Staphylococcus aureus‐mediated blood–brain barrier injury: an in vitro human brain microvascular endothelial cell model

Blood–brain barrier (BBB) disruption constitutes a hallmark event during pathogen‐mediated neurological disorders such as bacterial meningitis. As a prevalent opportunistic pathogen, Staphylococcus aureus (SA) is of particular interest in this context, although our fundamental understanding of how SA disrupts the BBB is very limited. This paper employs in vitro infection models to address this. Human brain microvascular endothelial cells (HBMvECs) were infected with formaldehyde‐fixed (multiplicity of infection [MOI] 0–250, 0–48 hr) and live (MOI 0–100, 0–3 hr) SA cultures. Both Fixed‐SA and Live‐SA could adhere to HBMvECs with equal efficacy and cause elevated paracellular permeability. In further studies employing Fixed‐SA, infection of HBMvECs caused dose‐dependent release of cytokines/chemokines (TNF‐α, IL‐6, MCP‐1, IP‐10, and thrombomodulin), reduced expression of interendothelial junction proteins (VE‐Cadherin, claudin‐5, and ZO‐1), and activation of both canonical and non‐canonical NF‐κB pathways. Using N‐acetylcysteine, we determined that these events were coupled to the SA‐mediated induction of reactive oxygen species (ROS) within HBMvECs. Finally, treatment of HBMvECs with Fixed‐ΔSpA (MOI 0–250, 48 hr), a gene deletion mutant of Staphylococcal protein A associated with bacterial infectivity, had relatively similar effects to Newman WT Fixed‐SA. In conclusion, these findings provide insight into how SA infection may activate proinflammatory mechanisms within the brain microvascular endothelium to elicit BBB failure.     

Glycation of fibronectin inhibits VEGF‐induced angiogenesis by uncoupling VEGF receptor‐2‐c‐Src crosstalk

Glycation of extracellular matrix proteins has been demonstrated to contribute to the pathogenesis of vascular complications. However, no previous report has shown the role of glycated fibronectin (FN) in vascular endothelial growth factor (VEGF)‐induced angiogenesis. Thus, this study aimed to investigate the effects of glycated FN on VEGF signalling and to clarify the molecular mechanisms involved. FN was incubated with methylglyoxal (MGO) in vitro to synthesize glycated FN, and human umbilical vein endothelial cells (HUVECs) were seeded onto unmodified and MGO‐glycated FN. Then, VEGF‐induced angiogenesis and VEGF‐induced VEGF receptor‐2 (VEGFR‐2) signalling activation were measured. The results demonstrated that normal FN‐positive bands (260 kD) vanished and advanced glycation end products (AGEs) appeared in MGO‐glycated FN and glycated FN clearly changed to a higher molecular mass. The glycation of FN inhibited VEGF‐induced VEGF receptor‐2 (VEGFR‐2), Akt and ERK1/2 activation and VEGF‐induced cell migration, proliferation and tube formation. The glycation of FN also inhibited the recruitment of c‐Src to VEGFR‐2 by sequestering c‐Src through receptor for AGEs (RAGE) and the anti‐RAGE antibody restored VEGF‐induced VEGFR‐2, Akt and ERK1/2 phosphorylation, endothelial cell migration, proliferation and tube formation. Furthermore, the glycation of FN significantly inhibited VEGF‐induced neovascularization in the Matrigel plugs implanted into subcutaneous tissue of mice. Taken together, these data suggest that the glycation of FN may inhibit VEGF signalling and VEGF‐induced angiogenesis by uncoupling VEGFR‐2‐c‐Src interaction. This may provide a novel mechanism for the impaired angiogenesis in diabetic ischaemic diseases.     

D1 receptor‐mediated endogenous tPA upregulation contributes to blood‐brain barrier injury after acute ischaemic stroke

Blood‐brain barrier (BBB) integrity injury within the thrombolytic time window is becoming a critical target to reduce haemorrhage transformation (HT). We have previously reported that BBB damage was initially damaged in non‐infarcted striatum after acute ischaemia stroke. However, the underlying mechanism is not clear. Since acute ischaemic stroke could induce a significant increase of dopamine release in striatum, in current study, our aim is to investigate the role of dopamine receptor signal pathway in BBB integrity injury after acute ischaemia using rat middle cerebral artery occlusion model. Our data showed that 2‐h ischaemia induced a significant increase of endogenous tissue plasminogen activator (tPA) in BBB injury area and intra‐striatum infusion of tPA inhibitor neuroserpin, significantly alleviated 2‐h ischaemia‐induced BBB injury. In addition, intra‐striatum infusion of D1 receptor antagonist SCH23390 significantly decreased ischaemia‐induced upregulation of endogenous tPA, accompanied by decrease of BBB injury and occludin degradation. More important, inhibition of hypoxia‐inducible factor‐1 alpha with inhibitor YC‐1 significantly decreased 2‐h ischaemia‐induced endogenous tPA upregulation and BBB injury. Taken together, our data demonstrate that acute ischaemia disrupted BBB through activation of endogenous tPA via HIF‐1α upregulation, thus representing a new therapeutic target for protecting BBB after acute ischaemic stroke.     

Spontaneous fibroblast‐derived pericyte recruitment in a human tissue‐engineered angiogenesis model in vitro

Cooperation between endothelial cells and pericytes is essential to the stabilization and maturation of blood microvessels. We developed a unique in vitro tissue‐engineered model to study angiogenesis. The human endothelialized reconstructed connective tissue model promotes the formation of a three‐dimensional branching network of capillary‐like tubes (CLT) with closed lumens. The purpose of this work was to investigate whether pericytes were spontaneously recruited around CLT in the model. We demonstrated that smooth muscle α‐actin (SMA)‐positive cells were found closely associated with PECAM‐1‐positive capillaries in the model. Twelve percent (±2.6) of SMA‐positive cells were detected along with 15% (±1.64) von Willebrand factor‐positive endothelial cells in the culture system after 31 days of in vitro maturation. Conversely, no SMA‐positive cells were detected in reconstructed connective tissues made solely of fibroblasts. Knowing that PDGF is a major factor in the recruitment of pericytes, we showed that blockade of the PDGFB receptor using the inhibitor AG1296 induced an overall 5, 2.6, and 2.4‐fold decrease in the SMA‐positive cells, von Willebrand factor‐positive cells, and number of capillaries, respectively. Using combinations of human GFP‐positive fibroblasts and endothelial cells, we demonstrated that pericytes were recruited from the fibroblast population in the model. In conclusion, our tissue‐engineered culture system promotes the spontaneous formation of a network of capillaries and the recruitment of pericytes derived from fibroblasts. Since pericytes are essential components of the blood microvasculature, this culture system is a powerful model to study angiogenesis and endothelial cell/pericyte interactions in vitro. J. Cell. Physiol. 227: 2130–2137, 2012. © 2011 Wiley Periodicals, Inc.     

Mutagenicity induced by UVC in Escherichia coli cells: reactive oxygen species involvement

We previously demonstrated that reactive oxygen species (ROS) could be involved in the DNA damage induced by ultraviolet-C (UVC). In this study, we evaluated singlet oxygen ((1)O(2)) involvement in UVC-induced mutagenesis in Escherichia coli cells. First, we found that treatment with sodium azide, an (1)O(2) chelator, protected cells against UVC-induced lethality. The survival assay showed that the fpg mutant was more resistant to UVC lethality than the wild-type strain. The rifampicin mutagenesis assay showed that UVC mutagenesis was inhibited five times more in cells treated with sodium azide, and stimulated 20% more fpg mutant. These results suggest that (1)O(2) plays a predominant role in UVC-induced mutagenesis. (1)O(2) generates a specific mutagenic lesion, 8-oxoG, which is repaired by Fpg protein. This lesion was measured by GC-TA reversion in the CC104 strain, its fpg mutant (BH540), and both CC104 and BH540 transformed with the plasmid pFPG (overexpression of Fpg protein). This assay showed that mutagenesis was induced 2.5-fold in the GC-TA strain and 7-fold in the fpg mutant, while the fpg mutant transformed with pFPG was similar to GC-TA strain. This suggests that UVC can also cause ROS-mediated mutagenesis and that the Fpg protein may be involved in this repair.     

Transendothelial permeability changes induced by free radicals in an in vitro model of the blood-brain barrier.

In the present study, we investigated the changes in blood-brain barrier (BBB) permeability following brain endothelial cell exposure to different xenobiotics able to promote free radical generation during their metabolism. Our in vitro BBB model consisted of confluent monolayers of immortalized rat brain capillary endothelial cells (RBE4) grown on collagen-coated filters in the presence of C6 glioma cells grown in the lower compartment. We have recently shown that a range of xenobiotics, including menadione, nitrofurazone, and methylviologen (paraquat) may undergo monoelectronic redox cycling in isolated brain capillaries, giving rise to reactive oxygen species. In this study, addition of 100 microM menadione to the culture medium for 30 min significantly increased the permeability of endothelial cell monolayers to radiolabeled sucrose. The effect on endothelial permeability induced by menadione was dose-dependent and reversible. These permeability changes preceded the onset of cell death, as assessed by the Trypan blue exclusion method. Pre-incubation with superoxide dismutase and catalase blocked changes in sucrose permeability to control levels in a dose-dependent manner, suggesting the involvement of reactive oxygen species in menadione-induced BBB opening.     

The m.3243A>G mtDNA mutation is pathogenic in an in vitro model of the human blood brain barrier

MELAS is a common mitochondrial disease frequently associated with the m.3243A>G point mutation in the tRNA^Leu(UUR) of mitochondrial DNA and characterized by stroke-like episodes with vasogenic edema and lactic acidosis. The pathogenic mechanism of stroke and brain edema is not known. Alterations in the blood brain barrier (BBB) caused by respiratory chain defects in the cortical microvessels could explain the pathogenesis. To test this hypothesis we developed a tissue culture model of the human BBB. The MELAS mutation was introduced into immortalized brain capillary endothelial cells and astrocytes. Respiratory chain activity and transendothelial electrical resistance, TEER was measured. Severe defects of respiratory chain complex I and IV activities, and a moderate deficiency of complex II activity in cells harboring the MELAS mutation were associated with low TEER, indicating that the integrity of the BBB was compromised. These data support our hypothesis that respiratory chain defects in the components of the BBB cause changes in permeability.     

Biosynthesis in vitro of tryptophanase by polyribosomes from induced cultures of Escherichia coli

1. Polyribosomes were isolated from Escherichia coli grown in media in which tryptophanase is induced and in which it is repressed. The polyribosomes from the induced bacteria had a small amount of tryptophanase activity associated with them. 2. A portion of the enzyme activity remained bound to polyribosomes during centrifuging in sucrose gradients. 3. Incubation of tryptophanase-containing polyribosomes with puromycin released enzyme activity. 4. The binding of the enzyme to the polyribosomes did not depend on the presence of DNA. 5. When the polyribosomes were incubated under conditions of protein synthesis with supernatant fraction obtained from repressed bacteria, a small but statistically significant increase in enzyme activity was produced. 6. When a radioactive amino acid was included in the incubation mixture for the tryptophanase system a radioactive protein was obtained whose chromatographic, electrophoretic and sedimentation properties were identical with those of tryptophanase. 7. The amount of incorporation was consistent with the amount of new enzyme synthesis predicted by the increase in enzyme activity. Both radioactive incorporation and increase in enzyme activity were shown to be energy-dependent and also negative controls were obtained by using zero-time incubations or polyribosomes isolated from either repressed cells or a mutant lacking the ability to produce tryptophanase. 8. The distribution of radioactive leucine in the carboxyl region of the newly labelled tryptophanase was examined by digesting the labelled protein with carboxypeptidases. It was shown that the radioactivity was more highly concentrated towards the carboxyl terminus when the incubation times for protein synthesis were shorter (implying that, with longer incubation times, longer lengths of polypeptide chain contained radioactive amino acid residues).     

Rapid microglial activation induced by traumatic brain injury is independent of blood brain barrier disruption

Following CNS injury, microglia respond and transform into reactive species exhibiting characteristic morphological changes that have been termed "activated" or "ameboid" microglia. In an attempt to establish that microglial reactions induced immediately after injury are caused by intrinsic mechanisms rather than infiltration of blood and its constituents, oxygenized Ringer's solution was perfused into the cerebral circulation of rats so that the circulating blood could be eliminated prior to injury induction. Under artificial respiration, a catheter was inserted from the cardiac apex into the ascending aorta, and oxygenized Ringer's solution was immediately perfused with a pulsatile blood pump, resulting in wash out of the circulating blood from the brain within 1 min. Subsequently, a cortical contusion was induced in the unilateral parietal cortex using a controlled cortical impact (CCI) device. At 5 min following the injury, the brain was fixed by perfusion of fixative through the catheter and removed. Coronal vibratome sections were then processed for CR3 immunohistochemistry to examine the microglial activation. It appeared that microglial activation with both morphological transformation and an increase in CR3 immunoreactivity was induced throughout the hemisphere ipsilateral to the injury side exclusively, even in rats with elimination of circulating blood. The microglial reactions did not differ substantially from those observed in the control rats with extensive BBB disruption. The present results thus provide direct evidence that the microglial activation induced immediately after injury is independent of infiltration of circulating blood induced by concurrent BBB disruption.     

Aβ1–42 oligomer‐induced leakage in an in vitro blood–brain barrier model is associated with up‐regulation of RAGE and metalloproteinases,and down‐regulation of tight junction scaffold proteins

Accumulating evidence indicates that abnormal deposition of amyloid‐β (Aβ) peptide in the brain is responsible for endothelial cell damage and consequently leads to blood–brain barrier (BBB) leakage. However, the mechanisms underlying BBB disruption are not well described. We employed an monolayer BBB model comprising bEnd.3 cell and found that BBB leakage was induced by treatment with Aβ_1–42, and the levels of tight junction (TJ) scaffold proteins (ZO‐1, Claudin‐5, and Occludin) were decreased. Through comparisons of the effects of the different components of Aβ_1–42, including monomer (Aβ_1–42‐Mono), oligomer (Aβ_1–42‐Oligo), and fibril (Aβ_1–42‐Fibril), our data confirmed that Aβ_1–42‐Oligo is likely to be the most important damage factor that results in TJ damage and BBB leakage in Alzheimer's disease. We found that the incubation of bEnd.3 cells with Aβ_1–42 significantly up‐regulated the level of receptor for advanced glycation end‐products (RAGE). Co‐incubation of a polyclonal antibody to RAGE and Aβ_1–42‐Oligo in bEnd.3 cells blocked RAGE suppression of Aβ_1–42‐Oligo‐induced alterations in TJ scaffold proteins and reversed Aβ_1–42‐Oligo‐induced up‐regulation of RAGE, matrix metalloproteinase (MMP)‐2, and MMP‐9. Furthermore, we found that these effects induced by Aβ_1–42‐Oligo treatment were effectively suppressed by knockdown of RAGE using small interfering RNA (siRNA) transfection. We also found that GM 6001, a broad‐spectrum MMP inhibitor, partially reversed the Aβ_1–42‐Oligo‐induced inhibitor effects in bEnd.3 cells. Thus, these results suggested that RAGE played an important role in Aβ‐induced BBB leakage and alterations of TJ scaffold proteins, through a mechanism that involved up‐regulation of MMP‐2 and MMP‐9.

     

Preparation of extracellular domain 3 of human VEGF receptor‐2 and the monitoring of its real‐time binding to VEGF by biosensors

Vascular endothelial growth factor receptor‐2 (VEGFR‐2) plays an important role in stimulating the proliferation of endothelial cells and improving the permeability of blood vessels, which is involved in tumor angiogenesis, a process that is essential for tumor growth and metastasis. In this study, we describe a method for high yield of recombinant extracellular domain 3 (KDR3) of human VEGFR‐2 in an Escherichia coli system with further purification by cation exchange chromatography and immobilized metal affinity chromatography (IMAC). The biological activity of recombinant KDR3 was performed by sequestering VEGF in HUVEC proliferation assay. The real‐time binding of human VEGF to immobilized KDR3 was monitored by a label‐free biosensor, Optical waveguide lightmode spectroscopy (OWLS). Under the given experimental conditions, the association rate constant k_a was 4.2 × 10³ M^?1 s^?1 and the dissociation rate k_d was 5.1 × 10^?3 s^?1. The dissociation constant K_D was then calculated to be 1.2 × 10^?6 M. The obtained values will serve as baseline parameters for the design of improved versions of recombinant soluble VEGF receptors and the evaluation of developed anti‐KDR antibodies. In addition, such a scenario established by the use of OWLS will potentiate the kinetic study of ligand/receptor and antigen/antibody. The receptor discussed here, which block VEGF binding to cell membrane KDR, have potential clinical application in the treatment of cancer and other diseases where pathological angiogenesis is involved. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Effect of mild hypothermia on blood brain barrier disruption induced by oleic acid in rats

The blood-brain barrier (BBB) is essential for the normal function of the central nervous system. The pathological conditions induced by brain diseases including cerebral ischemia result in the alteration of BBB integrity. This alteration of BBB is relieved by mild hypothermia that has been regarded as an effective therapy for brain injury. Experimental fat embolism by intra-arterial administration of fatty acid induces reversible dysfunction of BBB and is considered as a beneficial method for the research on BBB disruption. However, the implication of hypothermia on the fatty acid-induced BBB disruption is not clear yet. In this study, we aim to investigate the effect of mild hypothermia on BBB disruption by comparing the changes of brain inflammation, free radical production, and matrix metalloproteinases (MMPs) caused by cerebral fatty acid infusion between normothermic (37°C) and hypothermic (33°C) groups. Oleic acid infusion into the carotid artery induced the increase of BBB permeability, which was inhibited by mild hypothermia. Neutrophils were infiltrated and intercellular adhesion molecule-1 (ICAM-1) expression was increased in the vascular structures in the affected brain tissue of normothermic rats at 24 hrs following oleic acid administration. Inducible nitric oxide synthase (iNOS) and nitro-tyrosine immunoreactivities were also observed in the normothermic group. The expression of matrix metalloproteinase (MMP)-2, 3, and 13 were upregulated predominantly in the oleic acid-treated brain of the normothermic rats. In mild hypothermic condition, neutrophil infiltration and ICAM-1 expression were attenuated, whereas the inductions of iNOS, nitrotyrosine and MMPs except MMP3 were not affected. Therefore, we suggest that mild hypothermia contributes to the protective effect on oleic acid-induced BBB damage via reducing neutrophil infiltration and brain inflammation.     

VIM‐1 carbapenemase‐producing Escherichia coli in gulls from southern France

Acquired carbapenemases currently pose one of the most worrying public health threats related to antimicrobial resistance. A NDM‐1‐producing Salmonella Corvallis was reported in 2013 in a wild raptor. Further research was needed to understand the role of wild birds in the transmission of bacteria resistant to carbapenems. Our aim was to investigate the presence of carbapenem‐resistant Escherichia coli in gulls from southern France. In 2012, we collected 158 cloacal swabs samples from two gull species: yellow‐legged gulls (Larus michahellis) that live in close contact with humans and slender‐billed gulls (Chroicocephalus genei) that feed at sea. We molecularly compared the carbapenem‐resistant bacteria we isolated through culture on selective media with the carbapenem‐susceptible strains sampled from both gull species and from stool samples of humans hospitalized in the study area. The genes coding for carbapenemases were tested by multiplex PCR. We isolated 22 carbapenem‐resistant E. coli strains from yellow‐legged gulls while none were isolated from slender‐billed gulls. All carbapenem‐resistant isolates were positive for bla_VIM‐1 gene. VIM‐1‐producing E. coli were closely related to carbapenem‐susceptible strains isolated from the two gull species but also to human strains. Our results are alarming enough to make it urgently necessary to determine the contamination source of the bacteria we identified. More generally, our work highlights the need to develop more bridges between studies focusing on wildlife and humans in order to improve our knowledge of resistant bacteria transmission routes.     

Afamin is synthesized by cerebrovascular endothelial cells and mediates α-tocopherol transport across an in vitro model of the blood–brain barrier

α-Tocopherol (αTocH), a member of the vitamin E family, is essential for normal neurological function. Despite the importance of αTocH transport into the CNS, transfer mechanisms across the blood–brain barrier (BBB) are not entirely clear. We here investigate whether afamin, a known αTocH-binding protein, contributes to αTocH transport across an in vitro model of the BBB consisting of primary porcine brain capillary endothelial cells (BCEC) and basolaterally cultured astrocytoma cells. Exogenously added afamin had no adverse effects on BCEC viability or barrier function and was transported across BCEC Transwell cultures. Furthermore, αTocH transport across polarized BCEC cultures to astrocytoma cells is facilitated by afamin, though to a lesser extent than by high-density lipoprotein-mediated transport, an essential and in vivo operating αTocH import pathway at the cerebrovasculature. We also demonstrate that porcine BCEC endogenously synthesize afamin. In line with these in vitro findings, afamin was detected by immunohistochemistry in porcine, human postmortem, and mouse brain, where prominent staining was observed almost exclusively in the cerebrovasculature. The demonstration of afamin mRNA expression in isolated brain capillaries suggests that afamin might be a new family member of binding/transport proteins contributing to αTocH homeostasis at the BBB in vivo .