Oxidativer Stress als Modulator der NMDA-Rezeptorexpression auf zerebrovaskulären Endothelzellen

Oxidativer Stress in Form reaktiver Sauerstoffspezies (ROS) und Exzitotoxizität durch supraphysiologische Konzentrationen des Neurotransmitters Glutamat sind nicht nur beteiligt an der Pathogenese vielzähliger neurodegenerativer Erkrankungen wie Schlaganfall, Hirntrauma, Alzheimer Demenz oder Multipler Sklerose, sondern spielen zudem eine Schlüsselrolle im dort beobachteten Zusammenbruch der Blut-Hirn-Schranke. Glutamat führt durch Stimulation neuronaler und endothelialer NMDA-Rezeptoren zu einer Generierung von ROS. Nicht verfolgt worden war bisher, welche Auswirkungen ROS umgekehrt auch auf den NMDA-Rezeptor haben könnten. Im Rahmen der vorliegenden Arbeit wurde daher untersucht, ob und in welcher Weise die Exposition gegenüber reaktiven Sauerstoffspezies einen Einfluss auf die Expression und Aktivierbarkeit von NMDA-Rezeptoren auf zerebrovaskulären Endothelzellen ausübt.rnEs konnte zunächst die Expression der funktionell obligaten NR-1 Untereinheit des NMDA-Rezeptors auf der verwendeten Zelllinie b.End3 mittels Immunfluoreszenz-Mikroskopie gesichert werden. Ein Nachweis von mRNA für die Untereinheiten NR1 und NR2B, C und D erfolgte mittels RT-PCR. In der Analyse der replizierten RNA zeigten sich Hinweise für eine heterogene Komposition der exprimierten endothelialen NMDA- Rezeptoren.rnEs konnte weiter mit Hilfe der In-Cell-Western-Technik gezeigt werden, dass die Expression des NMDA-Rezeptors durch transiente Stimulation mit reaktiven Sauerstoffspezies im Sinne einer Heraufregulation moduliert werden kann. Die Stimulation der Zellen mit den reaktiven Sauerstoffspezies O2-, ONOO- und H2O2 führte dabei im Experiment zu einer deutlichen Zunahme der NR1-Expression, die spätestens nach 72 Stunden höchst signifikant war.rnUm zu überprüfen, welche Bedeutung diese Überexpression für die Integrität der Blut-Hirn-Schranke unter den exzitotoxischen Bedingungen hoher Glutamatkonzentrationen haben könnte, wurde mit Hilfe des ECIS-Systems („Electrical Cell-Substrate Impedance Sensing“) die Impedanz ROS-präexponierter Endothelmonolayer gemessen. Auf Rezeptorstimulation mit dem spezifischen Agonisten NMDA reagierten die vorbehandelten Gruppen mit einem Abfall der Impedanz gegenüber der nicht vorbehandelten Kontrolle.rnrnDie vorliegenden Ergebnisse zeigen, dass ROS in der Lage sind, funktionelle endotheliale NMDA-Rezeptoren zu induzieren und auf diesem Weg zu einem verstärkten Abfall der BHS-Integrität unter den Bedingungen exzitotoxischen und oxidativen Stresses führen. Dies stellt einen neuen Mechanismus zur Erklärung der Pathogenese des Blut-Hirn-Schrankenversagens dar.Oxidative stress in the form of reactive oxygen species (ROS) and excitotoxicity caused by excessive concentrations of the neurotransmitter glutamate not only participate in the pathogenesis of diverse neurodegenerative diseases like stroke, brain trauma, Alzheimer dementia or multiple sclerosis but do also play a key role in the disruption of the blood-brain barrier observed in these pathologies. Glutamate leads to generation of ROS via stimulation of neuronal and endothelial NMDA receptors. As yet, no investigations have been conducted examining whether ROS might have influences on the NMDA receptor as well. The present study was therefore performed to establish if and in what manner exposition to ROS modulates the expression and activity of NMDA receptors on cerebrovascular endothelium.rnFirstly, expression of the NR1 subunit mandatory for functionality of NMDA the receptor was confirmed on the b.End3 cell line employed in the experiments with immunofluorescence microscopy. RT-PCR was performed to detect mRNA for the subunits NR1 as well as NR2B, C and D. Analysis of replicated RNA suggested a heterogeneous composition of expressed endothelial NMDA receptors.rnUsing the In-Cell Western technique, it was shown that transient stimulation of cells with ROS modulates NMDA receptor expression, resulting in an up-regulation of receptors. Stimulation with the reactive oxygen species O2-, ONOO- and H2O2 led to marked increase in NR1 expression, which was highly significant ( p < 0.001) after 72 hours.rnTo evaluate the impact of overexpression of NMDA receptors on the integrity of the blood-brain barrier, the ECIS system („Electrical Cell-Substrate Impedance Sensing“) was used to measure the impedance of an ROS pre-exposed monolayer. Pre-treated groups reacted to stimulation using the specific agonist NMDA with a decrease in impedance as opposed to non pre-treated controls.rnrnThe results presented in this study support the hypothesis that ROS induce functional endothelial NMDA receptors and thus further disruption of blood-brain barrier integrity under conditions of excitotoxic and oxidative stress. Upregulation of NMDA receptors through the action of oxidative stress represents a novel mechanism in the pathogenesis of blood-brain barrier dysfunction

CXCR-4 expression by circulating endothelial progenitor cells and SDF-1 serum levels are elevated in septic patients

Background: Endothelial progenitor cell (EPC) numbers are increased in septic patients and correlate with survival. In this study, we investigated, whether surface expression of chemokine receptors and other receptors important for EPC homing is upregulated by EPC from septic patients and if this is associated with clinical outcome. Methods: Peripheral blood mononuclear cells from septic patients (n = 30), ICU control patients (n = 11) and healthy volunteers (n = 15) were isolated by Ficoll density gradient centrifugation. FACS-analysis was used to measure the expression of the CXC motif chemokine receptors (CXCR)-2 and − 4, the receptor for advanced glycation endproducts (RAGE) and the stem cell factor receptor c-Kit. Disease severity was assessed via the Simplified Acute Physiology Score (SAPS) II. The serum concentrations of vascular endothelial growth factor (VEGF), stromal cell-derived factor (SDF)-1α and angiopoietin (Ang)-2 were determined with Enzyme linked Immunosorbent Assays. Results: EPC from septic patients expressed significantly more CXCR-4, c-Kit and RAGE compared to controls and were associated with survival-probability. Significantly higher serum concentrations of VEGF, SDF-1α and Ang-2 were found in septic patients. SDF-1α showed a significant association with survival. Conclusions: Our data suggest that SDF-1α and CXCR-4 signaling could play a crucial role in EPC homing in the course of sepsis

CXCR-4 expression by circulating endothelial progenitor cells and SDF-1 serum levels are elevated in septic patients

Background: Endothelial progenitor cell (EPC) numbers are increased in septic patients and correlate with survival. In this study, we investigated, whether surface expression of chemokine receptors and other receptors important for EPC homing is upregulated by EPC from septic patients and if this is associated with clinical outcome. Methods: Peripheral blood mononuclear cells from septic patients (n = 30), ICU control patients (n = 11) and healthy volunteers (n = 15) were isolated by Ficoll density gradient centrifugation. FACS-analysis was used to measure the expression of the CXC motif chemokine receptors (CXCR)-2 and − 4, the receptor for advanced glycation endproducts (RAGE) and the stem cell factor receptor c-Kit. Disease severity was assessed via the Simplified Acute Physiology Score (SAPS) II. The serum concentrations of vascular endothelial growth factor (VEGF), stromal cell-derived factor (SDF)-1α and angiopoietin (Ang)-2 were determined with Enzyme linked Immunosorbent Assays. Results: EPC from septic patients expressed significantly more CXCR-4, c-Kit and RAGE compared to controls and were associated with survival-probability. Significantly higher serum concentrations of VEGF, SDF-1α and Ang-2 were found in septic patients. SDF-1α showed a significant association with survival. Conclusions: Our data suggest that SDF-1α and CXCR-4 signaling could play a crucial role in EPC homing in the course of sepsis

CRISPR RNA-guided FokI nucleases repair a PAH variant in a phenylketonuria model

The CRISPR/Cas9 system is a recently developed genome editing technique. In this study, we used a modified CRISPR system, which employs the fusion of inactive Cas9 (dCas9) and the FokI endonuclease (FokI-dCas9) to correct the most common variant (allele frequency 21.4%) in the phenylalanine hydroxylase (PAH) gene - c.1222C>T (p.Arg408Trp) - as an approach toward curing phenylketonuria (PKU). PKU is the most common inherited diseases in amino acid metabolism. It leads to severe neurological and neuropsychological symptoms if untreated or late diagnosed. Correction of the disease-causing variants could rescue residual PAH activity and restore normal function. Co-expression of a single guide RNA plasmid, a FokI-dCas9-zsGreen1 plasmid, and the presence of a single-stranded oligodeoxynucleotide in PAH_c.1222C>T COS-7 cells - an in vitro model for PKU - corrected the PAH variant and restored PAH activity. Also in this system, the HDR enhancer RS-1 improved correction efficiency. This proof-of-concept indicates the potential of the FokI-dCas9 system for precision medicine, in particular for targeting PKU and other monogenic metabolic diseases

Oxidativer Stress als Modulator der NMDA-Rezeptorexpression auf zerebrovaskulären Endothelzellen

Oxidativer Stress in Form reaktiver Sauerstoffspezies (ROS) und Exzitotoxizität durch supraphysiologische Konzentrationen des Neurotransmitters Glutamat sind nicht nur beteiligt an der Pathogenese vielzähliger neurodegenerativer Erkrankungen wie Schlaganfall, Hirntrauma, Alzheimer Demenz oder Multipler Sklerose, sondern spielen zudem eine Schlüsselrolle im dort beobachteten Zusammenbruch der Blut-Hirn-Schranke. Glutamat führt durch Stimulation neuronaler und endothelialer NMDA-Rezeptoren zu einer Generierung von ROS. Nicht verfolgt worden war bisher, welche Auswirkungen ROS umgekehrt auch auf den NMDA-Rezeptor haben könnten. Im Rahmen der vorliegenden Arbeit wurde daher untersucht, ob und in welcher Weise die Exposition gegenüber reaktiven Sauerstoffspezies einen Einfluss auf die Expression und Aktivierbarkeit von NMDA-Rezeptoren auf zerebrovaskulären Endothelzellen ausübt.rnEs konnte zunächst die Expression der funktionell obligaten NR-1 Untereinheit des NMDA-Rezeptors auf der verwendeten Zelllinie b.End3 mittels Immunfluoreszenz-Mikroskopie gesichert werden. Ein Nachweis von mRNA für die Untereinheiten NR1 und NR2B, C und D erfolgte mittels RT-PCR. In der Analyse der replizierten RNA zeigten sich Hinweise für eine heterogene Komposition der exprimierten endothelialen NMDA- Rezeptoren.rnEs konnte weiter mit Hilfe der In-Cell-Western-Technik gezeigt werden, dass die Expression des NMDA-Rezeptors durch transiente Stimulation mit reaktiven Sauerstoffspezies im Sinne einer Heraufregulation moduliert werden kann. Die Stimulation der Zellen mit den reaktiven Sauerstoffspezies O2-, ONOO- und H2O2 führte dabei im Experiment zu einer deutlichen Zunahme der NR1-Expression, die spätestens nach 72 Stunden höchst signifikant war.rnUm zu überprüfen, welche Bedeutung diese Überexpression für die Integrität der Blut-Hirn-Schranke unter den exzitotoxischen Bedingungen hoher Glutamatkonzentrationen haben könnte, wurde mit Hilfe des ECIS-Systems („Electrical Cell-Substrate Impedance Sensing“) die Impedanz ROS-präexponierter Endothelmonolayer gemessen. Auf Rezeptorstimulation mit dem spezifischen Agonisten NMDA reagierten die vorbehandelten Gruppen mit einem Abfall der Impedanz gegenüber der nicht vorbehandelten Kontrolle.rnrnDie vorliegenden Ergebnisse zeigen, dass ROS in der Lage sind, funktionelle endotheliale NMDA-Rezeptoren zu induzieren und auf diesem Weg zu einem verstärkten Abfall der BHS-Integrität unter den Bedingungen exzitotoxischen und oxidativen Stresses führen. Dies stellt einen neuen Mechanismus zur Erklärung der Pathogenese des Blut-Hirn-Schrankenversagens dar.Oxidative stress in the form of reactive oxygen species (ROS) and excitotoxicity caused by excessive concentrations of the neurotransmitter glutamate not only participate in the pathogenesis of diverse neurodegenerative diseases like stroke, brain trauma, Alzheimer dementia or multiple sclerosis but do also play a key role in the disruption of the blood-brain barrier observed in these pathologies. Glutamate leads to generation of ROS via stimulation of neuronal and endothelial NMDA receptors. As yet, no investigations have been conducted examining whether ROS might have influences on the NMDA receptor as well. The present study was therefore performed to establish if and in what manner exposition to ROS modulates the expression and activity of NMDA receptors on cerebrovascular endothelium.rnFirstly, expression of the NR1 subunit mandatory for functionality of NMDA the receptor was confirmed on the b.End3 cell line employed in the experiments with immunofluorescence microscopy. RT-PCR was performed to detect mRNA for the subunits NR1 as well as NR2B, C and D. Analysis of replicated RNA suggested a heterogeneous composition of expressed endothelial NMDA receptors.rnUsing the In-Cell Western technique, it was shown that transient stimulation of cells with ROS modulates NMDA receptor expression, resulting in an up-regulation of receptors. Stimulation with the reactive oxygen species O2-, ONOO- and H2O2 led to marked increase in NR1 expression, which was highly significant ( p < 0.001) after 72 hours.rnTo evaluate the impact of overexpression of NMDA receptors on the integrity of the blood-brain barrier, the ECIS system („Electrical Cell-Substrate Impedance Sensing“) was used to measure the impedance of an ROS pre-exposed monolayer. Pre-treated groups reacted to stimulation using the specific agonist NMDA with a decrease in impedance as opposed to non pre-treated controls.rnrnThe results presented in this study support the hypothesis that ROS induce functional endothelial NMDA receptors and thus further disruption of blood-brain barrier integrity under conditions of excitotoxic and oxidative stress. Upregulation of NMDA receptors through the action of oxidative stress represents a novel mechanism in the pathogenesis of blood-brain barrier dysfunction

VCAM-1 expression is upregulated by CD34+/CD133+-stem cells derived from septic patients.

CD34+/CD133+- cells are a bone marrow derived stem cell population, which presumably contain vascular progenitor cells and are associated with improved vascular repair. In this study, we investigated whether the adhesion molecules ICAM-1 (intercellular adhesion molecule-1), VCAM-1 (vascular adhesion molecule-1), E-selectin und L-selectin, which are involved in homing of vascular stem cells, are upregulated by CD34+/CD133+-stem cells from septic patients and would be associated with improved clinical outcome. Peripheral blood mononuclear cells from intensive care unit (ICU) patients with (n = 30) and without sepsis (n = 10), and healthy volunteers (n = 15) were isolated using Ficoll density gradient centrifugation. The expression of VCAM-1, ICAM-1, E-selectin and L-selectin was detected on CD34+/CD133+-stem cells by flow cytometry. The severity of disease was assessed by the Simplified Acute Physiology Score (SAPS) II. Serum concentrations of vascular endothelial growth factor (VEGF) and angiopoietin (Ang)-2 were determined by Enzyme-linked immunosorbent assay. The expression of VCAM-1, ICAM-1, E-selectin and L-selectin by CD34+/CD133+-stem cells was significantly upregulated in septic patients, and correlated with sepsis severity. Furthermore, high expression of VCAM-1 by CD34+/CD133+-stem cells revealed a positive association with mortalitiy (p<0.05). Furthermore, significantly higher serum concentrations of VEGF and Ang-2 were found in septic patients, however none showed a strong association with survival. Our data suggest, that VCAM-1 upregulation on CD34+/CD133+-stem cells could play a crucial role in their homing in the course of sepsis. An increase in sepsis severity resulted in both and increase in CD34+/CD133+-stem cells and VCAM-1-expression by those cells, which might reflect an increase in need for vascular repair

Logistic regression analysis of VCAM-1 expression by CD34⁺/CD133⁺-stem cells and sepsis survival.

<p>Binary logistic regression analysis revealed a significant negative association of VCAM-1 expression by CD34⁺/CD133⁺-stem cells with sepsis survival probability (p = 0.05). Equation of regression-line: <b>p(100) = exp(1,56 - 0,0366 VCAM-1 expression)/(1 + exp(1,56 - 0,0366 VCAM-1 expression)).</b> <i>p(100)</i>, probability of survival, <i>VCAM-1</i>, vascular adhesion molecule-1.</p

Upregulation of mobilizing growth factors in serum.

<p>(<b>A</b>) Vascular endothelial growth factor (VEGF) and (<b>C</b>) angiopoietin (Ang)-2 concentrations were detected in the serum of healthy volunteers (n = 15), non-septic intensive care unit (ICU) patients (n = 10) and septic patients (n = 30). (<b>B</b> and <b>D</b>) The group of septic patients was also divided by survival and serum concentrations of both VEGF and Ang2 are indicated. The results are expressed as mean simple linear regression ± SEM; * p<i><</i>0.05 was considered to be statistically significant.</p