Transendothelial Electrical Resistance Measurements Research Articles

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 Blood-Brain Barrier Co-Culture Model Mimicking the Neurovascular Unit Using Induced Pluripotent Stem Cells.

Human blood-brain barrier (BBB) in vitro models pose a promising tool in drug development and understanding of mechanistic regulations during health and disease. Human-induced pluripotent stem cells (hiPS cells) represent an unlimited cell source to generate functional cells of the neurovascular unit (NVU), independent of variations or limitations during isolation and in vitro cultivation. This unit describes the standardized 2-D differentiation of adherent hiPS cells into BBB endothelial cells and neuronal stem cells (NSCs). Both cell types are combined with primary astrocytes and pericytes to develop complex, physiological BBB in vitro models. The endothelial cells in the apical compartment of the transwell models are separated from the basolateral seeded co-culture mixture by a synthetic membrane, simplifying analyses. The barrier integrity and functionality of the endothelium is improved by the specific mixture of NVU niche cells, determined here by decrease in the paracellular permeability of sodium-fluorescein and transendothelial electrical resistance (TEER) measurement. © 2018 by John Wiley & Sons, Inc.

Function-Related Protein Expression in Fuchs Endothelial Corneal Dystrophy Cells and Tissue Models

Fuchs endothelial corneal dystrophy (FECD) is a corneal pathology that affects the endothelial cell's ability to maintain deturgescence, resulting in a progressive loss of corneal transparency. In this study, we investigated the expression of function-related proteins in corneal endothelial cells using FECD or healthy corneal endothelial cells, either in a cell culture two-dimensional model or in an engineered corneal endothelium three-dimensional tissue model. No statistically significant difference in gene regulation was observed for the function-related families ATP1, SLC4, SLC16, AQP, TJP, and CDH between the FECD and the healthy cell models. Similarly, no difference in barrier integrity (transendothelial electrical resistance measurements and permeability assays) was observed invitro between FECD and healthy cultured cells. Protein expression of the key function-related families was decreased for Na+/K+-ATPase α1 subunit, monocarboxylate transporters 1 and 4 in native exvivo end-stage FECD specimens, whereas it returned to levels comparable to that of healthy tissues in the engineered FECD model. These results indicate that cell expansion and tissue engineering culture conditions can generate a corneal endothelium from pathologic FECD cells, with levels of function-related proteins similar to that of healthy tissues. Overall, these results explain why it is possible to reform a functional endothelium using corneal endothelial cells isolated from nonfunctional FECD pathologic specimens.

A Three-Dimensional Arrayed Microfluidic Blood-Brain Barrier Model With Integrated Electrical Sensor Array.

The blood-brain barrier (BBB) poses a unique challenge to the development of therapeutics against neurological disorders due to its impermeabi-lity to most of the chemical compounds. Most in vitro BBB models have limitations in mimicking in vivo conditions and functions. Here, we show a co-culture microfluidic BBB-on-a-chip that provides interactions between neurovascular endothelial cells and neuronal cells across a porous polycarbonate membrane, which better mimics the in vivo conditions, as well as allows in vivo level shear stress to be applied. A 4 × 4 intersecting microchannel array forms 16 BBB sites on a chip, with a multielectrode array integrated to measure the transendothelial electrical resistance (TEER) from all 16 different sites, which allows label-free real-time analysis of the barrier function. Primary mouse endothelial cells and primary astrocytes were co-cultured in the chip while applying in vivo level shear stress. The chip allows the barrier function to be analyzed through TEER measurement, dextran permeability, as well as immunostaining. Co-culture between astrocytes and endothelial cells, as well as in vivo level shear stress applied, led to the formation of tighter junctions and significantly lower barrier permeability. Moreover, drug testing with histamine showed increased permeability when using only endothelial cells compared to almost no change when using co-culture. Results show that the developed BBB chip more closely mimics the in vivo BBB environment. The developed multisite BBB chip is expected to be used for screening drug by more accurately predicting their permeability through BBB as well as their toxicity.

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.

Measurement of Microvascular Endothelial Barrier Dysfunction and Hyperpermeability In Vitro.

Loss of microvascular endothelial barrier integrity leads to vascular hyperpermeability and vasogenic edema in a variety of disease processes including trauma, ischemia and sepsis. Understanding these principles gives valuable information on pathophysiology and therapeutic drug development. While animal models of traumatic and ischemic injuries are useful to understand vascular dysfunctions associated with such injuries, in vitro barrier integrity assays are reliable and helpful adjuncts to understand the cellular and molecular changes and signaling mechanisms that regulate barrier function. We describe here the endothelial monolayer permeability assay and transendothelial electrical resistance (TEER) measurement as in vitro methods to test changes in microvascular integrity and permeability. These in vitro assays are based on either the measurement of electrical resistance of the monolayer or the quantitative evaluation of fluorescently tagged molecules (e.g., FITC-dextran) that pass through the monolayerwhen there is damage or breakdown.

Real-time acquisition of transendothelial electrical resistance in an all-human, in vitro, 3-dimensional, blood-brain barrier model exemplifies tight-junction integrity.

The blood–brain barrier (BBB) consists of endothelial cells, astrocytes, and pericytes embedded in basal lamina (BL). Most in vitro models use nonhuman, monolayer cultures for therapeutic-delivery studies, relying on transendothelial electrical resistance (TEER) measurements without other tight-junction (TJ) formation parameters. We aimed to develop reliable, reproducible, in vitro 3-dimensional (3D) models incorporating relevant human, in vivo cell types and BL proteins. The 3D BBB models were constructed with human brain endothelial cells, human astrocytes, and human brain pericytes in mono-, co-, and tricultures. TEER was measured in 3D models using a volt/ohmmeter and cellZscope. Influence of BL proteins—laminin, fibronectin, collagen type IV, agrin, and perlecan—on adhesion and TEER was assessed using an electric cell-substrate impedance–sensing system. TJ protein expression was assessed by Western blotting (WB) and immunocytochemistry (ICC). Perlecan (10 µg/ml) evoked unreportedly high, in vitro TEER values (1200 Ω) and the strongest adhesion. Coculturing endothelial cells with astrocytes yielded the greatest resistance over time. ICC and WB results correlated with resistance levels, with evidence of prominent occludin expression in cocultures. BL proteins exerted differential effects on TEER, whereas astrocytes in contact yielded higher TEER values and TJ expression.—Maherally, Z., Fillmore, H. L., Tan, S. L., Tan, S. F., Jassam, S. A., Quack, F. I., Hatherell, K. E., Pilkington, G. J. Real-time acquisition of transendothelial electrical resistance in an all-human, in vitro, 3-dimensional, blood–brain barrier model exemplifies tight-junction integrity.

Abstract 5216: Transient modulation of the blood-brain barrier with time dependent adenosine receptor agonism

Abstract Background: The blood-brain barrier (BBB), a dynamic, highly regulated and poorly permeable blockade, is the key obstacle to effective drug delivery to the central nervous system (CNS). Its main function is to protect the brain from toxic agents such as chemotherapy. The BBB regulates drug entry by the presence of tight junctions, adherens junctions and multidrug resistance proteins. Agonism of adenosine A2A receptor (A2AR) by Regadenoson, a FDA approved A2AR agonist, in non-tumor bearing rodents has been shown to transiently disrupt BBB permeability. Here, we studied the time dependent impact of Regadenoson on brain endothelial cells to transiently decrease tight and adherens junctions, while impairing ABC transporter function, thus increasing BBB permeability. Methods: Mouse brain endothelial cells (bEnd.3), were treated with 10μM Regadenoson on a time-course of 0-24 hours. Claudin-5, Occludin, ZO-1, VE-Cadherin, and PECAM-1 expression studies were assessed by flow cytometry, immunofluorescence and western blotting of subcellular fractions at varied time points. Additionally, tight and adherens junctions’ gene expression was assessed by qRT-PCR using SYBR green. Expression and function of ABCB1, ABCG2 was reported by western blotting of whole cell lysates, qRT-PCR and flow cytometry measurement of cellular uptake of MRP substrates. Transendothelial electrical resistance (TEER) was monitored by cellular impedance of endothelial cell monolayers with and without Regadenoson to further describe permeability changes. Results: Membrane expression of Claudin-5, Occludin, and ZO-1 was decreased approximately 30 minutes after Regadenoson treatment. Claudin-5 and Occludin expression returned to basal levels after 8 hours whereas, ZO-1 expression returned to baseline expression by 4 hours after treatment. Total protein expression was unchanged with Regadenoson treatment, but the cytoplasmic expression of tight and adherens junctions’ proteins were increased; suggesting that changes in protein localization is the cause of transient disruption. Eight hours after treatment, protein concentration and tRNA expression of ABCB1 and ABCG2 was decreased by 50%. TEER measurements demonstrated a 40% reduction in cell impedance after Regadenoson. Conclusion: Our results demonstrate that A2AR agonism causes transient changes in junctional and ABC transporter expression and functionality. The short duration of Regadenoson effects on BBB function may have important implications for its clinical use to enhance drug delivery of chemotherapy and other therapies for CNS diseases. Citation Format: Amelie Vezina, Matthew McCord McCord, Mark R. Gilbert, Sadhana Jackson. Transient modulation of the blood-brain barrier with time dependent adenosine receptor agonism [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 5216. doi:10.1158/1538-7445.AM2017-5216

Abstract 3910: Analysis of metastatic potential by breast cancer type through a microfluidic blood-brain niche

Abstract Metastasis from the primary tumor site to the brain is the most lethal complication from advanced cancer. 15% of breast cancers metastasize in the brain with a median survival of 5-14 months depending on the subtype. Therefore it is critical to identify when a tumor has the clonal potential to metastasize to the brain. Current detection methods and treatment therapies have continued to improve but do not shed light on clonal metastatic potential. Models for characterizing metastatic potential of clonal populations currently used include murine in vivo and simple in vitro systems. Murine models are costly, time intensive, slow to manifest metastasis and are not easy to analyze. On the other hand, in vitro systems are faster and more cost effective but currently do not recapitulate the complexity of the “live” micro-environment. We have developed a microfluidic device that mimics the cellular and physical components of the human blood-brain niche to study the brain metastatic process. The device is composed of two chambers separate by a porous membrane. The top chamber and apical side of the membrane is seeded with human brain endothelial cells and uses flow to mimic shear stress encountered within the vasculature. Cancer cells are introduced into this chamber in which they adhere to and migrate through the endothelium into the bottom chamber. The bottom chamber contains astrocytes suspended in a collagen gel to mimic the brain stroma and provide room for invading cancer cells to colonize and grow. Barrier integrity is monitored using TEER (trans-endothelial electrical resistance), and fluctuates as the tight junctions of the endothelium are compromised by invading cancer cells. This is characterized by IF and tight junction staining. Throughout all time points, from introduction into the flow chamber, adherence to the endothelium, extravasation through the barrier, migration into the stroma, and proliferation the cancer cells can be monitored via both microscopy and TEER. We have applied this microfluidic blood-brain niche model to compare brain-seeking subclones of breast cancer cell lines of known whole exome sequence and normal-like cell lines (MCF10A) in terms of their ability to extravasate, migrate and survive in the niche. We characterize their migratory behavior from live-cell microscopy and correlate it to the TEER measurements to establish a metastatic model. We then compare metastatic markers for ∝BBN traversing and non-traversing cells when appropriate. Citation Format: Christopher Ryan Oliver, Megan Altemus, Brendan Leung, Aki Morikawa, Michele Dziubinski, Maria Castro, Sofai Merajver. Analysis of metastatic potential by breast cancer type through a microfluidic blood-brain niche [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 3910. doi:10.1158/1538-7445.AM2017-3910

Effects of prostaglandin lipid mediators on agonist-induced lung endothelial permeability and inflammation.

Prostaglandins (PG), the products of cyclooxygenase-mediated conversion of arachidonic acid, become upregulated in many situations including allergic response, inflammation, and injury, and exhibit a variety of biological activities. Previous studies described barrier-enhancing and anti-inflammatory effects of PGE2 and PGI2 on vascular endothelial cells (EC). Yet, the effects of other PG members on EC barrier and inflammatory activation have not been systematically analyzed. This study compared effects of PGE2, PGI2, PGF2α, PGA2, PGJ2, and PGD2 on human pulmonary EC. EC permeability was assessed by measurements of transendothelial electrical resistance and cell monolayer permeability for FITC-labeled tracer. Anti-inflammatory effects of PGs were evaluated by analysis of expression of adhesion molecule ICAM1 and secretion of soluble ICAM1 and cytokines by EC. PGE2, PGI2, and PGA2 exhibited the most potent barrier-enhancing effects and most efficient attenuation of thrombin-induced EC permeability and contractile response, whereas PGI2 effectively suppressed thrombin-induced permeability but was less efficient in the attenuation of prolonged EC hyperpermeability caused by interleukin-6 or bacterial wall lipopolysaccharide, LPS. PGD2 showed a modest protective effect on the EC inflammatory response, whereas PGF2α and PGJ2 were without effect on agonist-induced EC barrier dysfunction. In vivo, PGE2, PGI2, and PGA2 attenuated LPS-induced lung inflammation, whereas PGF2α and PGJ2 were without effect. Interestingly, PGD2 exhibited a protective effect in the in vivo model of LPS-induced lung injury. This study provides a comprehensive analysis of barrier-protective and anti-inflammatory effects of different prostaglandins on lung EC in vitro and in vivo and identifies PGE2, PGI2, and PGA2 as prostaglandins with the most potent protective properties.

Ultrasound-mediated blood-brain barrier disruption: Correlation with acoustic emissions

Blood-brain barrier (BBB) disruption mediated by ultrasound and microbubbles (US-BBBD) is a promising strategy for non-invasive and targeted delivery of therapeutics to the brain. In US-BBBD, treatment control is achieved by externally monitoring acoustic emissions (AE) and adjusting ultrasound parameters in real-time to avoid AE associated with damage. Recent work suggests that AE may also provide insight regarding the extent of BBB opening and BBB recovery time. The mechanisms underlying BBB opening and recovery, however, are largely not understood. To investigate US-BBBD mechanisms with regard to AE, we developed an in vitro platform for monitoring both BBB integrity and AE during US-BBBD. Temporally resolved BBB integrity monitoring was achieved using a microfluidic BBB-on-a-chip device with integrated trans-endothelial electrical resistance (TEER) measurements. Well-characterized ultrasound exposure and AE monitoring were achieved using a focally aligned high-intensity focused ultrasound transducer and passive cavitation detector. In addition to recording TEER and AE data, our platform is compatible with fluorescence microscopy during ultrasound exposure, providing further insight into US-BBBD mechanisms. This work further demonstrates potential for in vitro screening of cavitation agents and/or therapeutics for novel US-BBBD applications and strategies.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier.

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

A Novel Functional Variant of nNOS Mediates Anoxic Injury in Brain Microvascular Endothelial Cells via Mitochondrial Superoxide Generation

Deletions of endothelial (eNOS) and neuronal (nNOS) nitric oxide synthase isoforms in mice have been shown to elicit opposite effects on ischemic brain injury following experimental stroke. Recently, we have demonstrated the expression of nNOS in human, rat, and mouse primary brain microvascular endothelial cells (MECs). The constitutively active endothelial nNOS (enNOS) was found to be functionally distinct from the nNOS expressed in neurons and the eNOS but sensitive to inhibition by pharmacological nNOS inhibitors. Under physiological conditions, enNOS was found to produce superoxide instead of nitric oxide (NO), unlike all known NOS isoforms. The objective of the present study was to examine the pathophysiological role of enNOS in endothelial injury. We hypothesized that enNOS mediates the anoxic injury underlying the blood‐brain barrier (BBB) dysfunction and cell death by instigating mitochondrial oxidative stress. We utilized primary human brain MECs (passage# 7–9) and employed inhibitors of nNOS (N‐ω‐Propyl‐L‐arginine or NPA and ARL‐17477; 1 μmol/L each) and eNOS (L‐N5‐(1‐Iminoethyl)ornithine or NIO, 1 μmol/L). Electron spin resonance spectrometry measurements with mitochondria‐targeted mito‐TEMPO‐H (50 μmol/L) showed that cells treated with enNOS inhibitor (ARL‐17477, 165±16 a.u, n=7; p &lt; 0.05) reduced the mitochondrial superoxide in compared to the untreated MECs (108±10 a.u, n=7). Oxygen consumption rate (OCR; pmol/minute/pg protein) measured in MECs by Seahorse Analyzer showed increase in maximal respiration and decreased non‐mitochondrial OCR with NPA treatment (355 ± 60 and 7 ± 4, respectively; n=11; p &lt; 0.05) compared to untreated cells (189 ± 49 and 19 ± 4, respectively; n=11). Transendothelial electrical resistance (TEER) measurements were made to quantitate the BBB integrity in confluent monolayers of hMECs grown in transwells and subjected to oxygen‐glucose deprivation (OGD; 6 h). Inhibition of enNOS with ARL‐17477 (50 ± 3 vs. 39 ± 2 untreated, n=6–8; p &lt; 0.05) and NPA (46 ± 2 vs. 36 ± 2 untreated, n=7–9; p &lt; 0.05) increased TEER (Ω.cm2) in MECs. In addition, ARL‐17477 and NPA (119 ± 15 and 116 ± 9, n=9 each; p&lt;0.05) sustained recovery of TEER (% change) at 30 hours following OGD compared to the eNOS inhibited (89 ± 5, n=9; p=NS) and untreated cells (75 ± 8, n=9). Western blot studies have shown that treatment with enNOS inhibitors increases the levels of the tight junction protein, Zona Occludens‐1, in MECs compared to untreated cells. In contrast, viability measurements using CCK‐8 assay (% viable cells) observed that MECs treated with each NOS inhibitor (NPA: 42 ± 3; ARL‐17477: 52 ± 3; NIO: 70 ± 5; n = 16, p &lt; 0.05) and exposed to OGD increased cell survival compared to untreated cells (26 ± 2; n = 16). Thus, enNOS mediates anoxic‐injury leading to the impairment of BBB integrity as well as cell death.Support or Funding InformationAHA Predoctoral (VNS: 16PRE31450006) and postdoctoral (IR: 15POST23040005) Fellowship Grant, AHA Scientist Development Grant (PVK: 14SDG20490359), and NIH grants (DWB: HL093554 and PVK: NS094834).

Abstract WP431: Functionally Distinct Neuronal Nitric Oxide Synthase Expressed in the Brain Microvascular Endothelial Cells Mediates Anoxic-injury to Blood-Brain Barrier

Objective: Mice with genetic deletion of endothelial (eNOS; protective) and neuronal (nNOS; detrimental) nitric oxide synthase isoforms exhibit dramatically opposite consequences of ischemic brain injury. nNOS has been identified recently in endothelial cells, however, its functional significance is unclear. Our objective was to identify nNOS and characterize its functional role in primary brain microvascular endothelial cells (MECs). Methods and Results: MECs from humans (hMECs), rats (rMECs), and mice (mMECs) along with cultured primary rat cortical neurons were used. In addition, rat brain microvessels were freshly isolated. Transendothelial electrical resistance (TEER) measurements of monolayers of hMECs cultured in transwells were used to quantitate in vitro blood-brain barrier (BBB) integrity. Immunocytochemistry identified von Willebrand factor, eNOS, and nNOS in MECs but stained negative for glial (GFAP) and neuronal (Neu1) markers. PCR studies confirmed the expression of eNOS and nNOS mRNA in MECs and microvessels. We utilized electron spin resonance spectrometry to measure reactive oxygen species (ROS) (1-Hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine; CMH) and NO (colloid Fe(DETC)2). Inhibition of nNOS (N-ω-Propyl-L-arginine and ARL-17477) reduced ROS but increased NO levels in MECs and rat brain microvessels. In contrast, eNOS inhibitor (L-N5-(1-Iminoethyl)ornithine) increased ROS but reduced NO levels. Inhibition of nNOS in neurons, similarly increased ROS and decreased NO levels. siRNA targeting rat nNOS in rMECs was able to knockdown nNOS mRNA as well as ROS levels. BBB studies of hMECs treated with NOS inhibitors followed by oxygen-glucose deprivation (OGD) revealed that nNOS inhibition increased TEER at baseline and promoted TEER recovery following OGD. In contrast, eNOS inhibition had no effect on TEER at baseline but weakly albeit transiently helps in the post-OGD recovery of BBB function. Conclusions: Thus, we identified a constitutively active nNOS in MECs that is functionally distinct from the nNOS isoform expressed in neurons and eNOS. In addition, nNOS inhibition enhances the BBB integrity and affords protection against anoxic-injury induced impairment of BBB function.

Construction and histological analysis of a 3D human arterial wall model containing vasa vasorum using a layer-by-layer technique.

There is considerable global demand for three-dimensional (3D) functional tissues which mimic our native organs and tissues for use as in vitro drug screening systems and in regenerative medicine. In particular, there has been an increasing number of patients who suffer from arterial diseases such as arteriosclerosis. As such, in vitro 3D arterial wall models that can evaluate the effects of novel medicines and a novel artificial graft for the treatment are required. In our previous study, we reported the rapid construction of 3D tissues by employing a layer-by-layer (LbL) technique and revealed their potential applications in the pharmaceutical fields and tissue engineering. In this study, we successfully constructed a 3D arterial wall model containing vasa vasorum by employing a LbL technique for the first time. The cells were coated with extracellular matrix nanofilms and seeded into a culture insert using a cell accumulation method. This model had a three-layered hierarchical structure: a fibroblast layer, a smooth muscle layer, and an endothelial layer, which resembled the native arterial wall. Our method could introduce vasa vasorum into a fibroblast layer in vitro and the 3D arterial wall model showed barrier function which was evaluated by immunostaining and transendothelial electrical resistance measurement. Furthermore, electron microscopy observations revealed that the vasa vasorum was composed of single-layered endothelial cells, and the endothelial tubes were surrounded by the basal lamina, which are known to promote maturation and stabilization in native blood capillaries. These models should be useful for tissue engineering, regenerative medicine, and pharmaceutical applications. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 814-823, 2017.

Role of Cingulin in Agonist-induced Vascular Endothelial Permeability

Agonist-induced activation of Rho GTPase signaling leads to endothelial cell (EC) permeability and may culminate in pulmonary edema, a devastating complication of acute lung injury. Cingulin is an adaptor protein first discovered in epithelium and is involved in the organization of the tight junctions. This study investigated the role of cingulin in control of agonist-induced lung EC permeability via interaction with RhoA-specific activator GEF-H1. The siRNA-induced cingulin knockdown augmented thrombin-induced EC permeability monitored by measurements of transendothelial electrical resistance and endothelial cell permeability for macromolecules. Increased thrombin-induced permeability in ECs with depleted cingulin was associated with increased activation of GEF-H1 and RhoA detected in pulldown activation assays. Increased GEF-H1 association with cingulin was essential for down-regulation of thrombin-induced RhoA barrier disruptive signaling. Using cingulin-truncated mutants, we determined that GEF-H1 interaction with the rod + tail domain of cingulin was required for inactivation of GEF-H1 and endothelial cell barrier preservation. The results demonstrate the role for association of GEF-H1 with cingulin as the mechanism of RhoA pathway inactivation and rescue of EC barrier after agonist challenge.

Optimization of electrospun fibrous membranes for in vitro modeling of blood-brain barrier.

The blood-brain barrier (BBB) plays a critical role in brain homeostasis at the cellular and global level. Mimicking the selective permeability and transport properties of the BBB to specific molecules and cells remains a significant challenge towards the development of a physiologically relevant in vitro BBB model. In this study, we developed electrospun poly (ε-caprolactone) (PCL) and polyethylene glycol (PEG) copolymer membranes that supported different cellular components of the neurovascular unit including human-derived endothelial cells, pericytes and astrocytes. Comparative analyses of thickness, morphology, biocompatibility and permeability of membranes were also conducted. We found that collagen coated 4%PEG-96%PCL membranes supported the growth of a confluent and tight endothelium confirmed by transendothelial electrical resistance measurements (TEER). Based on fabrication process and reported results, we finally discuss the adoption of these electrospun fiber membranes for in vitro and on-a-chip human BBB models.

Soluble VE‐cadherin contributes to loss of VE‐cadherin‐mediated adhesion and endothelial barrier function

Endothelial barrier breakdown in systemic inflammation and sepsis is a hallmark preceding organ failure and death in patients. Vascular endothelial (VE)‐cadherin‐mediated adhesion represents a crucial determinant of endothelial barrier integrity. Previously we demonstrated that under inflammatory conditions the disintegrin and metalloproteinase 10 (ADAM10) caused shedding of VE‐cadherin which resulted in the formation of soluble VE‐cadherin (sVE‐cadherin) in close temporal correlation of inflammation‐induced endothelial barrier disruption. Here we further characterized ADAM10‐induced VE‐cadherin shedding and tested whether recombinant sVE‐cadherin inferes VE‐cadherin mediated adhesion.First we identified possible cleavages sites of VE‐cadherin by mass spectrometry (MSM). Therefore, digestion of recombinant VE‐cadherin (rVE‐cadherin) with recombinant ADAM10 was performed. This led to the formation of sVE‐cadherin at 90 and 110kDa, as shown by coomassie stained gel an western blot, which correlated with several cleavage sites of the extracellular domain of VE‐cadherin. MSM showed only peptides up to 452 amino acids (aa) and one peptide at 503–530 aa, which also corresponds to the extracellular domains 1–5 of human VE‐cadherin. Additionally two other cleavage sites at 80–127aa and 160–175aa were observed. In the next step we performed atomic force microscopy measurements in a cell free sytem to test whether sVe‐cadherin blocked VE‐cadherin‐mediated adhesion. In these experiments both, sVE‐cadherin and rVE‐cadherin blocked VE‐cadherin‐mediated adhesion. Accordingly, application of rVE‐cadherin on Human dermal microvascular endothelial cells (HDMECs) resulted in a dose‐dependent loss of endothelial barrier functions as revealed by measurements of transendothelial electrical resistance (TER). In immunostaining of endothelial monolayers incubation with rVE‐cadherin led to intercellular gap formation.In summary our data demonstrate specific cleavage sites of VE‐cadherin by ADAM10. The resulting sVE‐cadherin fragments interfere VE‐cadherin mediated adhesion and thereby contribute to loss of endothelial barrier disruption. This may play a novel role in inflammation‐induced endothelial barrier breakdown.Support or Funding InformationThe present study was supported by grants from the German Research Foundation (DFG) to N.S. (Grant numbers: SCHL1962/2‐1 and SCHL 1962/4‐1).

The extracellular matrix protein laminin-10 promotes blood-brain barrier repair after hypoxia and inflammation in vitro.

BackgroundThe blood–brain barrier (BBB) of the central nervous system (CNS) is essential for normal brain function. However, the loss of BBB integrity that occurs after ischaemic injury is associated with extracellular matrix (ECM) remodelling and inflammation, and contributes to poor outcome. ECM remodelling also contributes to BBB repair after injury, but the precise mechanisms and contribution of specific ECM molecules involved are unknown. Here, we investigated the mechanisms by which hypoxia and inflammation trigger loss of BBB integrity and tested the hypothesis ECM changes could contribute to BBB repair in vitro.MethodsWe used an in vitro model of the BBB, composed of primary rat brain endothelial cells grown on collagen (Col) I-, Col IV-, fibronectin (FN)-, laminin (LM) 8-, or LM10-coated tissue culture plates, either as a single monolayer culture or on Transwell® inserts above mixed glial cell cultures. Cultures were exposed to oxygen-glucose deprivation (OGD) and/or reoxygenation, in the absence or the presence of recombinant interleukin-1β (IL-1β). Cell adhesion to ECM molecules was assessed by cell attachment and cell spreading assays. BBB dysfunction was assessed by immunocytochemistry for tight junction proteins occludin and zona occludens-1 (ZO-1) and measurement of trans-endothelial electrical resistance (TEER). Change in endothelial expression of ECM molecules was assessed by semi-quantitative RT-PCR.ResultsOGD and/or IL-1 induce dramatic changes associated with loss of BBB integrity, including cytoplasmic relocalisation of membrane-associated tight junction proteins occludin and ZO-1, cell swelling, and decreased TEER. OGD and IL-1 also induced gene expression of key ECM molecules associated with the BBB, including FN, Col IV, LM 8, and LM10. Importantly, we found that LM10, but not FN, Col IV, nor LM8, plays a key role in maintenance of BBB integrity and reversed most of the key hallmarks of BBB dysfunction induced by IL-1.ConclusionsOur data unravel new mechanisms of BBB dysfunction induced by hypoxia and inflammation and identify LM10 as a key ECM molecule involved in BBB repair after hypoxic injury and inflammation.

Flurbiprofen-loaded Nanoparticles Can Cross a Primary Porcine In vitro Blood-brain Barrier Model to Reduce Amyloid-β42 Burden

Elevated amyloid-β42 (Aβ42) in the brain is expected to cause Alzheimer’s Disease (AD). Reducing Aβ42 is therefore a cornerstone in causal drug development. Nevertheless, many promising substances failed in clinical trials, because reaching the target organ in vivo is difficult. The brain is protected by the Blood-Brain Barrier (BBB) that shields off most molecules to maintain the brain homeostasis. Brain-targeted nanoparticles are one successful tool to bypass this problem: by acting as Trojan horses they carry embedded drugs across the BBB for brain disorder treatment. Here, flurbiprofen, a γ-secretase modulator, was embedded in Poly(Lactic Acid) (PLA) nanoparticles. We tested if the drug-loaded nanoparticles affected the integrity of our advanced in vitro BBB model in transendothelial electrical resistance measurements and permeability assays, and investigated the nanoparticle-cell interaction in flow cytometry and confocal laser scanning microscopy. Furthermore, we assessed the drug transport capacity by highperformance liquid chromatography and the biological efficacy of the embedded drug in an Aβ42-detecing ELISA. We also verified the viability of the AD model cells by a cellular viability assay. After adding flurbiprofen-loaded nanoparticles to the blood compartment of a Transwell® model, the drug was detectable in the brain compartment, where it induced an Aβ42 lowering effect. Flurbiprofen from nanoparticles crossed the BBB without impairing barrier integrity, whereas the free drug was highly cytotoxic and destroyed the barrier. Ligand coupling of apolipoprotein E3 to the nanoparticles increased cellular uptake. Hence, we expect an even more pronounced Aβ42 reducing effect for apolipoprotein-modified, flurbiprofen-loaded nanoparticles. In conclusion, we enabled transport of a hardly permeable drug across an advanced in vitro BBB model, opening opportunities in the treatment and prevention of AD and other brain disorders. Using a primary porcine BBB model that displays excellent barrier characteristics, we show that flurbiprofen-loaded nanoparticles reduce Aβ42 burden without impairing barrier function.