Cells bEnd Research Articles

Endothelial Surface Glycocalyx Revealed by Stochastic Optical Reconstruction Microscopy (STORM)

On the luminal surface of our blood vessels, there is a thin layer called endothelial surface glycocalyx (ESG) which consists of proteoglycans, glycosaminoglycans (GAGs) and glycoproteins. The GAGs in the ESG are heparan sulfate (HS), hyaluronic acid (HA), chondroitin sulfate (CS), and sialic acid (SA). In order to play important roles in vascular functions, such as being a mechanosensor and transducer for the endothelial cells (ECs) to sense the blood flow, a molecular sieve to maintain normal microvessel permeability and a barrier between the circulating cells and endothelial cells forming the vessel wall, the ESG should have an organized structure at the molecular level. Due to the limitations of optical and electrical microscopy, the ultra-structure of ESG has not been revealed until recent development of super high resolution fluorescence optical microscope, STORM. We used newly acquired Nikon-STORM system to observe the ESG on in vitro EC (bEnd3, mouse brain microvascular endothelial cells) monolayers. After confluent, the bEnd3 cells were immunolabeled with anti-HS, followed by an ATTO488 conjugated goat anti-mouse IgG, and with biotinylated HA binding protein, followed by an AF647 conjugated anti-biotin. The ESG was then imaged by the STORM with a 100 x/1.49 oil immersed lens. Multiple Reporters of ATTO488 and AF647 with alternating illumination were used to acquire the 3D images of HS and HA. The field of 256 × 256 (40 × 40 μm2) of HS and HA at the surface of ECs was obtained based on totally 40,000 of EM-CCD captured images for each reporter at a capturing speed of 19 ms/frame. The figure demonstrates the STORM images for the HA (top view, red, top row on the right) and HS (green, top row in the middle) and overlay (top row on the left) of the ESG focused at the bottom surface of the bEnd3 cells. The EC plasma membrane is located at the bottom plane of the 3D images (bottom row). The color bars in the 3D side views are the scale bars in nm. The figure shows that HA is a long molecule weaving into a network, which is horizontal to the EC cell surface. In contrast, HS is a shorter molecule, which is perpendicular to the cell surface. The height of the HS is ~600 nm. HA and HS seem to overlap with each other at the EC surface. The revealed ultra-structure of ESG by STORM suggests that HS plays a major role in mechanosensing and HA plays a major role in forming the molecular sieve. Support or Funding Information NIH 1SC1CA153325-01 and NSF MRI CBET 1337746 STORM images showing distribution of ATTO 488 labeled anti-HS (green, middle) and Alexa Fluor 647 labeled anti-HA (red, right) and overlay (left) in the ESG of bEnd3 cell monolayer, focused on the bottom surface of the cells. The EC plasma membrane is located at the bottom plane of the 3D images (bottom row). The color bars in the 3D side views are the scale bars in nm. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Tetrahydrocurcumin ameliorates homocysteine-mediated mitochondrial remodeling in brain endothelial cells.

Homocysteine (Hcy) causes endothelial dysfunction by inducing oxidative stress in most neurodegenerative disorders. This dysfunction is highly correlated with mitochondrial dynamics such as fusion and fission. However, there are no strategies to prevent Hcy-induced mitochondrial remodeling. Tetrahydrocurcumin (THC) is an anti-inflammatory and anti-oxidant compound. We hypothesized that THC may ameliorates Hcy-induced mitochondria remodeling in mouse brain endothelial cells (bEnd3) cells. bEnd3 cells were exposed to Hcy treatment in the presence or absence of THC. Cell viability and autophagic cell death were measured with MTT and MDC staining assay. Reactive oxygen species (ROS) production was determined using DCFH-DA staining by confocal microscopy. Autophagy flux was assessed using a conventional GFP-microtubule-associated protein 1 light chain 3 (LC3) dot assay. Interaction of phagophore marker LC-3 with mitochondrial receptor NIX was observed by confocal imaging. Mitochondrial fusion and fission were evaluated by western blot and RT-PCR. Our results demonstrated that Hcy resulted in cell toxicity in a dose-dependent manner and supplementation of THC prevented the detrimental effects of Hcy on cell survival. Furthermore, Hcy also upregulated fission marker (DRP-1), fusion marker (Mfn2), and autophagy marker (LC-3). Finally, we observed that Hcy activated mitochondrial specific phagophore marker (LC-3) and co-localized with the mitochondrial receptor NIX, as viewed by confocal microscopy. Pretreatment of bEnd3 with THC (15 μM) ameliorated Hcy-induced oxidative damage, mitochondrial fission/fusion, and mitophagy. Our studies strongly suggest that THC has beneficial effects on mitochondrial remodeling and could be developed as a potential therapeutic agent against hyperhomocysteinemia (HHcy) induced mitochondrial dysfunction.

Identification of two immortalized cell lines, ECV304 and bEnd3, for in vitro permeability studies of blood-brain barrier.

To identify suitable cell lines for a mimetic system of in vivo blood-brain barrier (BBB) for drug permeability assessment, we characterized two immortalized cell lines, ECV304 and bEnd3 in the respect of the tightness, tight junction proteins, P-glycoprotein (P-gp) function and discriminative brain penetration. The ECV304 monoculture achieved higher transendothelial electrical resistance (TEER) and lower permeability to Lucifer yellow than bEnd3. However, co-culture with rat glioma C6 cells impaired the integrity of ECV304 and bEnd3 cell layers perhaps due to the heterogeneity among C6 cells in inducing BBB characteristics. The immunostaining of ZO-1 delivered distinct bands along cell borders on both cell lines while those of occludin and claudin-5 were diffused and weak. P-gp functionality was only proved in bEnd3 by Rhodamine 123 (R123) uptake assay. A permeability test of reference compounds displayed a similar rank order (digoxin < R123 < quinidine, verapamil < propranolol) in ECV304 and bEnd3 cells. In comparison with bEnd3, ECV304 developed tighter barrier for the passage of reference compounds and higher discrimination between transcellular and paracellular transport. However, the monoculture models of ECV304 and bEnd3 fail to achieve the sufficient tightness of in vitro BBB permeability models with high TEER and evident immunostaining of tight junction proteins. Further strategies to enhance the paracellular tightness of both cell lines to mimic in vivo BBB tight barrier deserve to be conducted.

Targeting of AUF1 to vascular endothelial cells as a novel anti-aging therapy.

BackgroundInhibition of aging of vascular endothelial cells (VECs) may delay aging and prolong life. The goal of this study was to prepare anti-CD31 monoclonal antibody conjugated PEG-modified liposomes containing the AU-rich region connecting factor 1 (AUF1) gene (CD31-PILs-AUF1) and to explore the effects of targeting CD31-PILs-AUF1 to aging VECs.MethodsThe mean particle sizes of various PEGylated immunoliposomes (PILs) were measured using a Zetasizer Nano ZS. Gel retardation assay was used to confirm whether PILs had encapsulated the AUF1 plasmid successfully. Fluorescence microscopy and flow cytometry were used to quantify binding of CD31-PILs-AUF1 to target cells. Flow cytometry was also used to analyze the cell cycles of aging bEnd3 cells treated with CD31-PILs-AUF1. We also developed an aging mouse model by treating mice with D-galactose. Enzyme-linked immunosorbent assay (ELISA) was used to evaluate the levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). The malondialdehyde (MDA) and the superoxide dismutase (SOD) levels were detected by commercial kits. Hematoxylin-eosin (HE) staining was used to determine whether treatment with CD31-PILs-AUF1 was toxic to the mice.ResultsCD31-PILs-AUF1 specifically could targeted bEnd3 VECs and increased the percentage of cells in the S and G2/M phases of aging bEnd3 cells. ELISA showed that content of the IL-6 and TNF-α decreased in CD31-PILs-AUF1 group. The level of SOD increased, whereas MDA decreased in the CD31-PILs-AUF1 group. Additionally, CD31-PILs-AUF1 was not toxic to the mice.ConclusionCD31-PILs-AUF1 targets VECs and may delay their senescence.

Acetylcholine induces intracellular Ca2+ oscillations and nitric oxide release in mouse brain endothelial cells

Basal forebrain neurons increase cortical blood flow by releasing acetylcholine (Ach), which stimulates endothelial cells (ECs) to produce the vasodilating gasotransmitter, nitric oxide (NO). Surprisingly, the mechanism whereby Ach induces NO synthesis in brain microvascular ECs is unknown. An increase in intracellular Ca2+ concentration recruits a multitude of endothelial Ca2+-dependent pathways, such as Ca2+/calmodulin endothelial NO synthase (eNOS). The present investigation sought to investigate the role of intracellular Ca2+ signaling in Ach-induced NO production in bEND5 cells, an established model of mouse brain microvascular ECs, by conventional imaging of cells loaded with the Ca2+-sensitive dye, Fura-2/AM, and the NO-sensitive fluorophore, DAF-DM diacetate. Ach induced dose-dependent Ca2+ oscillations in bEND5 cells, 300 μM being the most effective dose to generate a prolonged Ca2+ burst. Pharmacological manipulation revealed that Ach-evoked Ca2+ oscillations required metabotropic muscarinic receptor (mAchR) activation and were patterned by a complex interplay between repetitive ER Ca2+ release via inositol-1,4,5-trisphosphate receptors (InsP3Rs) and store-operated Ca2+ entry (SOCE). A comprehensive real time-polymerase chain reaction analysis demonstrated the expression of the transcripts encoding for M3-mAChRs, InsP3R1 and InsP3R3, Stim1-2 and Orai2. Next, we found that Ach-induced NO production was hindered by L-NAME, a selective NOS inhibitor, and BAPTA, a membrane permeable intracellular Ca2+ buffer. Moreover, Ach-elicited NO synthesis was blocked by the pharmacological abrogation of the accompanying Ca2+ spikes. Overall, these data shed novel light on the molecular mechanisms whereby neuronally-released Ach controls neurovascular coupling in blood microvessels.

103 Blockade of hippo pathway effector YAP1 with shRNA attenuates vascular development in Hemangioma Bend3 cells in vivo

103 Blockade of hippo pathway effector YAP1 with shRNA attenuates vascular development in Hemangioma Bend3 cells in vivo

Selection and Characterization of Novel of RNA Aptamer Recognizing Homocysteine which Ameliorates Oxidative Stress in Mouse Brain Endothelial (bEND3) Cells

Neurodegenerative diseases are characterized by a progressive deterioration of brain function, with a significant consequent decrease in the quality of patient's lives. Recently pathological mechanisms operating in cerebrovascular diseases mediated via homocysteine (Hcy) have gained increased attention. It has also been known that Hcy causes endothelial dysfunction by inducing oxidative stress in most cerebrovascular disorders. This dysfunction is highly correlated with reactive oxygen generation (ROS). Therefore, it is crucial to develop potential novel measures to slow down or prevent the onset of these diseases mediated by increased level of Hcy. Therefore, we proposed to select and characterize novel RNA aptamer recognizing Hcy to inhibit and modulates the Hcy activities involved in cerebrovascular disease. RNA Aptamers are specific bio‐molecules selected by the process called Systematic Evolution of Ligands by Exponential Enrichment (SELEX). Aptamer folds into 3‐dimensional shapes which are capable of binding non‐covalently with high affinity and specificity to their target molecule. We have selected and characterized Hcy RNA aptamers using SELEX technology. The structural characterization was done by bioinformatics tools such as Clustal W, Mfold, IFold and HADDOCK. Binding characterization were done by CD and RNA EMSA studies. The best Hcy RNA aptamers were further investigated for their biological and functional characterization. Our results demonstrate that the Hcy RNA aptamer undergoes a conformation change upon ligand binding. These aptamers exhibit high specificity to ligand over nonspecific competitor. In our studies, bEND3 cells were exposed to Hcy treatment in the presence or absence of Hcy RNA aptamers for 24 hrs. We also demonstrated that these aptamers are novel class of RNA aptamer which ameliorates the effects of ROS thereby reducing the oxidative stress in bEND cell lines. Apoptotic and autophagic cell death were measured with 3‐(4, 5‐Dimethylthiazol‐2‐yl)‐2, 5‐ Diphenyltetrazolium Bromide (MTT) and monodansylcadaverine (MDC) staining assay. ROS production was determined using fluorescent probes 2, 7‐dichlorofluorescein acetate (DCFH‐DA. Autophagy flux was assessed using a conventional GFP‐microtubule‐associated protein 1 light chain 3 (LC3) dot assay. Mitochondrial fusion and fission was evaluated using Mfn‐2 and Drp‐1 RT‐PCR. Treatment of bEnd3 with Hcy RNA aptamer attenuated the production of free radicals and protected the cells from oxidative damage. Altogether, our studies strongly suggest that Hcy‐RNA aptamer has beneficial effects on cerebrovascular diseases and could be developed as a potential therapeutic agent against hyperhomocysteinemia (HHcy).Support or Funding InformationFinancial support from National Institute of health grant AR‐067667 and HL‐107640 are greatly acknowledged.

Acetylcholine induces intracellular Ca2+ oscillations and nitric oxide release in mouse brain endothelial cells

Basal forebrain neurons increase cortical blood flow by releasing acetylcholine (Ach), which stimulates endothelial cells (ECs) to produce the vasodilating gasotransmitter, nitric oxide (NO). Surprisingly, the mechanism whereby Ach induces NO synthesis in brain microvascular ECs is unknown. An increase in intracellular Ca2 + concentration recruits a multitude of endothelial Ca2 +-dependent pathways, such as Ca2 +/calmodulin endothelial NO synthase (eNOS). The present investigation sought to investigate the role of intracellular Ca2 + signaling in Ach-induced NO production in bEnd5 cells, an established model of mouse brain microvascular ECs, by conventional imaging of cells loaded with the Ca2+-sensitive dye, Fura-2/AM, and the NO-sensitive fluorophore, DAF-DM diacetate. Overall, our data shed novel light on the molecular mechanisms whereby neuronally-released Ach controls neurovascular coupling in blood microvessels.

Calpain-1 resident in lipid raft/caveolin-1 membrane microdomains plays a protective role in endothelial cells

We are here reporting that calpain-1 is a constitutive component of a distinct lipid raft/caveolin-1 microdomain isolated from bEnd5 cells in association with endothelial nitric oxide synthase (eNOS) and heat shock protein 90 (HSP90). Perturbations in intracellular calcium concentration by Ca2+-ionophore A23187 or prolonged cell exposure to high glucose induce a significant decrease in the level of eNOS accompanied by a recruitment of additional HSP90 molecules at this site. In these conditions the cells are more resistant to cell death by Ca2+ overload. The decrease of eNOS has been due not only to its Ca2+-mediated release from the caveolin-1 aggregates but also to its digestion by calpain-1. The specific involvement of calpain-1 in digestion of eNOS is supported by the preventive effect of a synthetic calpain inhibitor (CI-2) and by the absence of calpain-2 and calpastatin in the caveolin-1 microdomain.These results suggest that the protein adjustments observed in lipid raft/caveolin-1 microdomains could be visualized as a process required to protect the cells against NO overproduction and aberrant calpain activation. Alterations in eNOS, calpain-1 and HSP90 levels have been observed in aorta of Zucker Diabetic Rats (ZDR). The loss of HSP90 occurring in these animals indicates an aberrant activation of calpain and thereby the transition from a physiological to a pathological cell condition.

Metformin reduces morphine tolerance by inhibiting microglial-mediated neuroinflammation.

BackgroundTolerance seriously impedes the application of morphine in clinical medicine. Thus, it is necessary to investigate the exact mechanisms and efficient treatment. Microglial activation and neuroinflammation in the spinal cord are thought to play pivotal roles on the genesis and maintaining of morphine tolerance. Activation of adenosine monophosphate-activated kinase (AMPK) has been associated with the inhibition of inflammatory nociception. Metformin, a biguanide class of antidiabetic drugs and activator of AMPK, has a potential anti-inflammatory effect. The present study evaluated the effects and potential mechanisms of metformin in inhibiting microglial activation and alleviating the antinociceptive tolerance of morphine.MethodsThe microglial cell line BV-2 cells and mouse brain-derived endothelial cell line bEnd3 cells were used. Cytokine expression was measured using quantitative polymerase chain reaction. Cell signaling was assayed by western blot and immunohistochemistry. The antinociception and morphine tolerance were assessed in CD-1 mice using tail-flick tests.ResultsWe found that morphine-activated BV-2 cells, including the upregulation of p38 mitogen-activated protein kinase (p38 MAPK) phosphorylation, pro-inflammatory cytokines, and Toll-like receptor-4 (TLR-4) mRNA expression, which was inhibited by metformin. Metformin suppressed morphine-induced BV-2 cells activation through increasing AMPK phosphorylation, which was reversed by the AMPK inhibitor compound C. Additionally, in BV-2 cells, morphine did not affect the cell viability and the mRNA expression of anti-inflammatory cytokines. In bEnd3 cells, morphine did not affect the mRNA expression of interleukin-1β (IL-1β), but increased IL-6 and tumor necrosis factor-α (TNF-α) mRNA expression; the effect was inhibited by metformin. Morphine also did not affect the mRNA expression of TLR-4 and chemokine ligand 2 (CCL2). Furthermore, systemic administration of metformin significantly blocked morphine-induced microglial activation in the spinal cord and then attenuated the development of chronic morphine tolerance in mice.ConclusionsMetformin significantly attenuated morphine antinociceptive tolerance by suppressing morphine-induced microglial activation through increasing AMPK phosphorylation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0754-9) contains supplementary material, which is available to authorized users.

Nanoparticles of alkylglyceryl dextran and poly(ethyl cyanoacrylate) for applications in drug delivery: Preparation and characterization

ABSTRACTToward the development of drug carriers that are capable of crossing biological membranes, controlled emulsion polymerization has been utilized to produce nanoparticulate carriers from the combination of poly(alkyl cyanoacrylate) and alkylglyceryl dextran to a molecular structure designed to combine the nonimmunogenic and stabilizing properties of dextran with the demonstrated permeation enhancing ability of alkylglycerols. To this aim, a systematic series of alkylglyceryl dextrans have been synthesized and functionalized with ethyl or butyl cyanoacrylates to form stable polymeric nanocarriers (100–500 nm). Results of investigations into their capability to act as controlled-release devices and their cytotoxicity against bEnd3 cells are reported.

Plant-derived triterpene celastrol ameliorates oxygen glucose deprivation-induced disruption of endothelial barrier assembly via inducing tight junction proteins

BackgroundThe integrity and functions of blood-brain barrier (BBB) are regulated by the expression and organization of tight junction proteins. ObjectiveThe present study was designed to explore whether plant-derived triterpenoid celastrol could regulate tight junction integrity in murine brain endothelial bEnd3 cells. MethodsWe disrupted the tight junctions between endothelial bEnd3 cells by oxygen glucose deprivation (OGD). We investigated the effects of celastrol on the permeability of endothelial monolayers by measuring transepithelial electrical resistance (TEER). To clarify the tight junction composition, we analyzed the expression of tight junction proteins by RT-PCR and Western blotting techniques. ResultsWe found that celastrol recovered OGD-induced TEER loss in a concentration-dependent manner. Celastrol induced occludin, claudin-5 and zonula occludens-1 (ZO-1) in endothelial cells. As a result, celastrol effectively maintained tight junction integrity and inhibited macrophage migration through endothelial monolayers against OGD challenge. Further mechanistic studies revealed that celastrol induced the expression of occludin and ZO-1) via activating MAPKs and PI3K/Akt/mTOR pathway. We also observed that celastrol regulated claudin-5 expression through different mechanisms. ConclusionThe present study demonstrated that celastrol effectively protected tight junction integrity against OGD-induced damage. Thus, celastrol could be a drug candidate for the treatment of BBB dysfunction in various diseases.

Evaluation of various block copolymers for micelle formation and brain drug delivery: In vitro characterization and cellular uptake studies

The treatment of brain associated diseases is limited due to rapid drug release into blood circulation after administration of conventional dosage forms and the blood–brain barrier. Polymeric micelles have opened up new horizons for improving the drug delivery to brain particularly due to their small size, long circulation time, good stability and targetability. This study aims to evaluate the micelle forming ability of copolymers with varying block compositions and investigate their potential for brain drug delivery. Block copolymers from poly(styrene)-poly(acrylic acid) (PS-PAA), poly(ethylene glycol)-b-poly(lactic acid) (PEG-PLA) and distearyl-sn-glycero-3-phosphoethanolamine-N-methoxy poly(ethylene glycol) (PEG-DSPE) groups were evaluated. The critical micelle concentration and crystallinity of the copolymers were determined. Furthermore, size, surface charge and morphologic structure of the micelles were characterized by dynamic light scattering and transmission electron microscopy. The cytotoxicity and cellular uptake of micelles were assessed on mouse brain endothelial (bEnd-3) cells. The results demonstrated the effectiveness of chemical composition and block lengths of copolymers on micelle formation and their interaction with bEnd-3 cells. Although micellization was observed for all block copolymers, polymeric micelles derived from PEG5000-PLA4500 were found to be worthy for further consideration as pharmaceutical carriers which could improve brain uptake and efficacy of hydrophobic drugs that they carry.

Cerebral Microvascular Endothelial Cell Apoptosis after Ischemia: Role of Enolase-Phosphatase 1 Activation and Aci-Reductone Dioxygenase 1 Translocation.

Enolase-phosphatase 1 (ENOPH1), a newly discovered enzyme of the methionine salvage pathway, is emerging as an important molecule regulating stress responses. In this study, we investigated the role of ENOPH1 in blood brain barrier (BBB) injury under ischemic conditions. Focal cerebral ischemia induced ENOPH1 mRNA and protein expression in ischemic hemispheric microvessels in rats. Exposure of cultured brain microvascular endothelial cells (bEND3 cells) to oxygen-glucose deprivation (OGD) also induced ENOPH1 upregulation, which was accompanied by increased cell death and apoptosis reflected by increased 3-(4, 5-Dimethylthiazol-2-yl)-2, 5- diphenyltetrazolium bromide formation, lactate dehydrogenase release and TUNEL staining. Knockdown of ENOPH1 expression with siRNA or overexpressing ENOPH1 with CRISPR-activated plasmids attenuated or potentiated OGD-induced endothelial cell death, respectively. Moreover, ENOPH1 knockdown or overexpression resulted in a significant reduction or augmentation of reactive oxygen species (ROS) generation, apoptosis-associated proteins (caspase-3, PARP, Bcl-2 and Bax) and Endoplasmic reticulum (ER) stress proteins (Ire-1, Calnexin, GRP78 and PERK) in OGD-treated endothelial cells. OGD upregulated the expression of ENOPH1’s downstream protein aci-reductone dioxygenase 1 (ADI1) and enhanced its interaction with ENOPH1. Interestingly, knockdown of ENOPH1 had no effect on OGD-induced ADI1 upregulation, while it potentiated OGD-induced ADI1 translocation from the nucleus to the cytoplasm. Lastly, knockdown of ENOPH1 significantly reduced OGD-induced endothelial monolayer permeability increase. In conclusion, our data demonstrate that ENOPH1 activation may contribute to OGD-induced endothelial cell death and BBB disruption through promoting ROS generation and the activation of apoptosis associated proteins, thus representing a new therapeutic target for ischemic stroke.

Abstract 2163: Targeting brain tumor and brain metastases using brain penetrating peptide and long circulating nanoemulsions

Abstract Purpose: Glioblastoma brain tumor and brain metastases from primary breast or lung cancer have limited treatment options, primarily due to the extremely invasive and aggressive nature of tumors; the largely impermeable blood-brain barrier (BBB); and presence of efflux transporters at BBB. We aimed to improve brain delivery of drugs by intravenous injection of a mixture of brain penetrating peptide K16ApoE and drug encapsulated long circulating nanoemulsions. We also explored the potential mechanisms that account for its brain delivery. Methods: Oil-in-water nanoemulsions (NE) containing medium-chain triglycerides oil and co-surfactants were prepared by a spontaneous self-emulsification method (SNNE) and conventional high speed homogenization and sonication method (CNE). The nanoemulsions were mixed and injected with 20 nmol of K16ApoE peptide that comprising 16 lysine residues and 20 amino acids corresponding to the low-density lipoprotein receptor binding sequence from apolipoprotein E in healthy and tumor bearing mice. Results: Nanoemulsions had mean particle size less than 200 nm (CNE: 190 nm,+35 mV; SNNE: 120 nm, -10 mV). In vitro, both NE was taken up by brain capillary endothelial cells bEnd.3 and glioblastoma cell U87-luc in a serum- dependent manner (10% FBS&amp;gt; 5%FBS&amp;gt; 2.5%FBS&amp;gt; serum free). Both the bEnd3 and U87-luc cells express high levels of an endocytic low density lipoprotein receptor-related protein 1 (LRP1) receptor. Using LRP1 antagonist receptor-associated protein (RAP) to block a ligand binding to LRP1 led to significant decrease of cellular uptake of NE. The addition of one of LRP1 ligand apolipoprotein E (12.5-25 μg/mL) in the serum free medium resulted in significantly higher cellular uptake of NE in bEnd3 cells and U87-luc cells, suggesting the NE can be imported into the brain and brain tumor cells by binding to apolipoprotein E via LRP1 receptor mediated transcytosis. In vivo, intravenous injection of near-infrared labeled NE significantly improved its brain accumulation in comparison to that of free dye. When 20 nmol of K16ApoE peptide and NE mixture was inject intravenously, the peptide transiently opened the BBB and allowed even higher amount of NE accumulation at brain tumor. Conclusions: Our study demonstrates that nanoemulsion is a promising nanocarrier to deliver imaging or therapeutic agents for the diagnosis or systemic treatment of brain tumor or brain metastasis. K16ApoE peptide is a brain penetrating peptide and can help transport nanoparticles to the brain tissue without the need for chemical modification of surface of nanoparticles. Binding of nanoemulsion to apolipoprotein E and involvement of LRP1 receptor-mediated transcytosis could be important steps in mediating the uptake of nanoemulsion in the brain endothelial cells and brain tumor cells. Citation Format: Xinli Liu, Tanvirul Hye. Targeting brain tumor and brain metastases using brain penetrating peptide and long circulating nanoemulsions. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2163.

EF1A1/HSC70 Cooperatively Suppress Brain Endothelial Cell Apoptosis via Regulating JNK Activity.

In our previous study, eEF1A1 was identified to be a new target for protecting brain ischemia injury, but the mechanism remains largely unknown. In this study, we screened the downstream cellular protein molecules interacted with eEF1A1 and found mechanism of eEF1A1 in brain ischemia protection. Through co-immunoprecipitation and mass spectrometry for searching the interaction of proteins with eEF1A1 in bEnd3 cells, HSC70 was identified to be a binding protein of eEF1A1, which was further validated by Western blot and immunofluorescence. eEF1A1 or HSC70 knockdown, respectively, increased OGD-induced apoptosis of brain vascular endothelial cells, which was detected by Annexin V-FITC/PI staining. HSC70 or eEF1A1 knockdown enhances phosphorylated JNK, phosphorylation of c-JUN (Ser63, Ser73), cleaved caspase-9, and cleaved caspase-3 expression, which could be rescued by JNK inhibitor. In summary, our data suggest that the presence of chaperone forms of interaction between eEF1A1 and HSC70 in brain vascular endothelial cells, eEF1A1 and HSC70 can play a protective role in the process of ischemic stroke by inhibiting the JNK signaling pathway activation.

Inhibition of cerebral vascular inflammation by brain endothelium-targeted oligodeoxynucleotide complex

The present study generated a novel DNA complex to specifically target endothelial NF-κB to inhibit cerebral vascular inflammation. This DNA complex (GS24-NFκB) contains a DNA decoy which inhibits NF-κB activity, and a DNA aptamer (GS-24), a ligand of transferrin receptor (TfR), which allows for targeted delivery of the DNA decoy into cells. The results indicate that GS24-NFκB was successfully delivered into a murine brain-derived endothelial cell line, bEND5, and inhibited inflammatory responses induced by tumor necrosis factor α (TNF-α) or oxygen-glucose deprivation/re-oxygenation (OGD/R) via down-regulation of the nuclear NF-κB subunit, p65, as well as its downstream inflammatory cytokines, inter-cellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule (VCAM-1). The inhibitory effect of the GS24-NFκB was demonstrated by a significant reduction in TNF-α or OGD/R induced monocyte adhesion to the bEND5 cells after GS24-NFκB treatment. Intravenous (i.v.) injection of GS24-‘NFκB (15mg/kg) was able to inhibit the levels of phoseph-p65 and VCAM-1 in brain endothelial cells in a mouse lipopolysaccharide (LPS)-induced inflammatory model in vivo. In conclusion, our approach using DNA nanotechnology for DNA decoy delivery could potentially be utilized for inhibition of inflammation in ischemic stroke and other neuro-inflammatory diseases affecting cerebral vasculature.

Energetics of ligand-receptor binding affinity on endothelial cells: An in vitro model

Targeted therapies represent a challenge in modern medicine. In this contest, we propose a rapid and reliable methodology based on Isothermal Titration Calorimetry (ITC) coupled with confluent cell layers cultured around biocompatible templating microparticles to quantify the number of overexpressing receptors on cell membrane and study the energetics of receptor-ligand binding in near-physiological conditions. In the in vitro model here proposed we used the bEnd3 cell line as brain endothelial cells to mimic the blood brain barrier (BBB) cultured on dextran microbeads ranging from 67μm to 80μm in size (Cytodex) and the primary human umbilical vein cells (HUVEC) for comparison. The revealed affinity between transferrin (Tf) and transferrin receptor (TfR) in both systems is very high, Kd values are in the order of nM. Conversely, the value of TfRs/cell reveals a 100-fold increase in the number of TfRs per bEnd3 cells compared to HUVEC cells. The presented methodology can represent a novel and helpful strategy to identify targets, to address drug design and selectively deliver therapeutics that can cross biological barriers such as the blood brain barrier.

Release of Matrix Metalloproteinases-2 and 9 by S-Nitrosylated Caveolin-1 Contributes to Degradation of Extracellular Matrix in tPA-Treated Hypoxic Endothelial Cells.

Intracranial hemorrhage remains the most feared complication in tissue plasminogen activator (tPA) thrombolysis for ischemic stroke. However, the underlying molecular mechanisms are still poorly elucidated. In this study, we reported an important role of caveolin-1 (Cav-1) s-nitrosylation in matrix metalloproteinase (MMP)-2 and 9 secretion from tPA-treated ischemic endothelial cells. Brain vascular endothelial cells (bEND3) were exposed to oxygen-glucose deprivation (OGD) for 2 h before adding recombinant human tPA for 6 h. This treatment induced a significant increase of MMP2 and 9 in the media of bEND3 cells and a simultaneous degradation of fibronectin and laminin β-1, the two main components of extracellular matrix (ECM). Inhibition of MMP2 and 9 with SB-3CT completely blocked the degradation of fibronectin and laminin β-1. ODG+tPA treatment led to Cav-1 shedding from bEND3 cells into the media. Notably, OGD triggered nitric oxide (NO) production and S-nitrosylationof Cav-1 (SNCav-1). Meanwhile tPA induced activation of ERK signal pathway and stimulates the secretion of SNCav-1. Pretreatment of bEND3 cells with C-PTIO (a NO scavenger) or U0126 (a specific ERK inhibitor) significantly reduced OGD-induced S-nitrosylation of Cav-1 in cells and blocked the secretion of Cav-1 and MMP2 and 9 into the media as well as the degradation of fibronectin and laminin β-1 in OGD and tPA-treated cells. These data indicate that OGD-triggered Cav-1 S-nitrosylation interacts with tPA-induced ERK activation to augment MMP2 and 9 secretion and subsequent ECM degradation, which may account for the exacerbation of ischemic blood brain barrier damage following tPA thrombolysis for ischemic stroke.

Sialic acid (SA)-modified selenium nanoparticles coated with B6 peptide for potential use in Alzheimer's disease

The blood–brain barrier (BBB) is a formidable gatekeeper toward exogenous substances, playing an important role in brain homeostasis and maintaining a healthy microenvironment for complex neuronal activities. However, it also greatly hinders drug permeability into the brain and limits the management of brain diseases. The development of new drugs that show improved transport across the BBB represents a promising strategy for Alzheimer's disease (AD) intervention. Whereas a previous study of receptor-mediated endogenous BBB transport systems has focused on a strategy of using transferrin to facilitate brain drug delivery system, a system that still suffers from limitations including synthesis procedure, stability and immunological response, in the present study, we synthetized sialic acid (SA)-modified selenium (Se) nanoparticles conjugated with an alternative peptide-B6 peptide (B6-SA-SeNPs, a synthetic selenoprotein analogue), which shows high permeability across the BBB and has the potential to serve as a novel nanomedicine for disease modification in AD. Laser-scanning confocal microscopy, flow cytometry analysis and inductively coupled plasma-atomic emission spectroscopy ICP-AES revealed high cellular uptake of B6-SA-SeNPs by cerebral endothelial cells (bEnd.3). The transport efficiency of B6-SA-SeNPs was evaluated in a Transwell experiment based on in vitro BBB model. It provided direct evidence for B6SA-SeNPs crossing the BBB and being absorbed by PC12 cells. Moreover, inhibitory effects of B6-SA-SeNPs on amyloid-β peptide (Aβ) fibrillation could be demonstrated in PC12 cells and bEnd3 cells. B6-SA-SeNPs could not only effectively inhibit Aβ aggregation but could disaggregate preformed Aβ fibrils into non-toxic amorphous oligomers. These results suggested that B6SA-SeNPs might provide a promising platform, particularly for the application of nanoparticles in the treatment of brain diseases.