Mechanismen der Aktivierung des NLRP3 Inflammasoms bei chronischer Nierenerkrankung und kardiovaskulären Erkrankungen

Eine sterile Inflammation spielt eine wichtige Rolle bei der Entstehung und Progression einer chronischen Nierenerkrankung als auch bei kardiovaskulären Erkrankungen. In Vorarbeiten konnte unsere Arbeitsgruppe zeigen, dass die Lipoproteine HDL und LDL auf unterschiedliche Weise das angeborene Immunsystem wie beispielsweise Toll-like Rezeptor-2 (TLR2) aktivieren und so eine systemische Inflammation induzieren. Das NLRP3 Inflammasom stellt einen weiteren wichtigen Bestandteil des angeborenen Immunsystems dar, welcher pro-IL-1 in dessen aktive Form prozessiert. Ziel dieser Arbeit war es zu untersuchen, welche Rolle die verschiedenen Lipoproteinklassen bei der Aktivierung des NLRP3 Inflammasoms spielen. In humanen Monozyten führte VLDL aber nicht HDL oder LDL zur Freisetzung von IL-1. Wir konnten nachweisen, dass die Hauptproteinkomponente in VLDL Apolipoprotein C3 hierfür verantwortlich ist. ApoC3 führt auf der Oberfläche humaner Monozyten zur Heterotrimerisierung von TLR2 und TLR4 zusammen mit dem Adapterprotein SCIMP. Dies aktiviert die Kinase Syk, führt zum Calcium-Einstrom in die Zelle über den TRPM2 Kanal und induziert die NADPH Oxidase-abhängige Produktion reaktiver Sauerstoffspezies (ROS). Diese aktivieren Caspase-8, was zur Zusammenlagerung des NLRP3 Inflammasomkomplexes aus seinen Komponenten NLRP3, ASC und Caspase-1 führt. Das aktive NLRP3 Inflammasom prozessiert in der Folge pro- IL-1 in matures IL-1, welches von den Monozyten sezerniert wird und eine systemische proinflammatorische Antwort auslöst. Wir konnten nachweisen, dass dieser Signalweg der alternativen NLRP3 Aktivierung auf das humane System beschränkt ist. Zur Überprüfung der pathophysiologischen Relevanz dieser experimentellen Befunde wurden humanisierte Mäuse generiert. D.h. in immuninkompentente NOD-SCID Mäuse wurden humane CD14+ Monozyten transplantiert. Im murinen Carotis-Schädigungsmodell führte die Injektion von ApoC3 in die humanisierten Mäuse zu einer deutlichen Hemmung der endothelialen Regeneration. Nach unilateraler Ureterligatur förderte ApoC3 die renale Schädigung. ApoC3 Plasmakonzentrationen sind bei Patienten mit chronischer Nierenerkrankung als auch bei Patienten nach akutem Myocardinfarkt im Vergleich zu gesunden Probanden signifikant erhöht und auch mit einer höheren Mortalität assoziiert. Somit konnte in dieser Arbeit erstmals gezeigt werden, dass Triglycerid-reiche Lipoproteine via ApoC3 das NLRP3 Inflammasom aktivieren. Gleichzeitig konnte die alternative NLRP3 Aktivierung als ein pathophysiologisch relevanter Signalweg aufgeklärt werden. Diese Ergebnisse liefern somit wichtige Erkenntnisse über die Regulation des NLRP3 Inflammasoms generell als auch über die Mechanismen einer kardiorenalen Schädigung. ApoC3 und das NLRP3 Inflammasom stellen somit ein neues therapeutisches Target dar.Sterile inflammation represents a hallmark in the initiation and progression of chronic kidney disease as well as cardoiovascular diseases. In our previous, our group could show that the lipoproteins HDL and LDL activate the innate immune system via several distinct pathways such as toll-like receptor-2 (TLR2), which leads to systemic inflammation. The NLRP3 inflammasome represents another important component of the innate immune system by processing pro-IL-1 into its biologically active form. The aim of the present thesis was to assess the effects of the different lipoprotein classes on the activation of the NLRP3 inflammasome. In human monocytes, VLDL but not HDL or LDL induced the release of IL-1. We found that the major protein constituent of VLDL ApoC3 was responsible for this effect. On the surface of human monocytes, ApoC3 triggers hetertrimerization of TLR2, TLR4, together with the adapter protein SCIMP. This activates Syk kinase, leading to calcium influx via TRPM2, which induces NADPH oxidase-dependent production of reactive oxygen species (ROS). ROS activate caspase-8, which facilitates the assembly of the functional NLRP3 inflammasome complex from its components NLRP3, ASC, and caspase-1. The active NLRP3 inflammasome then processes pro-IL-1 into mature IL-1, which is released from human monocytes and triggers a systemic pro-inflammatory response. We found that this pathway of alterative NLRP3 activation is restricted the human system. To prove the pathophysiological relevance of these experimental findings, humanized mice were generated. Therefore, immune incompetent NOD-SCID mice were transplanted with human CD14+ monocytes. In the murine carotid injury model in humanized mice, injection of ApoC3 substantially suppressed endothelial regeneration. After unilateral ureter ligation, ApoC3 promoted kidney injury. ApoC3 plasma levels were significantly higher in patients after acute myocardial infarction or chronic kidney disease as compared to healthy control subjects. Moreover, higher ApoC3 plasma concentrations were associated with higher mortality during follow-up. In summary, this work provides first evidence that triglyceride-rich lipoproteins via ApoC3 activate the NLRP3 inflammasome. Moreover, we could describe alternative NLRP3 activation as a pathophysiologically relevant pathway. These results provide novel insights in the regulation of NLRP3 inflammasome in general as well as in the mechanisms of cardiorenal injury. Therefore, ApoC3 and the NLRP3 inflammasome represent a novel therapeutic target

Relations between lipoprotein(a) concentrations, LPA genetic variants, and the risk of mortality in patients with established coronary heart disease: a molecular and genetic association study

Background: Lipoprotein(a) concentrations in plasma are associated with cardiovascular risk in the general population. Whether lipoprotein(a) concentrations or LPA genetic variants predict long-term mortality in patients with established coronary heart disease remains less clear. Methods: We obtained data from 3313 patients with established coronary heart disease in the Ludwigshafen Risk and Cardiovascular Health (LURIC) study. We tested associations of tertiles of lipoprotein(a) concentration in plasma and two LPA single-nucleotide polymorphisms ([SNPs] rs10455872 and rs3798220) with all-cause mortality and cardiovascular mortality by Cox regression analysis and with severity of disease by generalised linear modelling, with and without adjustment for age, sex, diabetes diagnosis, systolic blood pressure, BMI, smoking status, estimated glomerular filtration rate, LDL-cholesterol concentration, and use of lipid-lowering therapy. Results for plasma lipoprotein(a) concentrations were validated in five independent studies involving 10 195 patients with established coronary heart disease. Results for genetic associations were replicated through large-scale collaborative analysis in the GENIUS-CHD consortium, comprising 106 353 patients with established coronary heart disease and 19 332 deaths in 22 studies or cohorts. Findings: The median follow-up was 9·9 years. Increased severity of coronary heart disease was associated with lipoprotein(a) concentrations in plasma in the highest tertile (adjusted hazard radio [HR] 1·44, 95% CI 1·14–1·83) and the presence of either LPA SNP (1·88, 1·40–2·53). No associations were found in LURIC with all-cause mortality (highest tertile of lipoprotein(a) concentration in plasma 0·95, 0·81–1·11 and either LPA SNP 1·10, 0·92–1·31) or cardiovascular mortality (0·99, 0·81–1·2 and 1·13, 0·90–1·40, respectively) or in the validation studies. Interpretation: In patients with prevalent coronary heart disease, lipoprotein(a) concentrations and genetic variants showed no associations with mortality. We conclude that these variables are not useful risk factors to measure to predict progression to death after coronary heart disease is established. Funding: Seventh Framework Programme for Research and Technical Development (AtheroRemo and RiskyCAD), INTERREG IV Oberrhein Programme, Deutsche Nierenstiftung, Else-Kroener Fresenius Foundation, Deutsche Stiftung für Herzforschung, Deutsche Forschungsgemeinschaft, Saarland University, German Federal Ministry of Education and Research, Willy Robert Pitzer Foundation, and Waldburg-Zeil Clinics Isny

Myricetin: A Naturally Occurring Regulator of Metal-Induced Amyloid-beta Aggregation and Neurotoxicity

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The prokaryotic zinc-finger: Structure, function and comparison with the eukaryotic counterpart

Classical zinc finger (ZF) domains were thought to be confined to the eukaryotic kingdom until the transcriptional regulator Ros protein was identified in Agrobacterium tumefaciens. The Ros Cys2His2 ZF binds DNA in a peculiar mode and folds in a domain significantly larger than its eukaryotic counterpart consisting of 58 amino acids (the 9-66 region) arranged in a βββαα topology, and stabilized by a conserved, extensive, 15-residue hydrophobic core. The prokaryotic ZF domain, then, shows some intriguing new features that make it interestingly different from its eukaryotic counterpart. This review will focus on the prokaryotic ZFs, summarizing and discussing differences and analogies with the eukaryotic domains and providing important insights into their structure/function relationships. The prokaryotic domain, found for the first time in the protein Ros from A. tumefaciens, shows some interesting structural and functional features that differentiate it from its eukaryotic counterpart. The review summarizes and discusses differences and analogies with the eukaryotic domains providing important insights into their structure/function relationships