0000000000134273
AUTHOR
Christoph R.w. Kuhlmann
showing 19 related works from this author
The Functional Role of the Second NPXY Motif of the LRP1 β-Chain in Tissue-type Plasminogen Activator-mediated Activation of N-Methyl-D-aspartate Rec…
2008
The low density lipoprotein receptor-related protein 1 (LRP1) emerges to play fundamental roles in cellular signaling pathways in the brain. One of its prominent ligands is the serine proteinase tissue-type plasminogen activator (tPA), which has been shown to act as a key activator of neuronal mitogen-activated protein kinase pathways via the N-methyl-D-aspartate (NMDA) receptor. However, here we set out to examine whether LRP1 and the NMDA receptor might eventually act in a combined fashion to mediate tPA downstream signaling. By blocking tPA from binding to LRP1 using the receptor-associated protein, we were able to completely inhibit NMDA receptor activation. Additionally, inhibition of …
Oxidative stress upregulates the NMDA receptor on cerebrovascular endothelium.
2009
N-methyl-d-aspartate receptor (NMDA-R)-mediated oxidative stress has been implicated in blood-brain barrier (BBB) disruption in a variety of neuropathological diseases. Although some interactions between both phenomena have been elucidated, possible influences of reactive oxygen species (ROS) on the NMDA-R itself have so far been neglected. The objective of this study was to examine how the cerebroendothelial NMDA-R is affected by exposure to oxidative stress and to assess possible influences on BBB integrity. RT-PCR confirmed several NMDA-R subunits (NR1, NR2B-D) expressed in the bEnd3 cell line (murine cerebrovascular endothelial cells). NR1 protein expression after exposure to ROS was ob…
LRP1 mediates bidirectional transcytosis of amyloid-β across the blood-brain barrier.
2011
According to the "amyloid hypothesis", the amyloid-β (Aβ) peptide is the toxic intermediate driving Alzheimer's disease (AD) pathogenesis. Recent evidence suggests that the low density lipoprotein receptor-related protein 1 (LRP1) transcytoses Aβ out of the brain across the blood-brain barrier (BBB). To provide genetic evidence for LRP1-mediated transcytosis of Aβ across the BBB we analyzed Aβ transcytosis across primary mouse brain capillary endothelial cells (pMBCECs) derived from wild-type and LRP1 knock-in mice. Here, we show that pMBCECs in vitro express functionally active LRP1. Moreover, we demonstrate that LRP1 mediates transcytosis of [(125)I]-Aβ(1-40) across pMBCECs in both direct…
Pro-inflammatory effects of interleukin-17A on vascular smooth muscle cells involve NAD(P)H- oxidase derived reactive oxygen species.
2010
T cells are known for their contribution to the inflammatory element of atherosclerosis. Recently, it has been demonstrated that the Th17 derived cytokine IL-17 is involved in the pro-inflammatory response of vascular smooth muscle cells (VSMC). The aim of the present study was to examine whether reactive oxygen species (ROS) might be involved in this context. The effect of IL-17A on ROS generation was examined using the fluorescent dye 2′7′-dichlorodihydrofluorescein (H<sub>2</sub>DCF) in primary murine VSMC. IL-17A induced an increase in H<sub>2</sub>DCF fluorescence in VSMC, and this effect was blocked by the NAD(P)H-oxidase inhibitor apocynin and siRNA targeting …
Inhibition of the myosin light chain kinase prevents hypoxia-induced blood-brain barrier disruption
2007
Increased mortality after stroke is associated with development of brain edema. The aim of the present study was to examine the contribution of endothelial myosin light chain (MLC) phosphorylation to hypoxia-induced blood-brain barrier (BBB) opening. Measurements of trans-endothelial electrical resistance (TEER) were performed to analyse BBB integrity in an in vitro co-culture model (bovine brain microvascular endothelial cells (BEC) and rat astrocytes). Brain fluid content was analysed in rats after stroke induction using a two-vein occlusion model. Dihydroethidium was used to monitor intracellular generation of reactive oxygen species (ROS) in BEC. MLC phosphorylation was detected using i…
Fine-tuning DNA/albumin polyelectrolyte interactions to produce the efficient transfection agent cBSA-147.
2010
We present the preparation and isolation of different chemically modified BSA species with varying numbers of primary amino groups at the surface. Highly cationic albumin proteins with increased numbers of amino groups were achieved and complex formation with plasmid DNA was carefully investigated. We compare the transfection results, polyelectrolyte complexes morphologies with their impact on complex stabilities, cytotoxicities and DNA accessibility. This knowledge-driven approach led to the identification of the efficient non-viral DNA delivery agent cBSA-147, which showed high transfection efficacies and stability.
Caspase-3 contributes to ZO-1 and Cl-5 tight-junction disruption in rapid anoxic neurovascular unit damage.
2011
BACKGROUND: Tight-junction (TJ) protein degradation is a decisive step in hypoxic blood-brain barrier (BBB) breakdown in stroke. In this study we elucidated the impact of acute cerebral ischemia on TJ protein arrangement and the role of the apoptotic effector protease caspase-3 in this context. METHODOLOGY/PRINCIPAL FINDINGS: We used an in vitro model of the neurovascular unit and the guinea pig whole brain preparation to analyze with immunohistochemical methods the BBB properties and neurovascular integrity. In both methodological approaches we observed rapid TJ protein disruptions after 30 min of oxygen and glucose deprivation or middle cerebral artery occlusion, which were accompanied by…
Fluvastatin prevents glutamate-induced blood-brain-barrier disruption in vitro.
2008
Abstract Glutamate is an important excitatory amino acid in the central nervous system. Under pathological conditions glutamate levels dramatically increase. Aim of the present study was to examine whether the HMG-CoA inhibitor fluvastatin prevents glutamate-induced blood-brain-barrier (BBB) disruption. Measurements of transendothelial electrical resistance (TEER) were performed to analyze BBB integrity in an in vitro co-culture model of brain endothelial and glial cells. Myosin light chain (MLC) phosphorylation was detected by immunohistochemistry, or using the in-cell western technique. Intracellular Ca 2+ and reactive oxygen species (ROS) levels were analyzed using the fluorescence dyes …
Cellular mechanisms of IL-17-induced blood-brain barrier disruption.
2009
Recently T-helper 17 (Th17) cells were demonstrated to disrupt the blood-brain barrier (BBB) by the action of IL-17A. The aim of the present study was to examine the mechanisms that underlie IL-17A-induced BBB breakdown. Barrier integrity was analyzed in the murine brain endothelial cell line bEnd.3 by measuring the electrical resistance values using electrical call impedance sensing technology. Furthermore, in-cell Western blots, fluorescence imaging, and monocyte adhesion and transendothelial migration assays were performed. Experimental autoimmune encephalomyelitis (EAE) was induced in C57BL/6 mice. IL-17A induced NADPH oxidase- or xanthine oxidase-dependent reactive oxygen species (ROS)…
Studying the Neurovascular Unit: An Improved Blood–Brain Barrier Model
2009
The blood–brain barrier (BBB) closely interacts with the neuronal parenchyma in vivo. To replicate this interdependence in vitro, we established a murine coculture model composed of brain endothelial cell (BEC) monolayers with cortical organotypic slice cultures. The morphology of cell types, expression of tight junctions, formation of reactive oxygen species, caspase-3 activity in BECs, and alterations of electrical resistance under physiologic and pathophysiological conditions were investigated. This new BBB model allows the application of techniques such as laser scanning confocal microscopy, immunohistochemistry, fluorescent live cell imaging, and electrical cell substrate impedance se…
BDNF-induced nitric oxide signals in cultured rat hippocampal neurons: time course, mechanism of generation, and effect on neurotrophin secretion
2014
BDNF and nitric oxide signaling both contribute to plasticity at glutamatergic synapses. However, the role of combined signaling of both pathways at the same synapse is largely unknown. Using NO imaging with diaminofluoresceine in cultured hippocampal neurons we analyzed the time course of neurotrophin-induced NO signals. Application of exogenous BDNF, NT-4, and NT-3 (but not NGF) induced NO signals in the soma and in proximal dendrites of hippocampal neurons that were sensitive to NO synthase activity, TrkB signaling, and intracellular calcium elevation. The effect of NO signaling on neurotrophin secretion was analyzed in BDNF-GFP, and NT-3-GFP transfected hippocampal neurons. Exogenous ap…
Impaired calcium homeostasis in aged hippocampal neurons
2009
Abstract Development of neurodegenerative diseases such as Alzheimer's and Parkinson's disease is strongly age-associated. The impairment of calcium homeostasis is considered to be a key pathological event leading to neuronal dysfunction and cell death. However, the exact impact of aging on calcium homeostasis in neurons remains largely unknown. In the present work we have investigated intracellular calcium levels in cultured primary hippocampal neurons from young (2 months) and aged (24 months) rat brains. Upon stimulation with glutamate or hydrogen peroxide aged neurons in comparison to young neurons demonstrated an increased vulnerability to these disease-related toxins. Measurement of c…
Fluvastatin stabilizes the blood–brain barrier in vitro by nitric oxide-dependent dephosphorylation of myosin light chains
2006
Inhibition of the 3-hydroxy-3-methylglutaryl-coenzyme-A reductase and the downstream mevalonate pathway is in part responsible for the beneficial effects that statins exert on the cardiovascular system. In this study we aimed at analysing the stabilizing effects of fluvastatin on the blood-brain barrier (BBB) integrity, using an in vitro co-culture model of ECV304 and C6, or primary bovine endothelial cells and rat astrocytes. Fluvastatin dose-dependently (1-25 micromol/l) increased barrier integrity as analysed by measurements of transendothelial electrical resistance (TEER). This effect (117.4+/-2.6% at 25 micromol/l) was significantly reduced by the nitric oxide (NO) synthase inhibitor L…
Mechanisms of C-reactive protein-induced blood-brain barrier disruption.
2009
Background and Purpose— Increased mortality after stroke is associated with brain edema formation and high plasma levels of the acute phase reactant C-reactive protein (CRP). The aim of this study was to examine whether CRP directly affects blood–brain barrier stability and to analyze the underlying signaling pathways. Methods— We used a cell coculture model of the blood–brain barrier and the guinea pig isolated whole brain preparation. Results— We could show that CRP at clinically relevant concentrations (10 to 20 μg/mL) causes a disruption of the blood–brain barrier in both approaches. The results of our study further demonstrate CRP-induced activation of surface Fcγ receptors CD16/32 fo…
MK801 blocks hypoxic blood-brain-barrier disruption and leukocyte adhesion.
2008
The aim of the present study was to examine the signaling pathways of hypoxia followed by reoxygenation (H/R)-induced disruption of the blood-brain-barrier (BBB) in a co-culture of astrocytes and brain endothelial cells (BEC) in vitro. We analyzed the possible stabilizing effect of MK801, a highly selective N-methyl-d-aspartate receptor (NMDAR) antagonist, on BBB integrity. Levels of reactive oxygen species (ROS), glutamate (Glut) release and monocyte adhesion were measured under normoxia and H/R. BBB integrity was monitored measuring the trans-endothelial electrical resistance (TEER). TEER values dropped under H/R conditions which was abolished by MK801. Glut release from astrocytes, but n…
CRP-induced levels of oxidative stress are higher in brain than aortic endothelial cells
2010
C-reactive protein (CRP) has been demonstrated to induce blood-brain barrier disruption (BBB) involving NAD(P)H-oxidase dependent oxidative stress. It is unclear why CRP affects the BBB and not other vascular beds following stroke. Therefore we examined CRP receptor and NAD(P)H-oxidase expression levels in bovine brain- (BEC) and aortic endothelial cells. Dichlorodihydrofluorescein measurements revealed significantly higher CRP-induced reactive oxygen species (ROS) levels in BEC. Protein expression of the CRP-receptors CD16, CD32 and of the NAD(P)H-oxidase subunit p22phox were also significantly higher in BEC. In conclusion BEC show a higher vulnerability to CRP due to increased levels of C…
Inhibition of myosin light chain kinase reduces brain edema formation after traumatic brain injury.
2010
The role of the endothelial contractile apparatus in the process of brain edema formation after brain trauma is not characterized. Phosphorylation of myosin light chains by myosin light chain kinases (MLCK) activates endothelial contractile elements and results in a rearrangement of the cytoskeleton. This may enhance post-traumatic blood-brain barrier dysfunction. In order to investigate the role of the MLCK on brain edema formation and blood-brain barrier permeability after brain injury, mice were anesthetized and subjected to a controlled cortical impact (CCI). MLCK expression is significantly up-regulated after CCI with a maximum 12 h post-injury. Specific inhibition of MLCK by ML-7 resu…
Effects of cerivastatin on adrenergic pathways, hypertrophic growth and TGFbeta expression in adult ventricular cardiomyocytes.
2010
Abstract The effects of statin treatment in the setting of heart failure have already been shown. Nevertheless, there is little knowledge about its influence on adrenergic pathways in cardiomyocytes. Therefore, this study investigated the impact of cerivastatin on adrenoceptor-mediated signalling pathways in isolated adult ventricular cardiomyocytes. It focused on two endpoints: hypertrophic growth and TGFbeta expression. Cultured cardiomyocytes were used to study rac activation (analysed by its translocation into the membrane fraction), ROS formation (H 2 DCF fluorescence) and hypertrophic growth ( 14 C-phenylalanine incorporation). Alpha- and beta-adrenoceptor stimulation showed significa…
An Alternative Pathway of Imiquimod-Induced Psoriasis-Like Skin Inflammation in the Absence of Interleukin-17 Receptor A Signaling
2013
Topical application of imiquimod (IMQ) on the skin of mice induces inflammation with common features found in psoriatic skin. Recently, it was postulated that IL-17 has an important role both in psoriasis and in the IMQ model. To further investigate the impact of IL-17RA signaling in psoriasis, we generated IL-17 receptor A (IL-17RA)-deficient mice (IL-17RA(del)) and challenged these mice with IMQ. Interestingly, the disease was only partially reduced and delayed but not abolished when compared with controls. In the absence of IL-17RA, we found persisting signs of inflammation such as neutrophil and macrophage infiltration within the skin. Surprisingly, already in the naive state, the skin …