Transendothelial Electrical Resistance Values Research Articles

Derivation of Brain Capillary-like Endothelial Cells from Human Pluripotent Stem Cell-Derived Endothelial Progenitor Cells.

SummaryThe derivation of human brain capillary endothelial cells is of utmost importance for drug discovery programs focusing on diseases of the central nervous system. Here, we describe a two-step differentiation protocol to derive brain capillary-like endothelial cells from human pluripotent stem cells. The cells were initially differentiated into endothelial progenitor cells followed by specification into a brain capillary-like endothelial cell phenotype using a protocol that combined the induction, in a time-dependent manner, of VEGF, Wnt3a, and retinoic acid signaling pathways and the use of fibronectin as the extracellular matrix. The brain capillary-like endothelial cells displayed a permeability to lucifer yellow of 1 × 10−3 cm/min, a transendothelial electrical resistance value of 60 Ω cm2 and were able to generate a continuous monolayer of cells expressing ZO-1 and CLAUDIN-5 but moderate expression of P-glycoprotein. Further maturation of these cells required coculture with pericytes. The study presented here opens a new approach for the study of soluble and non-soluble factors in the specification of endothelial progenitor cells into brain capillary-like endothelial cells.

The use of bacterial cellulose as a basement membrane improves the plausibility of the static in vitro blood-brain barrier model

There are several blood-brain barrier (BBB) models available for pharmaceutical research, but none of those are able to properly imitate the permeability of this special barrier. In this study, it is aimed to produce different BBB models with different cellular combinations and different basement membrane polymers, such as polyethylene terephthalate (PET) and bacterial cellulose (BC), which has not been used for BBB models before, to compare their barrier properties. Primary human brain microvascular endothelial cells were seeded on the luminal side and primary human astrocytes and/or primary human brain microvascular pericytes were seeded on the abluminal side of the membranes. Immunofluorescence (IF) staining results indicate that the expression of tight and adherence junction proteins increases on the 5th day of the cultivation. In accordance with Live-Dead staining results, IF images show that cells in the model lose their viability before the 10th day. Transendothelial electrical resistance (TEER) measurements indicate that BC membrane leads to statistically higher (p < 0.05) TEER values than the standard Transwell PET insert membrane. Sucrose and caffeine permeability values of all models are close to in vivo values. BC shows potential to be used as a more reliable basement membrane for BBB models for pharmaceutical research.

Establishment of a Human iPSC- and Nanofiber-Based Microphysiological Blood-Brain Barrier System.

The blood-brain barrier (BBB) is an active and complex diffusion barrier that separates the circulating blood from the brain and extracellular fluid, regulates nutrient transportation, and provides protection against various toxic compounds and pathogens. Creating an in vitro microphysiological BBB system, particularly with relevant human cell types, will significantly facilitate the research of neuropharmaceutical drug delivery, screening, and transport, as well as improve our understanding of pathologies that are due to BBB damage. Currently, most of the in vitro BBB models are generated by culturing rodent astrocytes and endothelial cells, using commercially available transwell membranes. Those membranes are made of plastic biopolymers that are nonbiodegradable, porous, and stiff. In addition, distinct from rodent astrocytes, human astrocytes possess unique cell complexity and physiology, which are among the few characteristics that differentiate human brains from rodent brains. In this study, we established a novel human BBB microphysiologocal system, consisting of a three-dimensionally printed holder with a electrospun poly(lactic- co-glycolic) acid (PLGA) nanofibrous mesh, a bilayer coculture of human astrocytes, and endothelial cells, derived from human induced pluripotent stem cells (hiPSCs), on the electrospun PLGA mesh. This human BBB model achieved significant barrier integrity with tight junction protein expression, an effective permeability to sodium fluorescein, and higher transendothelial electrical resistance (TEER) comparing to electrospun mesh-based counterparts. Moreover, the coculture of hiPSC-derived astrocytes and endothielial cells promoted the tight junction protein expression and the TEER value. We further verified the barrier functions of our BBB model with antibrain tumor drugs (paclitaxel and bortezomib) and a neurotoxic peptide (amyloid β 1-42). The human microphysiological system generated in this study will potentially provide a new, powerful tool for research on human BBB physiology and pathology.

Functional brain-specific microvessels from iPSC-derived human brain microvascular endothelial cells: the role of matrix composition on monolayer formation

BackgroundTranswell-based models of the blood–brain barrier (BBB) incorporating monolayers of human brain microvascular endothelial cells (dhBMECs) derived from induced pluripotent stem cells show many of the key features of the BBB, including expression of transporters and efflux pumps, expression of tight junction proteins, and physiological values of transendothelial electrical resistance. The fabrication of 3D BBB models using dhBMECs has so far been unsuccessful due to the poor adhesion and survival of these cells on matrix materials commonly used in tissue engineering.MethodsTo address this issue, we systematically screened a wide range of matrix materials (collagen I, hyaluronic acid, and fibrin), compositions (laminin/entactin), protein coatings (fibronectin, laminin, collagen IV, perlecan, and agrin), and soluble factors (ROCK inhibitor and cyclic adenosine monophosphate) in 2D culture to assess cell adhesion, spreading, and barrier function.ResultsCell coverage increased with stiffness of collagen I gels coated with collagen IV and fibronectin. On 7 mg mL−1 collagen I gels coated with basement membrane proteins (fibronectin, collagen IV, and laminin), cell coverage was high but did not reliably reach confluence. The transendothelial electrical resistance (TEER) on collagen I gels coated with basement membrane proteins was lower than on coated transwell membranes. Agrin, a heparin sulfate proteoglycan found in basement membranes of the brain, promoted monolayer formation but resulted in a significant decrease in transendothelial electrical resistance (TEER). However, the addition of ROCK inhibitor, cAMP, or cross-linking the gels to increase stiffness, resulted in a significant improvement of TEER values and enabled the formation of confluent monolayers.ConclusionsHaving identified matrix compositions that promote monolayer formation and barrier function, we successfully fabricated dhBMEC microvessels in cross-linked collagen I gels coated with fibronectin and collagen IV, and treated with ROCK inhibitor and cAMP. We measured apparent permeability values for Lucifer yellow, comparable to values obtained in the transwell assay. During these experiments we observed no focal leaks, suggesting the formation of tight junctions that effectively block paracellular transport.

Effect of heterogeneous ribonucleoprotein A2/B1 on lipopolysaccharide-induced permeability of vascular endothelial cells

Objective To investigate the effect of heterogeneous ribonucleoprotein A2/B1 (hnRNP A2/B1) on the human umbilical vein endothelial cells (HUVECs) permeability induced by lipopolysaccharide(LPS). Methods HUVECs were used in this study, small interfering RNA (siRNA) with hnRNP A2/B1 gene silencing was screened. The samples were divided into four groups: NC siRNA group, hnRNP A2/B1 siRNA group, NC siRNA-LPS group and hnRNP A2/B1 siRNA-LPS group. The transendothelial electrical resistance (TEER) of HUVECs was detected at 0, 6, 12 h and 24 h. The expression of vascular endothelial cadherin (VE-cadherin) and intercellular adhesion molecule-1 (ICAM-1) were measured at 12 h. Results After LPS treatment, the TEER values of HUVECs in hnRNP A2/B1 siRNA-LPS group was significantly decreased compared with values of NC siRNA-LPS group at 12 h (P<0.05). The expression level of VE-cadherin in hnRNP A2/B1 siRNA-LPS group was lower than the level in the NC siRNA-LPS group, and the expression level of ICAM-1 was increased in hnRNP A2/B1 siRNA-LPS group compared with the level of NC siRNALPS group(P<0.05). Conclusions hnRNP A2/B1 may play an important role in vascular endothelial permeability via regulating the expression of VE-cadherin and ICAM-1, thus protecting vascular endothelial barrier function. Key words: Heterogeneous ribonucleoprotein A2/B1; Lipopolysaccharide; Endothelial permeability; Vascular endothelial cadherin

DT-13 Ameliorates TNF-α-Induced Vascular Endothelial Hyperpermeability via Non-Muscle Myosin IIA and the Src/PI3K/Akt Signaling Pathway.

DT-13(25(R,S)-ruscogenin-1-O-[β-d-glucopyranosyl-(1→2)][β-d-xylopyranosyl-(1→3)]-β-d-fucopyranoside) has been identified as an important factor in TNF-α-induced vascular inflammation. However, the effect of DT-13 on TNF-α-induced endothelial permeability and the potential molecular mechanisms remain unclear. Hence, this study was undertaken to elucidate the protective effect of DT-13 on TNF-α-induced endothelial permeability and the underlying mechanisms in vivo and in vitro. The in vivo results showed that DT-13 could ameliorate endothelial permeability in mustard oil-induced plasma leakage in the skin and modulate ZO-1 organization. In addition, the in vitro results showed that pretreatment with DT-13 could increase the transendothelial electrical resistance value and decrease the sodium fluorescein permeability coefficient. Moreover, DT-13 altered the mRNA and protein levels of ZO-1 as determined by real-time PCR, Western blotting, and immunofluorescence analyses. DT-13 treatment decreased the phosphorylations of Src, PI3K, and Akt in TNF-α-treated human umbilical vein endothelial cells (HUVECs). Further analyses with PP2 (10 µM, inhibitor of Src) indicated that DT-13 modulated endothelial permeability in TNF-α-induced HUVECs in an Src-dependent manner. LY294002 (10 µM, PI3K inhibitor) also had the same effect on DT-13 but did not affect phosphorylation of Src. Following decreased expression of non-muscle myosin IIA (NMIIA), the effect of DT-13 on the phosphorylations of Src, PI3K, and Akt was abolished. This study provides pharmacological evidence showing that DT-13 significantly ameliorated the TNF-α-induced vascular endothelial hyperpermeability through modulation of the Src/PI3K/Akt pathway and NMIIA, which play an important role in this process.

Hydrocortisone and Ascorbic Acid Synergistically Prevent and Repair Lipopolysaccharide-Induced Pulmonary Endothelial Barrier Dysfunction

Sepsis refers to the dysregulated host immune response elicited by microbial infections resulting in life-threatening organ dysfunction. Sepsis represents a medical challenge, since it is associated with a rate of death as high as 60%. Septic shock is strongly associated with vascular dysfunction and elevated pulmonary capillary permeability. We recently reported that the combination of hydrocortisone (HC), ascorbic acid (vitC), and thiamine dramatically improves outcomes and reduces mortality in patients with sepsis. In the present study, we provide experimental evidence in support of the hypothesis that the combination of HC and vitC enhances endothelial barrier function. Human lung microvascular endothelial cells were exposed to lipopolysaccharide (LPS) in the absence or presence of HC and vitC. LPS alone induced profound hyperpermeability, as reflected in decreased values oftransendothelial electrical resistance. vitC alone did not exhibit barrierenhancement properties nor did it affect the LPS-induced hyperpermeability. Similarly, HC alone exhibited only a minor barrier-enhancing and protective effect. Conversely, the combination of HC andvitC, either as before or after treatment, dramatically reversed the LPS-induced barrier dysfunction. The barrier-protective effects of HC andvitC were associated with reversal of LPS-induced p53 and phosphorylated cofilin downregulation and LPS-induced RhoA activation and myosin light chain phosphorylation. These data provide a novel mechanism of endothelial barrier protection and suggest one possible pathway that may contribute to the therapeutic effects of HC and vitC in patients with sepsis.

Abstract 4330: Astrocytic S1P3 regulates blood-brain/tumor barrier permeability

Abstract Introduction: Incidence of breast cancer brain metastasis is increasing owing to prolonged life-span and better detection techniques. Prognosis of patients with brain metastases is extremely poor with median survival time of one year. One of the major impediments in treating brain metastases is presence of blood-brain/tumor barrier that limits the permeability of chemotherapeutic drugs into the brain parenchyma. Understanding the mechanisms that regulate blood-brain/tumor barrier permeability in context of brain metastases is imperative to developing successful therapy. Methods: Mouse models with brain-tropic sublines of MDA-MB-231 (231), JIMT-1, and SUM190 were used to generate breast cancer brain metastases. Using laser capture microscopy, the permeable and non-permeable lesions from mouse brains were isolated and profiled for gene expression using microarray. Immortalized human brain endothelial cells, astrocytes, and pericytes were used for developing in vitro blood-brain/tumor barrier model along with spheres generated with 231-BR6 or JIMT-1-BR3 cells. Secreted cytokines were evaluated using human cytokine profiler. Transendothelial electrical resistance (TEER) was measured using EVOM2 volt/ohm meter. Results: Gene expression profiling and immunostaining of mouse brains, harboring breast cancer metastases showed that astrocytes at permeable regions express elevated S1P3. Pharmacological inhibition of S1P3 using antagonist TY-52156 (10mg/kg) in mice bearing 231-BR6 brain metastases showed reduction in 3KDa Texas red dextran (TRD) uptake. To investigate the role of S1P3 in regulating barrier permeability, we established in vitro blood-brain/tumor barrier models. Treatment of astrocytes with TY-52156 (2μM) significantly increased mean TEER values (33.9 to 55.8 Ω.cm2; p&amp;lt;0.001, after 24 hrs), while there was a decrease in permeability for TRD (1.9 fold; p&amp;lt;0.0001) and doxorubicin (1.3 folds; p&amp;lt;0.05). Immunostaining on endothelial monolayer showed increased membranous ZO-1 and VE-cadherin expression. The dynamics of increase in TEER was faster when 231-BR6 spheres were included. We observed similar results when S1P3 was knocked down using shRNA. Astrocytes with down-modulated S1P3 showed decreased secretion of various cytokines including IL-6, IL-8, CCL2, CXCL1, and GM-CSF. Inhibition of these cytokines individually using neutralizing antibodies recapitulated the effects of S1P3 inhibition, while treatment of endothelial monolayer with activated cytokines increased the permeability. This study provides a proof of concept for role of S1P3 and downstream cytokine signaling in regulating blood-brain/tumor barrier permeability in breast cancer brain metastases. Conclusion: Our study shows that astrocytic S1P3 regulates blood-brain/tumor barrier permeability in breast cancer brain metastases by modulating cytokine secretion. This observation might lead to discovery of novel strategies for augmenting drug efficacy. Citation Format: Anurag N. Paranjape, Brunilde Gril, Stephan Woditschka, Emily Hua, Jeffrey C. Hanson, Xiaolin Wu, Renata Duchnowska, Priscilla K. Brastianos, David L. Liewehr, Seth M. Steinberg, Cody Peer, William D. Figg, Gary T. Pauly, Christina Robinson, Joel P. Schneider, Patricia S. Steeg. Astrocytic S1P3 regulates blood-brain/tumor barrier permeability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4330. doi:10.1158/1538-7445.AM2017-4330

Abstract 628: Circulating Exosomes Isolated From Septic Mice Induce Endothelial Hyperpermeability Through Promoting Podosome Cluster Formation

Introduction: Septic shock increases vascular permeability, leading to multiple organ failure and higher mortality. Reactive oxygen species (ROS) have been shown to promote both actin cytoskeleton reorganization resulting in vascular leakage and the formation of podosome, an actin-based dynamic membrane structure. Interestingly, recent studies have shown that circulating exosomes from septic patients contained higher levels of ROS than healthy ones. In this study, we hypothesized that septic exosomes can transfer exosomal ROS to endothelial cell (EC) to promote the generation of podosomes, leading to hyperpermeability. Methods: C57BL/6 mice (20-25g) were subjected to CLP surgery or injection with LPS (25 μg/g). Sham-operated or PBS-treated mice were used as controls. Exosomes were isolated from sera, collected at 3 h post-CLP or post-LPS-injection. Podosomes were identified by co-immunostaining F-actin/cortactin following stimulation with PMA, thrombin and exosomes. Transendothelial electrical resistance (TEER) analysis was performed to monitor the change of EC monolayer permeability. The ROS levels in exosomes and ECs were measured by using a ROS-Glo H 2 O 2 Assay kit. Results: First, we observed that thrombin and PMA both stimulated podosome cluster formation at the cell periphery in (30-36%, 90-108 of 300) ECs, which correlated well with reduction (40.5-50.7%, 6-7.5 of 14.8) in TEER values (n=4, p&lt;0.01). Next, we discovered that septic exosomes collected at 3 h post-CLP or post-LPS-injection contained significantly higher amount of H 2 O 2 which can be transferred into ECs, leading to 1.5-fold increase of H 2 O 2 in ECs (n=6, p&lt;0.01). Moreover, treatment of ECs with septic exosomes remarkably increased the percentage of cells carrying podosome clusters (20.3%, 51 of 251) compared to controls (9.5%, 24 of 252 n=4, p&lt;0.01). Meanwhile, the TEER value was reduced by 27.6% (3.4 of 12.3) upon septic exosome stimulation (n=4, p&lt;0.01). By contrast, scavenging H 2 O 2 in septic exosomes significantly attenuated their effects on podosome cluster formation and EC monolayer TEER. Conclusion: septic exosomes can stimulate the formation of podosome clusters in ECs through transferring exosomal H 2 O 2, leading to endothelial hyperpermeability.

Microfluidic blood-brain barrier model provides in vivo-like barrier properties for drug permeability screening.

Efficient delivery of therapeutics across the neuroprotective blood-brain barrier (BBB) remains a formidable challenge for central nervous system drug development. High-fidelity in vitro models of the BBB could facilitate effective early screening of drug candidates targeting the brain. In this study, we developed a microfluidic BBB model that is capable of mimicking in vivo BBB characteristics for a prolonged period and allows for reliable in vitro drug permeability studies under recirculating perfusion. We derived brain microvascular endothelial cells (BMECs) from human induced pluripotent stem cells (hiPSCs) and cocultured them with rat primary astrocytes on the two sides of a porous membrane on a pumpless microfluidic platform for up to 10 days. The microfluidic system was designed based on the blood residence time in human brain tissues, allowing for medium recirculation at physiologically relevant perfusion rates with no pumps or external tubing, meanwhile minimizing wall shear stress to test whether shear stress is required for in vivo-like barrier properties in a microfluidic BBB model. This BBB-on-a-chip model achieved significant barrier integrity as evident by continuous tight junction formation and in vivo-like values of trans-endothelial electrical resistance (TEER). The TEER levels peaked above 4000 Ω · cm2 on day 3 on chip and were sustained above 2000 Ω · cm2 up to 10 days, which are the highest sustained TEER values reported in a microfluidic model. We evaluated the capacity of our microfluidic BBB model to be used for drug permeability studies using large molecules (FITC-dextrans) and model drugs (caffeine, cimetidine, and doxorubicin). Our analyses demonstrated that the permeability coefficients measured using our model were comparable to in vivo values. Our BBB-on-a-chip model closely mimics physiological BBB barrier functions and will be a valuable tool for screening of drug candidates. The residence time-based design of a microfluidic platform will enable integration with other organ modules to simulate multi-organ interactions on drug response. Biotechnol. Bioeng. 2017;114: 184-194. © 2016 Wiley Periodicals, Inc.

YiQiFuMai Powder Injection ameliorates the oxygen-glucose deprivation-induced brain microvascular endothelial barrier dysfunction associated with the NF-κB and ROCK1/MLC signaling pathways

Ethnopharmacological relevanceYiQiFuMai Powder Injection (YQFM) is a modern preparation derived from Sheng-mai San, a traditional Chinese prescription, widely used for the treatment of cardiovascular and cerebrovascular diseases. However, its potential molecular mechanism remains unclear. Aim of the studyThe present study was designed to observe the effect of YQFM on oxygen-glucose deprivation (OGD)-induced the brain microvascular endothelial barrier dysfunction and to explore the underlying pathways in vitro. MethodsA mouse brain microvascular endothelial cell line (bEnd.3) was subjected to OGD (2–9h) to examine the efficacy and molecular mechanisms in the presence or absence of YQFM (100, 200 and 400 μg/ml). ResultsThe results of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Trans-endothelial electrical resistance (TEER) assays demonstrated that treatment with YQFM increased the cell viability and TEER value, decreased even blue (EB) albumin leakage after OGD in bEnd.3 cells. Western blotting and immunofluorescence staining showed that YQFM reduced the breakage and translocation of Zonula occludens-1 (ZO-1) and claudin-5 after 4h of OGD and decreased the expression of these proteins after 9h of OGD. Moreover, YQFM significantly inhibited the expression, phosphorylation and nuclear translocation of NF-κB/p65 and decreased the expression of intercellular adhesionmolecule-1 (ICAM-1) and cyclooxygenase (COX-2) as well as production of nitric oxide (NO). In addition, real time-PCR results revealed that YQFM suppressed the mRNA levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) after 4h of OGD. Furthermore, YQFM markedly inhibited both the phosphorylation of myosin light chain (MLC) and cytoskeletal reorganization and reduced the expression of cleaved-ROCK1 in bEnd.3 cells subjected to OGD. ConclusionThese findings suggest that YQFM ameliorates the OGD-induced brain microvascular endothelial cell barrier disruption associated with the NF-κB/p65 and ROCK1/MLC signaling pathways. These data provide new insights into the use of this preparation for treating cerebrovascular diseases.

MiRNA-200b Regulates RMP7-Induced Increases in Blood-Tumor Barrier Permeability by Targeting RhoA and ROCKII.

The primary goals of this study were to investigate the potential roles of miR-200b in regulating RMP7-induced increases in blood-tumor barrier (BTB) permeability and some of the possible molecular mechanisms associated with this effect. Microarray analysis revealed 34 significantly deregulated miRNAs including miR-200b in the BTB as induced by RMP7 and 8 significantly up-regulated miRNAs in the BTB by RMP7. RMP7 induced tight junction (TJ) opening of the BTB, thereby increasing BTB permeability. Associated with this effect of RMP7 was a decrease in miR-200b expression within the human cerebral microvascular endothelial cells line hCMEC/D3 (ECs) of the BTB. Overexpression of miR-200b inhibited endothelial leakage and restored normal transendothelial electric resistance values. A simultaneous shift in occludin and claudin-5 distributions from insoluble to soluble fractions were observed to be significantly reduced. In addition, overexpression of miR-200b inhibited the relocation of occludin and claudin-5 from cellular borders into the cytoplasm as well as the production of stress fiber formation in GECs (ECs with U87 glioma cells co-culturing) of the BTB. MiR-200b silencing produced opposite results as that obtained from that of the miR-200b overexpression group. Overexpression of miR-200b was also associated with a down-regulation in RhoA and ROCKII expression, concomitant with a decrease in BTB permeability. Again, results which were opposite to that obtained with the miR-200b silencing group. We further found that miR-200b regulated BTB permeability by directly targeting RhoA and ROCKII. Collectively, these results suggest that miR-200b's contribution to the RMP7-induced increase in BTB permeability was associated with stress fiber formation and TJ disassembly as achieved by directly targeting RhoA and ROCKII.

Heat shock protein A12B protects against sepsis-induced impairment in vascular endothelial permeability

BackgroundAs a common and life-threatening infectious syndrome, sepsis contributes significantly to morbidity and mortality in clinical settings. Vascular endothelial injury and hyperpermeability play an important role in the development of sepsis-induced organ dysfunction. Heat shock protein A12B (HSPA12B) is one of the HSP70 superfamily members and is mainly expressed in vascular endothelial cells. The present study was performed to investigate the role of HSPA12B in endothelial barrier dysfunction during sepsis. MethodsHuman umbilical vein endothelial cells (HUVECs) were stimulated with 1 μg/mL of lipopolysaccharide (LPS) and harvested at 0, 3, 6, 9, 12, and 24 h. The messenger RNA and protein levels of HSPA12B were detected by Real Time-polymerase chain reaction and Western blot. Upregulation of HSPA12B was induced by transfection of pIRES2-EGFP plasmid carrying the HSPA12B complementary DNA. The in vitro effect of HSPA12B overexpression on endothelial permeability was manifested by the transendothelial electrical resistance value, expression of the adhesion molecules VE-cadherin, and the level of permeability-related kinase myosin light chain, SRC, and CDC42. Mice received cecal ligation and puncture surgery followed by nasal inhalation of nano-polymer–mediated siRNA. Lung endothelial permeability was assessed via intrajugular vein injection of Evans Blue 30 h after cecal ligation and puncture. ResultsAfter LPS induction, the messenger RNA and protein level of HSPA12B in HUVECs increased and peaked at 12 h, whereas they returned to the baseline level at 24 h. Overexpression of HSPA12B can reduce the permeability of HUVEC stimulated by LPS in vitro, while increasing the expression of VE-Cadherin, myosin light chain, and CDC42. On the other hand, downregulating the expression of HSPA12B can significantly increase lung permeability in mice with sepsis-induced vascular injury. ConclusionsHSPA12B plays a protective role in vascular endothelial barrier dysfunction by preserving the endothelial permeability during sepsis.

Roles of NMDARs in maintenance of the mouse cerebrovascular endothelial cell-constructed tight junction barrier

Glutamate can activate NMDA receptor (NMDAR) and subsequently induces excitotoxic neuron loss. However, roles of NMDARs in the blood-brain barrier (BBB) are little known. This study used a mouse cerebrovascular endothelial cell (MCEC) model to evaluate the effects of NMDAR activation on maintenance of the BBB and its possible mechanisms. Analysis of confocal microscopy revealed expressions of NMDAR subunits, GluN1 and GLUN2B, in MCECs. An immunoblot assay further showed the existence of GluN1 in plasma membranes of MCECs. In brain tissues, a confocal microscopic analysis demonstrated co-localization of GluN1 and factor VIII, a biomarker of MCECs. In addition, GluN1 mRNA was detected in MCECs and the brain. Functional assays showed that exposure of MCECs to NMDA increased calcium influx. Separately, NMDA suppressed transendothelial electrical resistance values, levels of occludin, and occludin tight junctions. As to the mechanism, NMDA stimulated sequential phosphorylations of extracellular signal-regulated kinase (ERK)1/2 and mitogen-activated ERK (MEK)1. Interestingly, amounts of matrix metalloproteinase (MMP)2 and MMP9 in MCECs were augmented by NMDA. The NMDA-induced alterations in ERK1/2 phosphorylation and occludin levels were reversed by pretreatment with PD98059, a MEK inhibitor, and MK-801, a NMDAR antagonist, respectively. Therefore, this study shows the functional presence of NMDARs in MCECs, and NMDAR activation can disrupt the MCEC-constructed tight junction barrier via activation of the MEK1/2-ERK1/2 signaling pathway and upregulation of MMP2/9 expressions.

The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on the development and function of the blood–brain barrier

Prenatal exposure to environmental chemicals such as dioxins is known to have adverse effects on the developing central nervous system (CNS) in mammals. Because the fetal blood–brain barrier (BBB) is immature, dioxins are thought to exert their toxic effects on the CNS by crossing the BBB and acting on neural cells directly. However, little is known whether dioxins alter the BBB. In this study, to investigate the effects of dioxins on BBB function, we exposed an in vitro BBB system comprising rat endothelial cells, astrocytes, and pericytes to the toxicant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) either before or after BBB formation. We assessed BBB permeability and the function of tight junctions by measuring transendothelial electric resistance (TEER) values following exposure. Subsequently, total RNA and proteins were obtained from the cells for analysis. TEER values following TCDD exposure before but not after BBB formation were lower than those of the control group. We also observed that the expression of the tight junction proteins ZO-1 and claudin-5 was suppressed following TCDD exposure. To examine the cause of this reduction in protein levels, we performed a real-time quantitative polymerase chain reaction assay and observed low expression of the glial cell line-derived neurotrophic factor (GDNF) mRNA in the exposed groups. Moreover, to rescue the effects of TCDD, we applied extrinsic GDNF with TCDD. The several disruptions caused by TCDD were rescued by the GDNF addition. Our findings suggest that exposure to TCDD during BBB formation disrupts and impairs BBB function in part by the suppression of GDNF action, which may contribute to the adverse effects of TCDD on the fetal CNS.

CBIO-21INFLUENCES OF BLOOD BRAIN BARRIER (BBB) BASEMENT MEMBRANE ECM MOLECULES ON HUMAN BRAIN VASCULAR PERICYTES

BACKGROUND: Pericytes are specialised cells found within the blood brain barrier (BBB) ensheathed by a basement membrane (BM) containing numerous extracellular matrix (ECM) proteins which regulate vessel stabilisation and barrier formation. Knowledge on ECM influence on pericyte behaviour is limited. This study aimed to determine the effects of BM-ECM components on pericyte proliferation, adhesion, motility and cell-to-cell contact. METHODS: Human brain vascular pericytes (HBVP) were used. Cell seeding number was optimised and cells were plated on different human-derived, previously optimised, ECM molecules including Fibronectin (5 µg/ml), Laminin (75 µg/ml), Collagen IV (10 µg/ml), Agrin (1 µg/ml) and Perlecan (10 µg/ml) and assessed for proliferation and both quantitative and qualitative adhesion. Motility assays were performed using live cell (time-lapse) imaging. Resistance values between pericytes were collated using an Electric Cell-Substrate Impedance Sensing (ECIS) system. Trans-Endothelial Electrical Resistance (TEER) changes when pericytes were seeded onto human endothelial cells under the different ECM culture conditions were also analysed by voltometer and ECIS. RESULTS: Cells adhered best when seeded on Perlecan and least well on Agrin (p < 0.05). Motility assays showed that pericytes travelled the longest distance and fastest on Agrin and least on Perlecan (p < 0.05). Higher resistance values were reached when cells were plated on Perlecan (∼400 Ω/cm2) and least on Agrin (∼300 Ω/cm2). A tighter barrier was noted when pericytes were co-cultured with endothelial cells (1200 Ω/cm2) compared to when pericytes were added to endothelial cell monolayers (1000 Ω/cm2). CONCLUSIONS: Results demonstrate that different BBB basement molecules have varied effects on TEER values, one indicator of BBB function. Findings also suggest that Agrin may function to recruit pericytes by migration while Perlecan may function to serve as an anchor. This study highlights the importance of BM-ECM molecules and suggests a concentration dependant role of the perlecan/agrin axis in BBB establishment and function.

Silver nanoparticles induce tight junction disruption and astrocyte neurotoxicity in a rat blood-brain barrier primary triple coculture model.

BackgroundSilver nanoparticles (Ag-NPs) can enter the brain and induce neurotoxicity. However, the toxicity of Ag-NPs on the blood–brain barrier (BBB) and the underlying mechanism(s) of action on the BBB and the brain are not well understood.MethodTo investigate Ag-NP suspension (Ag-NPS)-induced toxicity, a triple coculture BBB model of rat brain microvascular endothelial cells, pericytes, and astrocytes was established. The BBB permeability and tight junction protein expression in response to Ag-NPS, NP-released Ag ions, and polystyrene-NP exposure were investigated. Ultrastructural changes of the microvascular endothelial cells, pericytes, and astrocytes were observed using transmission electron microscopy (TEM). Global gene expression of astrocytes was measured using a DNA microarray.ResultsA triple coculture BBB model of primary rat brain microvascular endothelial cells, pericytes, and astrocytes was established, with the transendothelial electrical resistance values >200 Ω·cm2. After Ag-NPS exposure for 24 hours, the BBB permeability was significantly increased and expression of the tight junction (TJ) protein ZO-1 was decreased. Discontinuous TJs were also observed between microvascular endothelial cells. After Ag-NPS exposure, severe mitochondrial shrinkage, vacuolations, endoplasmic reticulum expansion, and Ag-NPs were observed in astrocytes by TEM. Global gene expression analysis showed that three genes were upregulated and 20 genes were downregulated in astrocytes treated with Ag-NPS. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that the 23 genes were associated with metabolic processes, biosynthetic processes, response to stimuli, cell death, the MAPK pathway, and so on. No GO term and KEGG pathways were changed in the released-ion or polystyrene-NP groups. Ag-NPS inhibited the antioxidant defense of the astrocytes by increasing thioredoxin interacting protein, which inhibits the Trx system, and decreasing Nr4a1 and Dusp1. Meanwhile, Ag-NPS induced inflammation and apoptosis through modulation of the MAPK pathway or B-cell lymphoma-2 expression or mTOR activity in astrocytes.ConclusionThese results draw our attention to the importance of Ag-NP-induced toxicity on the neurovascular unit and provide a better understanding of its toxicological mechanisms on astrocytes.

Effects of Muscone on the Expression of P-gp, MMP-9 on Blood-Brain Barrier Model In Vitro.

Muscone is the main chemical ingredient in Musk which is main crude drug in Tongqiaohuoxue decoction (TQHXD), and TQHXD has a protective effect on damaged neurons, so we hypothesize that muscone can alter blood-brain barrier (BBB) permeability via the modulation of P-glycoprotein (P-gp) and matrix metalloproteinase-9 (MMP-9) expression. In this study, astrocytes (AC) and human umbilical vein endothelial cells (ECV304) were co-cultured to simulate the BBB model in vitro. Leak testing, transmembrane resistance experiments, and BBB-specific enzyme testing were used to test whether the model was successful. Different concentrations of muscone permeating the BBB were detected by gas chromatography (GC). The change of the transendothelial electrical resistance (TEER) on the BBB in vitro after treating with muscone was detected by Millicell-ERS. The protein expression of P-gp, MMP-9 in normal, and oxygen/glucose deprivation (OGD) BBB model was determined by western blotting to inquire that the mechanism of muscone penetrates the BBB model in vitro. The results show that muscone was detected in the lower medium of the BBB model by GC; the values of TEER were no significant difference before and after muscone (8 μM) was added to the BBB model; the expression of P-gp significantly decreased after the BBB model treatment with muscone (4, 8, and 16 μM) for 24 h; the expression of P-gp and MMP-9 in different concentrations of muscone groups had different degrees of reduction compared with the BBB in the state of OGD. In conclusion, muscone could permeate the BBB model, and it was associated with the inhibition of P-gp and MMP-9 expression. An understanding of the mechanisms of muscone across the BBB is crucial to the development of therapeutic modalities for cerebral vascular diseases.

Fingolimod prevents blood-brain barrier disruption induced by the sera from patients with multiple sclerosis.

ObjectiveEffect of fingolimod in multiple sclerosis (MS) is thought to involve the prevention of lymphocyte egress from lymphoid tissues, thereby reducing autoaggressive lymphocyte infiltration into the central nervous system across blood-brain barrier (BBB). However, brain microvascular endothelial cells (BMECs) represent a possible additional target for fingolimod in MS patients by directly repairing the function of BBB, as S1P receptors are also expressed by BMECs. In this study, we evaluated the effects of fingolimod on BMECs and clarified whether fingolimod-phosphate restores the BBB function after exposure to MS sera.MethodsChanges in tight junction proteins, adhesion molecules and transendothelial electrical resistance (TEER) in BMECs were evaluated following incubation in conditioned medium with or without fingolimod/fingolimod-phosphate. In addition, the effects of sera derived from MS patients, including those in the relapse phase of relapse-remitting (RR) MS, stable phase of RRMS and secondary progressive MS (SPMS), on the function of BBB in the presence of fingolimod-phosphate were assessed.ResultsIncubation with fingolimod-phosphate increased the claudin-5 protein levels and TEER values in BMECs, although it did not change the amount of occludin, ICAM-1 or MelCAM proteins. Pretreatment with fingolimod-phosphate restored the changes in the claudin-5 and VCAM-1 protein/mRNA levels and TEER values in BMECs after exposure to MS sera.ConclusionsPretreatment with fingolimod-phosphate prevents BBB disruption caused by both RRMS and SPMS sera via the upregulation of claudin-5 and downregulation of VCAM-1 in BMECs, suggesting that fingolimod-phosphate is capable of directly modifying the BBB. BMECs represent a possible therapeutic target for fingolimod in MS patients.

Roundabout 4 regulates blood-tumor barrier permeability through the modulation of ZO-1, Occludin, and Claudin-5 expression.

The blood-tumor barrier (BTB) restricts the delivery of chemotherapeutic drug molecules to tumor tissues. We found that the endothelial cell (EC) receptor molecule Roundabout 4 (Robo4) is endogenously expressed in human brain microvascular ECs and that it is upregulated in a BTB model of glioma cocultured ECs. Knockdown of Robo4 in this BTB model increased permeability; short hairpin RNA targeting Robo4 (shRobo4) led to decreased transendothelial electric resistance values, increased BTB permeability, and downregulated expression of the EC tight junction proteins ZO-1, occludin, and claudin-5. Roundabout 4 influenced BTB permeability via binding with its ligand, Slit2. Short hairpin RNA targeting Robo4 also increased matrix metalloproteinase-9 (MMP-9) activity and expression in glioma cocultured ECs; pretreatment with the MMP inhibitor GM6001 partially blocked the effects of shRobo4 on the transendothelial electric resistance values and ZO-1 and occludin expression. Short hairpin RNA targeting Robo4 also upregulated the phosphorylation of Src and Erk1/2; the Src inhibitor PP2 and the Erk1/2 inhibitor PD98059 blocked shRobo4-mediated alteration in ZO-1 and occludin expression. Together, our results indicate that knockdown of Robo4 increased BTB permeability by reducing EC tight junction protein expression, and that the Src-Erk1/2-MMP-9 signal pathways are involved in this process. Thus, Robo4 may represent a useful future therapeutic target for enhancing BTB permeability.