Inhibition of Advanced Glycation and Absence of Galectin-3 Prevent Blood-Retinal Barrier Dysfunction during Short-Term Diabetes

Breakdown of the inner blood-retinal barrier (iBRB) occurs early in diabetes and is central to the development of sight-threatening diabetic macular edema (DME) as retinopathy progresses. In the current study, we examined how advanced glycation end products (AGEs) forming early in diabetes could modulate vasopermeability factor expression in the diabetic retina and alter inter-endothelial cell tight junction (TJ) integrity leading to iBRB dysfunction. We also investigated the potential for an AGE inhibitor to prevent this acute pathology and examined a role of the AGE-binding protein galectin-3 (Gal-3) in AGE-mediated cell retinal pathophysiology. Diabetes was induced in C57/BL6 wild-type (WT) mice and in Gal-3(-/-) transgenic mice. Blood glucose was monitored and AGE levels were quantified by ELISA and immunohistochemistry. The diabetic groups were subdivided, and one group was treated with the AGE-inhibitor pyridoxamine (PM) while separate groups of WT and Gal-3(-/-) mice were maintained as nondiabetic controls. iBRB integrity was assessed by Evans blue assay alongside visualisation of TJ protein complexes via occludin-1 immunolocalization in retinal flat mounts. Retinal expression levels of the vasopermeability factor VEGF were quantified using real-time RT-PCR and ELISA. WT diabetic mice showed significant AGE -immunoreactivity in the retinal microvasculature and also showed significant iBRB breakdown (P < .005). These diabetics had higher VEGF mRNA and protein expression in comparison to controls (P < .01). PM-treated diabetics had normal iBRB function and significantly reduced diabetes-mediated VEGF expression. Diabetic retinal vessels showed disrupted TJ integrity when compared to controls, while PM-treated diabetics demonstrated near-normal configuration. Gal-3(-/-) mice showed significantly less diabetes-mediated iBRB dysfunction, junctional disruption, and VEGF expression changes than their WT counterparts. The data suggests an AGE-mediated disruption of iBRB via upregulation of VEGF in the diabetic retina, possibly modulating disruption of TJ integrity, even after acute diabetes. Prevention of AGE formation or genetic deletion of Gal-3 can effectively prevent these acute diabetic retinopathy changes.

Involvement of MAPKs in endostatin-mediated regulation of blood-retinal barrier function

Purpose: This study aimed to evaluate the effects of endostatin on tight junction (TJ) integrity in retinal microvascular endothelial cells (RMECs) in vitro and in vivo. Moreover, it was hypothesized that endostatin-induced occludin upregulation regulated VEGF(165)-mediated increases in endothelial cell permeability and involved activation of the MAPK signaling cascade. Endostatin is a 20-kDa fragment of collagen XVIII that has been shown to be efficacious in the eye by preventing retinal neovascularization. Endostatin is a specific inhibitor of endothelial cell proliferation, migration, and angiogenesis and has been reported to reverse VEGF-mediated increases in vasopermeability and to promote integrity of the blood-retinal barrier (BRB). In order to determine the mechanism of endostatin action on BRB integrity, we have examined the effects of endostatin on a number of intracellular pathways implicated in endothelial cell physiology. Methods: C57/Bl6 mice were injected with VEGF(165) and/or endostatin, and the distribution of occludin staining was determined using retinal flatmounts. Western blot analysis of RMECs treated with VEGF(165) and/or endostatin was used to determine changes in occludin expression and p38 MAPK and extracellular regulated kinase (ERK1/ERK2 MAPK) activation, while FD-4 flux across the RMEC monolayer was used to determine changes in paracellular permeability. Results: Endostatin prevented the discontinuous pattern of occludin staining observed at the retinal blood vessels of mice administered an intraocular injection of VEGF(165). It was shown that endostatin activated p38 MAPK 5 min after addition to RMECs and continued to do so for approximately 30 min. Endostatin was also shown to activate ERK1/ERK2 5 min after addition and continued to do so, albeit with less potency, up to and including 15 min after addition. Inhibition of p38 MAPK and ERK1/ERK2 prevented endostatin's ability to upregulate levels of occludin expression. Inhibition of these key signaling molecules was shown to prevent endostatin's ability to protect against VEGF(165)- mediated increases in paracellular permeability in vitro. However, it appears that p38 MAPK may play a more important role in VEGF-mediated permeability, as inhibition of ERK1/ERK2 will not prevent VEGF(165)- mediated permeability compared with control ( untreated) cells or cells treated with both a p38 MAPK inhibitor and VEGF(165). Conclusions: Occludin is important for the maintenance of tight junction integrity in vivo. In a p38 MAPK and ERK1/ERK2 dependent manner, endostatin was shown to upregulate the levels of expression of the tight junction protein occludin. Inhibition of these key MAPK components may prevent endostatin's ability to decrease VEGF(165)-induced paracellular permeability.

In vitro blood-brain barrier models to predict the permeation of gene therapy vectors into the brain

A terapia génica tem-se revelado uma alternativa relevante no tratamento de doenças neurodegenerativas (DN). Contudo, a entrega de vetores para transferência génica no cérebro representa ainda um enorme desafio devido à presença da barreira hemato-encefálica (BHE). A BHE é uma interface dinâmica e seletiva entre o sangue e o cérebro, constituída pelas células endoteliais cerebrais, astrócitos e pericitos, desempenhando um importante papel na regulação da homeostasia cerebral. A BHE representa um dos maiores obstáculos no tratamento de DN, uma vez que esta barreira impede o transporte para o cérebro da maioria das moléculas terapêuticas, incluindo os vetores para terapia génica. Embora tenham sido desenvolvidos diferentes modelos in vitro da BHE de forma a avaliar o transporte de fármacos através da BHE, muito poucos foram criados com o intuito de testar a permeabilidade desta barreira a vetores de terapia génica. O presente trabalho teve como objetivo principal o desenvolvimento e a avaliação de modelos in vitro de BHE que permitam a investigação da capacidade dos vetores de terapia génica de penetrarem no cérebro. No nosso estudo, foram testados diferentes modelos in vitro de BHE em monocultura, constituídos por células endoteliais de rato ou murganho (RBE4 e bEnd3, respetivamente), e modelos de co-cultura, que combinam células endoteliais com células neuronais (Neuro2a) ou astrócitos primários, cultivados num sistema transwell. Para caraterizar estes modelos foram realizados testes de permeabilidade e de resistência elétrica transendotelial, bem como estudos baseados na técnica de PCR quantitativo e na imunocitoquímica das proteínas das junções intercelulares. Verificámos que os modelos baseados na cultura de células bEnd3 e células neuronais ou astrócitos apresentavam as melhores propriedades de barreira. Posteriormente foi avaliada nos modelos selecionados a penetração de um vetor não-viral que reconhecidamente tem a capacidade de atravessar in vivo a BHE: o peptídeo da glicoproteína do vírus da raiva (RGV-9r). Os siRNAs marcados com um fluoróforo e acoplados ao peptídeo RVG-9r foram capazes de penetrar eficientemente as células bEnd3, localizadas no lado luminal do insert, via endocitose mediada por recetores, e ainda de penetrar os astrócitos ou células neuronais, previamente cultivadas no lado abluminal. Estes resultados correlacionam-se, de forma clara, com os resultados previamente descritos em estudos in vivo. Em conclusão, os modelos in vitro de BHE baseados na co-cultura de células bEnd3 com células Neuro2a ou astrócitos, têm grande potencial na seleção de candidatos a vetores de terapia génica para o cérebro, uma vez que apresentam importantes características da BHE e se baseiam num método fácil e reprodutível. Tal facto representa uma promessa significativa para a identificação de novas estratégias de terapia génica não invasiva para o tratamento de doenças neurológicas.

The importance of the Hedgehog signaling pathway at the level of the blood-brain barrier

La barrière hémato-encéphalique (BHE) protège le système nerveux central (SNC) en contrôlant le passage des substances sanguines et des cellules immunitaires. La BHE est formée de cellules endothéliales liées ensemble par des jonctions serrées et ses fonctions sont maintenues par des astrocytes, celles ci sécrétant un nombre de facteurs essentiels. Une analyse protéomique de radeaux lipidiques de cellules endothéliales de la BHE humaine a identifié la présence de la voie de signalisation Hedgehog (Hh), une voie souvent liées à des processus de développement embryologique ainsi qu’au niveau des tissus adultes. Suite à nos expériences, j’ai déterminé que les astrocytes produisent et secrètent le ligand Sonic Hh (Shh) et que les cellules endothéliales humaines en cultures primaires expriment le récepteur Patched (Ptch)-1, le co-récepteur Smoothened (Smo) et le facteur de transcription Gli-1. De plus, l’activation de la voie Hh augmente l’étanchéité des cellules endothéliales de la BHE in vitro. Le blocage de l’activation de la voie Hh en utilisant l’antagoniste cyclopamine ainsi qu’en utilisant des souris Shh déficientes (-/-) diminue l’expression des protéines de jonctions serrées, claudin-5, occcludin, et ZO-1. La voie de signalisation s’est aussi montrée comme étant immunomodulatoire, puisque l’activation de la voie dans les cellules endothéliales de la BHE diminue l’expression de surface des molécules d’adhésion ICAM-1 et VCAM-1, ainsi que la sécrétion des chimiokines pro-inflammatoires IL-8/CXCL8 et MCP-1/CCL2, créant une diminution de la migration des lymphocytes CD4+ à travers une monocouche de cellules endothéliales de la BHE. Des traitements avec des cytokines pro-inflammatoires TNF-α and IFN-γ in vitro, augmente la production de Shh par les astrocytes ainsi que l’expression de surface de Ptch-1 et de Smo. Dans des lésions actives de la sclérose en plaques (SEP), où la BHE est plus perméable, les astrocytes hypertrophiques augmentent leur expression de Shh. Par contre, les cellules endothéliales de la BHE n’augmentent pas leur expression de Ptch-1 ou Smo, suggérant une dysfonction dans la voie de signalisation Hh. Ces résultats montrent que la voie de signalisation Hh promeut les propriétés de la BHE, et qu’un environnement d’inflammation pourrait potentiellement dérégler la BHE en affectant la voie de signalisation Hh des cellules endothéliales.

Tempol ameliorates murine viral encephalomyelitis by preserving the blood-brain barrier, reducing viral load, and lessening inflammation

Multiple sclerosis (MS) is a progressive inflammatory and/or demyelinating disease of the human central nervous system (CNS). Most of the knowledge about the pathogenesis of MS has been derived from murine models, such as experimental autoimmune encephalomyelitis and vital encephalomyelitis. Here, we infected female C57BL/6 mice with a neurotropic strain of the mouse hepatitis virus (MHV-59A) to evaluate whether treatment with the multifunctional antioxidant tempol (4-hydroxy-2,2,6,6-tetramethyl-1-piperidinyloxy) affects the ensuing encephalomyelitis. In untreated animals, neurological symptoms developed quickly: 90% of infected mice died 10 days after virus inoculation and the few survivors presented neurological deficits. Treatment with tempol (24 mg/kg, ip, two doses on the first day and daily doses for 7 days plus 2 mM tempol in the drinking water ad libitum) profoundly altered the disease outcome: neurological symptoms were attenuated, mouse survival increased up to 70%, and half of the survivors behaved as normal mice. Not Surprisingly, tempol substantially preserved the integrity of the CNS, including the blood-brain barrier. Furthermore, treatment with tempol decreased CNS vital titers, macrophage and T lymphocyte infiltration, and levels of markers of inflammation, such as expression of inducible nitric oxide synthase, transcription of tumor necrosis factor-alpha and interferon-gamma, and protein nitration. The results indicate that tempol ameliorates murine viral encephalomyelitis by altering the redox status of the infectious environment that contributes to an attenuated CNS inflammatory response. overall, our study supports the development of therapeutic strategies based on nitroxides to manage neuroinflammatory diseases, including MS. (C) 2009 Elsevier Inc. All rights reserved.

Enhancement of blood-tumor barrier permeability by Sar-[D-Phe8]des-Arg9BK, a metabolically resistant bradykinin B1 agonist, in a rat C6 glioma model

Abstract Background While it is well known that bradykinin B2 agonists increase plasma protein extravasation (PPE) in brain tumors, the bradykinin B1 agonists tested thus far are unable to produce this effect. Here we examine the effect of the selective B1 agonist bradykinin (BK) Sar-[D-Phe8]des-Arg9BK (SAR), a compound resistant to enzymatic degradation with prolonged activity on PPE in the blood circulation in the C6 rat glioma model. Results SAR administration significantly enhanced PPE in C6 rat brain glioma compared to saline or BK (p < 0.01). Pre-administration of the bradykinin B1 antagonist [Leu8]-des-Arg (100 nmol/Kg) blocked the SAR-induced PPE in the tumor area. Conclusions Our data suggest that the B1 receptor modulates PPE in the blood tumor barrier of C6 glioma. A possible role for the use of SAR in the chemotherapy of gliomas deserves further study.

A fragment-based approach for the in silico prediction of blood-brain barrier permeation

Blood-brain barrier (BBB) permeation is an essential property for drugs that act in the central nervous system (CNS) for the treatment of human diseases, such as epilepsy, depression, Alzheimer's disease, Parkinson disease, schizophrenia, among others. In the present work, quantitative structure-property relationship (QSPR) studies were conducted for the development and validation of in silico models for the prediction of BBB permeation. The data set used has substantial chemical diversity and a relatively wide distribution of property values. The generated QSPR models showed good statistical parameters and were successfully employed for the prediction of a test set containing 48 compounds. The predictive models presented herein are useful in the identification, selection and design of new drug candidates having improved pharmacokinetic properties.

Volatile anesthetics influence blood-brain barrier integrity by modulation of tight junction protein expression in traumatic brain injury

Disruption of the blood-brain barrier (BBB) results in cerebral edema formation, which is a major cause for high mortalityrnafter traumatic brain injury (TBI). As anesthetic care is mandatory in patients suffering from severe TBI it may be importantrnto elucidate the effect of different anesthetics on cerebral edema formation. Tight junction proteins (TJ) such as zonularnoccludens-1 (ZO-1) and claudin-5 (cl5) play a central role for BBB stability. First, the influence of the volatile anestheticsrnsevoflurane and isoflurane on in-vitro BBB integrity was investigated by quantification of the electrical resistance (TEER) inrnmurine brain endothelial monolayers and neurovascular co-cultures of the BBB. Secondly brain edema and TJ expression ofrnZO-1 and cl5 were measured in-vivo after exposure towards volatile anesthetics in native mice and after controlled corticalrnimpact (CCI). In in-vitro endothelial monocultures, both anesthetics significantly reduced TEER within 24 hours afterrnexposure. In BBB co-cultures mimicking the neurovascular unit (NVU) volatile anesthetics had no impact on TEER. In healthyrnmice, anesthesia did not influence brain water content and TJ expression, while 24 hours after CCI brain water contentrnincreased significantly stronger with isoflurane compared to sevoflurane. In line with the brain edema data, ZO-1 expressionrnwas significantly higher in sevoflurane compared to isoflurane exposed CCI animals. Immunohistochemical analysesrnrevealed disruption of ZO-1 at the cerebrovascular level, while cl5 was less affected in the pericontusional area. The studyrndemonstrates that anesthetics influence brain edema formation after experimental TBI. This effect may be attributed tornmodulation of BBB permeability by differential TJ protein expression. Therefore, selection of anesthetics may influence thernbarrier function and introduce a strong bias in experimental research on pathophysiology of BBB dysfunction. Futurernresearch is required to investigate adverse or beneficial effects of volatile anesthetics on patients at risk for cerebral edema.

Differential roles for endothelial ICAM-1, ICAM-2, and VCAM-1 in shear-resistant T cell arrest, polarization, and directed crawling on blood-brain barrier endothelium

Endothelial ICAM-1 and ICAM-2 were shown to be essential for T cell diapedesis across the blood-brain barrier (BBB) in vitro under static conditions. Crawling of T cells prior to diapedesis was only recently revealed to occur preferentially against the direction of blood flow on the endothelial surface of inflamed brain microvessels in vivo. Using live cell-imaging techniques, we prove that Th1 memory/effector T cells predominantly crawl against the direction of flow on the surface of BBB endothelium in vitro. Analysis of T cell interaction with wild-type, ICAM-1-deficient, ICAM-2-deficient, or ICAM-1 and ICAM-2 double-deficient primary mouse brain microvascular endothelial cells under physiological flow conditions allowed us to dissect the individual contributions of endothelial ICAM-1, ICAM-2, and VCAM-1 to shear-resistant T cell arrest, polarization, and crawling. Although T cell arrest was mediated by endothelial ICAM-1 and VCAM-1, T cell polarization and crawling were mediated by endothelial ICAM-1 and ICAM-2 but not by endothelial VCAM-1. Therefore, our data delineate a sequential involvement of endothelial ICAM-1 and VCAM-1 in mediating shear-resistant T cell arrest, followed by endothelial ICAM-1 and ICAM-2 in mediating T cell crawling to sites permissive for diapedesis across BBB endothelium.

Comparison of immortalized bEnd5 and primary mouse brain microvascular endothelial cells as in vitro blood-brain barrier models for the study of T cell extravasation

Important insights into the molecular mechanism of T cell extravasation across the blood-brain barrier (BBB) have already been obtained using immortalized mouse brain endothelioma cell lines (bEnd). However, compared with bEnd, primary brain endothelial cells have been shown to establish better barrier characteristics, including complex tight junctions and low permeability. In this study, we asked whether bEnd5 and primary mouse brain microvascular endothelial cells (pMBMECs) were equally suited as in vitro models with which to study the cellular and molecular mechanisms of T cell extravasation across the BBB. We found that both in vitro BBB models equally supported both T cell adhesion under static and physiologic flow conditions, and T cell crawling on the endothelial surface against the direction of flow. In contrast, distances of T cell crawling on pMBMECs were strikingly longer than on bEnd5, whereas diapedesis of T cells across pMBMECs was dramatically reduced compared with bEnd5. Thus, both in vitro BBB models are suited to study T cell adhesion. However, because pMBMECs better reflect endothelial BBB specialization in vivo, we propose that more reliable information about the cellular and molecular mechanisms of T cell diapedesis across the BBB can be attained using pMBMECs.

Alpha4beta1 integrin mediates the recruitment of immature dendritic cells across the blood-brain barrier during experimental autoimmune encephalomyelitis

Dendritic cells (DCs) within the CNS are recognized to play an important role in the effector phase and propagation of the immune response in experimental autoimmune encephalomyelitis (EAE), a mouse model for multiple sclerosis. However, the mechanisms regulating DC trafficking into the CNS still need to be characterized. In this study, we show by performing intravital fluorescence videomicroscopy of the inflamed spinal cord white-matter microvasculature in SJL mice with EAE that immature, and to a lesser extent, LPS-matured, bone marrow-derived DCs efficiently interact with the CNS endothelium by rolling, capturing, and firm adhesion. Immature but not LPS-matured DCs efficiently migrated across the wall of inflamed parenchymal microvessels into the CNS. Blocking alpha4 integrins interfered with the adhesion but not the rolling or capturing of immature and LPS-matured DCs to the CNS microvascular endothelium, inhibiting their migration across the vascular wall. Functional absence of beta1 integrins but not of beta7 integrins or alpha4beta7 integrin similarly reduced the adhesion of immature DCs to the CNS microvascular endothelium, demonstrating that alpha4beta1 but not alpha4beta7 integrin mediates this step of immature DCs interaction with the inflamed blood-brain barrier during EAE. Our study shows that during EAE, especially immature DCs migrate into the CNS, where they may be crucial for the perpetuation of the CNS-targeted autoimmune response. Thus therapeutic targeting of alpha4 integrins affects DC trafficking into the CNS and may therefore lead to the resolution of the CNS autoimmune inflammation by reducing the number of CNS professional APCs.