glioblastomas and other CNS malignancies. In addition, even CAR-T therapies targeting non-CNS antigens, such as the well-known CD19-CAR-T therapies, are known to trigger CNS side-effects including brain swelling due to BBB disruption. In this study, we used iPSC-derived brain endothelial-like cell (iBEC) transwell co-culture model to assess BBB extravasation of CAR-T based immunotherapies targeting U87MG human glioblastoma (GBM) cells overexpressing the tumor-specific mutated protein EGFRvIII (U87vIII). Two types of anti-EGFRvIII targeting CAR-T cells, with varying tonic signaling profiles (CAR-F263 and CAR-F269), and control Mock T cells were applied on the luminal side of BBB model in vitro. CAR-F263 and CAR-F269 T cells triggered a decrease in transendothelial electrical resistance (TEER) and an increase in BBB permeability. CAR-T cell extravasation and U87vIII cytotoxicity were assessed from the abluminal compartment using flow cytometry and Incucyte real-time viability imaging, respectively. A significant decrease in U87vIII cell viability was observed over 48 h, with the most robust cytotoxicity response observed for the constitutively activated CAR-F263. CAR-F269 T cells showed a similar cytotoxic profile but were approximately four fold less efficient at killing the U87vIII cells compared to CAR-F263, despite similar transmigration rates. Visualization of CAR-T cell extravasation across the BBB was further confirmed using BBTB-on-CHIP models. The described BBB assay was able to discriminate the cytotoxic efficacies of different EGFRvIII-CARs and provide a measure of potential alterations to BBB integrity. Collectively, we illustrate how BBB models in vitro can be a valuable tool in deciphering the mechanisms of CAR-T–induced BBB disruption, accompanying toxicity and effector function on post-barrier target cells.

Ultrasound-targeted microbubble cavitation increases paracellular gaps in an in vitro blood brain barrier model

Background: Ultrasound-targeted microbubble cavitation (UTMC) can transiently open the blood brain barrier (BBB). We sought to determine the timeline of paracellular gap formation after UTMC in an in vitro model of the BBB. Methods: We utilized a transwell model with murine brain endothelial cells (EC) and astrocytes on opposite sides of a support membrane. Ultrasound (1 MHz, 10 μs duration, 10 ms pulse interval) at 250 kPa was applied to lipid microbubbles in contact with ECs for 20 s. Z-stacks of transwells acquired by confocal microscopy were converted to maximum intensity projections to quantify number and size of paracellular gaps (NIS-Elements). Endothelial barrier function was assessed using transendothelial electrical resistance (TEER). One-way ANOVA with post-hoc t-testing was performed. Results: Compared to no UTMC, there was an increase in the number and size of paraceullar gaps two minutes after UTMC that lasted at least 60 min (p < 0.05). The increase in gaps was associated with a decrease in TEER (p < 0.05). The number of gaps started to decrease 15 min after UTMC, and total gap area started to decrease 30 min after UTMC (p < 0.05). Conclusions: In our model of the BBB, UTMC induces endothelial hyperpermeability, which appears mediated by dynamic paracellular gap formation.

ArhGEF12 activates Rap1A and not RhoA in human dermal microvascular endothelial cells to reduce tumor necrosis factor-induced leak.

Overwhelming inflammation in the setting of acute critical illness induces capillary leak resulting in hypovolemia, edema, tissue dysoxia, organ failure and even death. The tight junction (TJ)-dependent capillary barrier is regulated by small GTPases, but the specific regulatory molecules most active in this vascular segment under such circumstances are not well described. We set out to identify GTPase regulatory molecules specific to endothelial cells (EC) that form TJs. Transcriptional profiling of confluent monolayers of TJ-forming human dermal microvascular ECs (HDMECs) and adherens junction only forming-human umbilical vein EC (HUVECs) demonstrate ARHGEF12 is basally expressed at higher levels and is only downregulated in HDMECs by junction-disrupting tumor necrosis factor (TNF). HDMECs depleted of ArhGEF12 by siRNA demonstrate a significantly exacerbated TNF-induced decrease in trans-endothelial electrical resistance and disruption of TJ continuous staining. ArhGEF12 is established as a RhoA-GEF in HUVECs and its knock down would be expected to reduce RhoA activity and barrier disruption. Pulldown of active GEFs from HDMECs depleted of ArhGEF12 and treated with TNF show decreased GTP-bound Rap1A after four hours but increased GTP-bound RhoA after 12h. In cell-free assays, ArhGEF12 immunoprecipitated from HDMECs is able to activate both Rap1A and RhoA, but not act on Rap2A-C, RhoB-C, or even Rap1B which shares 95% sequence identity with Rap1A. We conclude that in TJ-forming HDMECs, ArhGEF12selectively activates Rap1A to limit capillary barrier disruption in a mechanism independent of cAMP-mediated Epac1 activation.

Differential Effects of Normoxic versus Hypoxic Derived Breast Cancer Paracrine Factors on Brain Endothelial Cells

Simple SummaryThe potential of breast cancer to spread to the brain increases the clinical complications of the disease; breast cancer is considered to have the second-highest capacity to spread to the brain after lung cancer. The brain is protected by highly specialized endothelial cells, forming a barrier against the entry of circulating molecules and cells. The ability of breast cancer cells to penetrate the protective endothelial barrier is still not completely understood. Here, we aimed to investigate the effect of breast cancer cells on the brain’s endothelial cells. We showed that breast cancer cells induce changes in endothelial cells by releasing factors that target the mitochondria, affecting the endothelial cells and their attachment to each other and, therefore, their function as a protective barrier of the brain. Understanding the mechanism that breast cancer cells utilize to affect endothelial cells under normoxic and hypoxic conditions contributes to the development of treatments to prevent the metastasis of cancer cells to the brain.Background: The blood-brain barrier (BBB) is a central nervous system protective barrier formed primarily of endothelial cells that regulate the entry of substances and cells from entering the brain. However, the BBB integrity is disrupted in disease, including cancer, allowing toxic substances, molecules, and circulating cells to enter the brain. This study aimed to determine the mitochondrial changes in brain endothelial cells co-cultured with cancer cells. Method: Brain endothelial cells (bEnd.3) were co-cultivated with various concentrations of breast cancer (MCF7) conditioned media (CM) generated under normoxic (21% O2) and hypoxic conditions (5% O2). The mitochondrial activities (including; dehydrogenases activity, mitochondrial membrane potential (ΔΨm), and ATP generation) were measured using Polarstar Omega B.M.G-Plate reader. Trans-endothelial electrical resistance (TEER) was evaluated using the EVOM system, followed by quantifying gene expression of the endothelial tight junction (ETJs) using qPCR. Results: bEnd.3 cells had reduced cell viability after 72 h and 96 h exposure to MCF7CM under hypoxic and normoxic conditions. The ΔΨm in bEnd.3 cells were hyperpolarized after exposure to the hypoxic MCF7CM (p < 0.0001). However, the normoxic MCF7CM did not significantly affect the state of ΔΨm in bEnd.3 cells. ATP levels in bEnd.3 co-cultured with hypoxic and normoxic MCF7CM was significantly reduced (p < 0.05). The changes in brain endothelial mitochondrial activity were associated with a decrease in TEER of bEnd.3 monolayer co-cultured with MCF7CM under hypoxia (p = 0.001) and normoxia (p < 0.05). The bEnd.3 cells exposed to MCF7CM significantly increased the gene expression level of ETJs (p < 0.05). Conclusions: MCF7CM modulate mitochondrial activity in brain endothelial cells, affecting the brain endothelial barrier function.

Abstract 11809: Trans-Endothelial Electrical Resistance to Assess Cell Layer Integrity in Coronary Artery Endothelial Cells After Hypoxia/Reoxygenation Injury

Introduction: Cardiovascular disease remains one of the leading causes of complications and death worldwide. Therefore, accurate and reliable methods of mimicking ischemia/reperfusion (IR) injury in cardiac cells in vivo are crucial when testing drugs/substances for prevention and treatment. Coronary artery endothelial cells play a critical role in not only supplying blood to myocardial cells but protecting them from insult as well. However, the endothelial layer can be compromised by ischemic injury, heightening damage to the heart during reperfusion. Hypothesis: Varied ischemic insult of mouse coronary artery endothelial cells (MCAECs) affects cell layer integrity as measured by Trans-Endothelial Electrical Resistance (TEER). Methods: MCAECs were cultured on Grenier Bio-One ThinCert™-cell culture inserts for 72 hrs to allow for adequate confluency. Cells were then subjected to either continued normoxic conditions or hypoxia for 3, 6, 12, or 24 hrs, with a 2-hr reperfusion period immediately following. TEER was used to measure the integrity of the endothelial cell layer on the insert. Results: Our data showed a significant decrease in TEER between control and hypoxic groups after 6 hrs (p = 0.0400), 12 hrs (p = 0.0179) and 24 hrs (p = 0.0103), but not after 3 hrs (p = 0.4453) of hypoxia. Conclusion: This indicates that titrating the hypoxia time to a sufficient duration is necessary to achieve an adequate H/R injury which can then be used for potential cardioprotective agents and/or strategies to be tested. Furthermore, TEER is a reliable and reproducible method to assess the role of endothelial cell membrane integrity in cardioprotection.

The Effect of ABO Immune Complexes on Endothelial Cell Integrity As Measured By Electric Cell-Substrate Impedance Sensing (ECIS) and Confocal Microscopy

Background: ABO non-identical transfusions have been associated with increased risk of hemolytic transfusion reactions. We previously reported that the ABO immune complexes (ABO-IC) formed in vitro as a result of ABO non-identical exposure affect platelet function, red blood cell integrity, and clot kinetics (Vox Sang. 2016;110:219-26). We developed an in vitro model as a means to investigate the interaction between ABO-IC and endothelial cells (EC).Methods: The ABO-IC were prepared by combining equal volumes of concentrated O plasma (high titers of anti-A and anti-B) with A plasma (high concentration of A antigens). Anti-A titers were measured before and after mixing to confirm the presence of ABO-IC. ABO-known human pulmonary vessel EC were then seeded onto gold microarray plates to prepare for electric cell-substrate impedance sensing (ECIS). The trans-endothelial electrical resistance (TER) was recorded for 48 hours after the addition of media, PBS, thrombin, O, A, B, and/or AB plasmas added 1:1 with media, as well as various concentrations of ABO-IC. Statistics were performed using the chi-square test to compare TER values at various timepoints to baseline. Human pulmonary EC were cultured on glass-bottomed petri dishes for confocal microscopy. The cells were stained with fluorescent green calcein AM dye and assessed for viability at 100x magnification at baseline and again 15 minutes following the addition of ABO-IC.Results: Substantial decreases in TER were observed over the course of 2 hours in O and A EC when treated with non-identical plasma, and sustained over 10 hours (p<0.05 for all). Significant decreases in TER were also observed in cells treated with various concentrations of ABO-IC, over 2 hours and were found to be dose-dependent and independent of ABO type (p<0.05 for all). Unlike the ABO-IC, exposure to thrombin revealed a marked decrease in TER after 2 hours, although the final TER measurement revealed complete recovery (Table 1a-c). Incubation with ABO-identical plasma showed slight decreases in TER over time that were not statistically significant when compared to incubation with cell media (Figure 1a-b). Physical damage to the EC membrane were observed via confocal microscopy after 15 minutes of exposure to ABO-IC (Figures 2a-b).Discussion: The exposure of ABO-IC to human pulmonary vessel EC revealed significantly decreased monolayer integrity via ECIS and confocal microscopy. The decreases seen in the TER of EC incubated with non-identical plasma and ABO-IC indicate the potential for capillary leak as a possible reaction to ABO non-identical transfusion. We have shown that the substantial decrease in TER when exposed to plasma is ABO type-dependent, and that the damage caused to the cells happens within a few hours and is irreversible over 48 hours. The confocal images reveal a significant change in the morphology of the EC membrane within 15 minutes of exposure to ABO-IC. These preliminary data suggest that anti-A and ABO-IC may significantly affect EC viability and function. This in vitro model provides potential novel mechanisms for the detrimental effects of ABO non-identical transfusion on EC. Further investigation of biomarkers for cell viability and integrity in response to exposure to ABO-IC is underway. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Inflammatory Immune Cytokine TNF-α Modulates Ezrin Protein Activation via FAK/RhoA Signaling Pathway in PMVECs Hyperpermeability

Background: One of the important pathogenesis of acute respiratory distress syndrome (ARDS) is the dysfunction of pulmonary microvascular endothelial barrier induced by a hyperinflammatory immune response. However, the potential mechanisms of such an imbalance in pulmonary microvascular endothelial cells (PMVECs) are not yet understood. Purpose: Explore the molecular mechanism of endothelial barrier dysfunction induced by inflammatory immune cytokines in ARDS, and find a therapeutic target for this syndrome. Methods: Rat PMVECs were cultured to form a monolayer. Immunofluorescence, flow cytometry, and Western blotting were selected to detect the distribution and the expression level of phosphorylated Ezrin protein and Ezrin protein. Transendothelial electrical resistance (TER) and transendothelial fluxes of fluorescein isothiocyanate (FITC)-labeled bovine serum albumin (BSA) were utilized to measure the permeability of the cell monolayer. Ezrin short hairpin RNA (shRNA) and Ezrin 567-site threonine mutant (EzrinT567A) were used to examine the role of Ezrin protein and phosphorylated Ezrin protein in endothelial response induced by tumor necrosis factor-alpha (TNF-α), respectively. The function of focal adhesion kinase (FAK) and Ras homolog gene family, member A (RhoA) signaling pathways were estimated by inhibitors and RhoA/FAK shRNA in TNF-α-stimulated rat PMVECs. The activation of FAK and RhoA was assessed by Western blotting or pull-down assay plus Western blotting. Results: The TER was decreased after TNF-α treatment, while the Ezrin protein phosphorylation was increased in a time- and dose-dependent manner. The phosphorylated Ezrin protein was localized primarily at the cell periphery, resulting in filamentous actin (F-actin) rearrangement, followed by a significant decrease in TER and increase in fluxes of FITC-BSA. Moreover, FAK and RhoA signaling pathways were required in the phosphorylation of Ezrin protein, and the former positively regulated the latter. Conclusion: The phosphorylated Ezrin protein was induced by TNF-α via the FAK/RhoA signaling pathway leading to endothelial hyperpermeability in PMVECs.

Propofol-induced damage to the blood-brain barrier endothelium evinces a vascular endothelial growth factor (VEGF) -dependent mechanism.

Background and Hypothesis: &#x0D; Propofol is an anesthetic commonly used to induce general anesthesia for a myriad of medical procedures. However, a growing corpus of evidence suggests that propofol-induced increases in VEGF may contribute to blood-brain barrier (BBB) leakiness in varying animal models. The BBB is a neurovascular structure which protects the central nervous system from pathogens, toxins, and other deleterious metabolites; therefore, considerations regarding BBB integrity in humans are indispensable to the practice of anesthesia. We hypothesize that propofol-induced BBB dysfunction in human models is partially mediated by an increase in VEGF expression. &#x0D; &#x0D; Methods: &#x0D; We utilized human induced pluripotent stem cells (hiPSC) to derive brain microvascular endothelial cells (BMECs)—the barrier forming cell type of the BBB. BMECs were then subjected to clinically relevant doses of propofol for 3 hours, and barrier integrity was monitored via transendothelial electrical resistance (TEER) and para-cellular permeability for up to 72 hours. Propofol-induced VEGF levels were determined with an ELISA assay. Axitinib, a VEGF receptor blocker, was further utilized to assess the role of VEGF in propofol-induced BBB breakdown. &#x0D; &#x0D; Results: &#x0D; Prior works, including this study, have shown that propofol induces BBB damage, as demonstrated by decreases in TEER; here, preliminary work with ELISA assays further suggest that BMECs treated with propofol demonstrate an upregulation of VEGF. Pretreatment of BMECs with Axitinib before the addition of propofol partially rescues TEER and thus attenuates the propofol-mediated diminution of TEER. These observations thereby implicate VEGF as a damage mediator after propofol treatment. &#x0D; &#x0D; Conclusion and Potential Impact: &#x0D; This study utilized an in vitro model to demonstrate that propofol may mediate, in part, damage to blood- brain barrier endothelium via a VEGF dependent mechanism; thus, this work may guide future investigations to facilitate the development of safer anesthetic alternatives, or towards additional pharmacologic interventions that counteract propofol-mediated damage during anesthetic induction.

CXCL13 Is Involved in the Lipopolysaccharide-Induced Hyperpermeability of Umbilical Vein Endothelial Cells

Sepsis is a disease that is characterized by a severe systemic inflammatory response to microbial infection and lipopolysaccharide (LPS) and is a well-known inducer of sepsis, as well as endothelial cell hyperpermeability. In the present study, we confirm the elevation of CXC chemokine ligand 13 (CXCL13) in sepsis patients. We also show that LPS exposure increases the release of CXCL13, as well as the mRNA and protein expression of CXCL13 and its receptor, CXC chemokine receptor 5 (CXCR5) in human umbilical vein endothelial cells (HUVECs) in a dose- and time-dependent manner. We also examined the effects of CXCL13 knockdown on LPS-mediated endothelial hyperpermeability and tight junction (TJ) protein expression in HUVECs. Our results show that HUVECs exposed to LPS result in a significant decrease in transendothelial electrical resistance (TER) and TJ protein (Zonula occluden-1, occludin, and claudin-4) expression, and a notable increase in fluorescein isothiocyanate (FITC)-dextran flux and p38 phosphorylation, which was partially reversed by CXCL13 knockdown. Recombinant CXCL13 treatment had a similar effect as LPS exposure, which was attenuated by a p38 inhibitor, SB203580. Moreover, the CXCL13-neutralizing antibody significantly increased the survival rate of LPS-induced sepsis mice. Collectively, our results show that CXCL13 plays a key role in LPS-induced endothelium hyperpermeability via regulating p38 signaling and suggests that therapeutically targeting CXCL13 may be beneficial for the treatment of sepsis.

Outgrowth endothelial cells form a functional cerebral barrier and restore its integrity after damage.

Breakdown of blood-brain barrier, formed mainly by brain microvascular endothelial cells (BMECs), represents the major cause of mortality during early phases of ischemic strokes. Hence, discovery of novel agents that can effectively replace dead or dying endothelial cells to restore blood-brain barrier integrity is of paramount importance in stroke medicine. Although endothelial progenitor cells (EPCs) represent one such agents, their rarity in peripheral blood severely limits their adequate isolation and therapeutic use for acute ischemic stroke which necessitate their ex vivo expansion and generate early EPCs and outgrowth endothelial cells (OECs) as a result. Functional analyses of these cells, in the present study, demonstrated that only OECs endocytosed DiI-labelled acetylated low-density lipoprotein and formed tubules on matrigel, prominent endothelial cell and angiogenesis markers, respectively. Further analyses by flow cytometry demonstrated that OECs expressed specific markers for stemness (CD34), immaturity (CD133) and endothelial cells (CD31) but not for hematopoietic cells (CD45). Like BMECs, OECs established an equally tight in vitro model of human BBB with astrocytes and pericytes, suggesting their capacity to form tight junctions. Ischemic injury mimicked by concurrent deprivation of oxygen and glucose (4 hours) or deprivation of oxygen and glucose followed by reperfusion (20 hours) affected both barrier integrity and function in a similar fashion as evidenced by decreases in transendothelial electrical resistance and increases in paracellular flux, respectively. Wound scratch assays comparing the vasculoreparative capacity of cells revealed that, compared to BMECs, OECs possessed a greater proliferative and directional migratory capacity. In a triple culture model of BBB established with astrocytes, pericytes and BMEC, exogenous addition of OECs effectively repaired the damage induced on endothelial layer in serum-free conditions. Taken together, these data demonstrate that OECs may effectively home to the site of vascular injury and repair the damage to maintain (neuro)vascular homeostasis during or after a cerebral ischemic injury.

Sphingosine 1-Phosphate Signaling Is Involved in Impaired Blood-Brain Barrier Function in Ischemia-Reperfusion Injury.

Sphingosine 1-phosphate (S1P) is a major bioactive lipid mediator in the vascular and immune system. Here, we have shown that inhibition of S1P signaling prevents blood-brain barrier (BBB) dysfunction after ischemia both in vitro and in vivo. In the in vitro BBB models, oxygen-glucose deprivation and reoxygenation (OGD/R) enhanced the expression of an S1P synthesizing enzyme (Sphk1) and S1P transporters (Abca1, Spns2), increasing S1P in culture media. Inhibitors of Sphk1 (SKI-II) or Abca1 (probucol) attenuated the decrease in transendothelial electrical resistance and the increase in permeability caused by OGD/R. In the middle cerebral artery occlusion and reperfusion (MCAO/R) model of mice, probucol administration after MCAO operation reduced the infarction area and vascular leakage, preserving the integrity of tight junction proteins. Furthermore, MCAO/R caused activation of STAT3, a downstream mediator of S1P signaling, which was suppressed by postoperative probucol administration. Accordingly, S1P activated STAT3, both in cultured vascular endothelial cells and pericytes, and STAT3 signaling inhibitor (Stattic) protected BBB dysfunction in OGD/R-treated in vitro BBB models. These results suggest that inhibition of S1P signaling is a strategy to treat BBB impairment after cerebral ischemia and highlight the potential alternative use of probucol, a classical anti-hyperlipidemic drug, for emergency treatment of stroke.

Panax notoginseng saponins suppress lipopolysaccharide-induced barrier disruption and monocyte adhesion on bEnd.3 cells via the opposite modulation of Nrf2 antioxidant and NF-κB inflammatory pathways.

Dysfunction of the blood-brain barrier (BBB) is a prerequisite for the pathogenesis of many cerebral diseases. Oxidative stress and inflammation are well-known factors accounting for BBB injury. Panax notoginseng saponins (PNS), a clinical commonly used drug against cerebrovascular disease, possess efficient antioxidant and anti-inflammatory activity. In the present study, the protective effects of PNS on lipopolysaccharide (LPS)-insulted cerebral microvascular endothelial cells (bEnd.3) were assessed and the underlying mechanisms were investigated. The results showed that PNS mitigated the decrease of Trans-Endothelial Electrical Resistance, increase of paracellular permeability, and loss of tight junction proteins in bEnd.3 BBB model. Meanwhile, PNS suppressed the THP-1 monocytes adhesion on bEnd.3 monolayer. Moreover, PNS prevented the pro-inflammatory cytokines secretion and reactive oxygen species generation in bEnd.3 cells stimulated with LPS. Mechanism investigations suggested that PNS promoted the Akt phosphorylation, activated Nrf2 antioxidant signaling, and inhibited the NF-κB activation. All the effects of PNS could be abolished by PI3K inhibition at different levels. Taken together, these observations suggest that PNS may act as an extrinsic regulator that activates Nrf2 antioxidant defense system depending on PI3K/Akt and inhibits NF-κB inflammatory signaling to attenuate LPS-induced BBB disruption and monocytes adhesion on cerebral endothelial cells in vitro.

Pericytes Suppress Brain Metastasis from Lung Cancer In Vitro.

Metastasis of lung cancer to the brain is associated with poor outcomes and limited therapeutic options. The blood-brain barrier (BBB) plays a major role in brain metastasis. However, little is known about the role of pericytes in brain metastasis formation. This study aimed to reveal the interaction between pericytes and cancer cells. We established in vitro BBB models with rat primary cultured BBB-related cells (endothelial cells, astrocytes, and pericytes) and investigated the relationship between BBB-related cells and metastatic cancer cell lines. We observed a significant decrease in transendothelial electrical resistance with metastatic cancer cells in monolayer and coculture models with astrocytes. In contrast, the coculture model with pericytes showed inhibition of the decrease in transendothelial electrical resistance with metastatic cancer cells. In addition, the expression of tight junction protein was preserved only in the coculture model with pericytes. The conditioned medium of pericytes with metastatic cancer cells suppressed the proliferation of the cancer cells significantly. This study revealed that brain pericytes are the major regulators of the resistance of the BBB to lung cancer metastasis to the brain. Pericytes exert an anti-metastatic effect and thus have potential for the preventive treatment of brain metastasis.