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Molecular mechanism of intracellular signal transduction by the angiotensin-converting enzyme
(2007)
The angiotensin converting enzyme (ACE) is an important component of the renin-angiotensin system (RAS) and is crucially involved in the homeostasis of fluid and electrolyte balance and thus in the regulation of blood pressure. The zinc metallopeptidase is involved in the generation of angiotensin II, a potent vasoconstrictor and in the degradation of bradykinin, a potent vasodilator. It is worth noting that ACE more readily hydrolyzes bradykinin than it does angiotensin I thus culminating in the net physiological effect of the production of a vasoconstrictor and the decrease in the availability of a vasodilator. ACE inhibitors have become one of the most successful therapeutic approaches as a first line of therapy in hypertension, and are also widely used in treating heart failure, myocardial infarction, stroke, coronary artery disease and impaired left ventricular function. However, one unexpected clinically relevant finding related to ACE inhibitors is their ability to delay the onset of type II diabetes that was revealed by various large clinical trials. However, the mechanisms underlying these beneficial effects of ACE inhibitor therapy are currently unclear and cannot be explained by the prevention of angiotensin II formation or the attenuated degradation of bradykinin. Thus the potential beneficial effects attributed to ACE inhibitors may occur independent of reductions in blood pressure paving way for new and/or unknown mechanism. Our group has recently redefined ACE as a signal transduction molecule which upon binding to ACE inhibitor turns on a signalling cascade leading to phosphorylation of Ser1270 by CK2, activation of JNK and changes in gene expression in endothelial cells. However the mechanism by which ACE inhibitor initiates the signalling cascade was not clear. It was hypothesized that ACE, which is anchored to the membrane with a single transmembrane domain should dimerize prior to initiating further downstream signalling events in endothelial cells. Therefore, we sought to explore whether or not ACE forms dimers in endothelial cells and whether ACE dimerization is essential for the initiation of ACE signalling in endothelial cells. Using native gel electrophoresis, we found that ACE forms dimers in endothelial cells and that there is an increase in the dimer formation upon treatment of endothelial cells with ACE inhibitors. ACE homodimerization was also demonstrated using the split-ubiquitin system and chemical cross-linking experiments. ACE dimers are also formed in endothelial cells overexpressing the non-phosphorylatable ACE, wherein ACE signalling was abolished indicating that dimerization process is not influenced by the phosphorylation of the serine residue residing in the cytoplasmic tail. Monosaccharides like glucose, galactose and mannitol did not have any influence on ACE-inhibitor induced dimerization. Making use of different monoclonal antibodies directed to the epitopes of N-domain which harbours carbohydrate recognizing domain, also did not affect dimerization. However, inactivation of the C-domain active site by introducing mutation of the key histidine residues in HEMGH consensus sequences, which complexes the zinc ions, abolished enzyme dimerization both in the basal state and in response to ramiprilat. Mutation of the C-domain also resulted in the loss of ACE inhibitor-induced ACE signalling, that is we failed to observe ramiprilat-induced increase in the phosphorylation of the Ser1270 and the subsequent JNK activation. ACE-inhibitor induced dimerization precedes the phosphorylation of Ser1270 and activation of JNK. Thus the ACE-inhibitor induced dimerization via the C-domain of ACE represents the initial step in the ACE signalling pathway which involves the activation of JNK/c-Jun pathway and leading to the changes in the gene expression in endothelial cells. Our group previously identified ACE itself as well as cyclooxygenase-2 (COX-2) as two “ACE signalling-regulated” genes. To screen for additional genes regulated in a similar manner we used DNA microarray technology, to assess ramiprilat-induced changes in the endothelial cell gene expression. 21 genes were identified to be differentially regulated of which, 7 were upregulated and 14 were downregulated by ramiprilat. However, when screened at the protein level, we found no significant differences between the untreated control cells and those treated with ramiprilat. As several other cells and tissues possess a fully functional RAS we screened plasma samples from healthy volunteers as well as from patients with coronary artery disease for the proteins identified in the microarray. We observed that the cellular retinal binding protein-1 (CRBP-1) was detectable at low levels in plasma from patients and that ramipril markedly increased serum levels of this protein. Endothelial cells overexpressing CRBP-1 demonstrated increased RXRE and PPRE activity when stimulated with 9-cis retinoic acid and rosiglitazone respectively suggesting that CRBP-1 might affect gene expression via heterodimerization of PPAR elements with RXR elements by virtue of its function as a transport protein of retinoic acid. Studies aimed at determining the consequences of elevated CRBP-1 expression on endothelial cell homeostasis are ongoing. Although the RAS has been described in many other tissues apart from endothelial cells, ACE signalling has not yet been addressed in tissues such as monocytes/macrophages, which have an increased ACE expression in an atherosclerotic setting. We observed that upon stimulation of cultured ACE expressing monocytes with ramiprilat, JNK is activated suggesting the occurrence of ACE signalling in human monocytes. It is worth noting that ACE inhibitors delay the onset of type II diabetes in spite of moderate decrease in blood pressure. To further elucidate the mechanism underlying this effect, we found that ACE inhibitors increase the PPARgamma levels in the nuclear extracts of ACE expressing monocytes which were also reproduced in human endothelial cells overexpressing human somatic ACE. However, ramiprilat did not have any direct effect on the activity of a luciferase-coupled promoter containing several copies of the PPRE in human endothelial cells. These results contrasted with the actions of the PPARgamma agonist suggesting that ramiprilat enhances PPARgamma levels through an indirect mechanism. We next hypothesized that ramiprilat might increase the levels of 15-deoxy-D12,14-prostaglandin J2 (15dPGJ2) which is a natural ligand for PPARgamma via COX enzymes in monocytes. We observed that ramiprilat was able to decrease the diminution of COX-2 levels upto 48 hours of treatment but the levels of 15dPGJ2 were too low to be detected by ELISA. However ramiprilat enhanced the plasma levels of adiponectin, a downstream target of PPARgamma, which is a anti-atherogenic and anti-inflammatory adipokine, in patients with coronary artery disease. Though adiponectin is a PPARgamma-regulated gene, the observed increase in adiponectin might be attributed to the increase in RXR rather than via PPARgamma. Taken together, the results of this investigation have revealed that ACE inhibitors initiate ACE signalling by eliciting the dimerization of the enzyme, more specifically via its C-domain active centers. The ACE signalling cascade when activated leads to the enhanced expression of ACE, COX-2 and CRBP-1 which in turn favours the heterodimerization of PPARgamma with RXR and thus results in the increased expression of “PPARgamma regulated” genes such as adiponectin. The latter results provide a molecular basis for the observation that ACE inhibitors can delay the onset of type 2 diabetes in as much as it was possible to link ramipril with CRBP-1, RXR activity and the expression of adiponectin, an adipokine associated with improved insulin sensitivity. Further work is however required to elucidate the consequences of ACE inhibitors in monocytes and adipocytes as well as in intact animals.
All living organisms exhibit daily fluctuations in biochemical, physiological and behavioural parameters driven by endogenous oscillators, residing in the organism itself. In mammals, the core circadian oscillator is located in the paired suprachiasmatic nuclei (SCN) of the hypothalamus. Circadian rhythm generation in the SCN depends upon the expression of clock genes interacting in positive and negative transcriptional/translational feedback loops. The SCN governs the timing of peripheral circadian oscillators by neuronal pathways and by neuroendocrine mechanisms. An important neuroendocrine hand of the core circadian oscillator is melatonin, which is produced in and secreted from the pineal gland night by night. The adenohypophysis represents a peripheral circadian oscillator and the secretion of one of its hormones, prolactin, is known to be regulated by melatonin. The aim of the present study was to analyze a putative influence of melatonin on the activity state and diurnal variations of identified cell types in the hypophysis. Particular attention was paid to lactotroph, gonadotroph and pars intermedia cells. Experiments were performed with young male mice of different strains: melatonin-proficient C3H, melatonin-deficient C57BL, melatonin-proficient C3H with targeted deletions of the Mel1a receptor (MelaaBB), Mel1b receptor (MelAAbb) or both receptors (Melaabb). Cells producing prolactin (PRL), follicle stimulating hormone (FSH) were immunocytochemically identified and the presence of phosphorylated CREB protein (pCREB) and clock gene protein PER1 was demonstrated by double immunolabeling at different time points during the light/dark cycle in melatonin deficient, melatonin proficient and melatonin receptor knockout mice. Melatonin influence on Prl mRNA levels was investigated by means of in situ hybridization. At night the percentage of lactotroph cells showing a positive nuclear pCREB- and PER1-immunoreaction is significantly smaller in C57BL than in C3H mice. In both mouse strains, the percentage of pCREB –immunoreactive cells is minimal in the early morning and gradually increases to reach a maximum in the late night. PER1 levels show a parallel temporal variation in C3H, but in C57BL, they are drastically reduced in the early afternoon. The percentage of FSH-immunoreactive cells showing pCREB immunoreaction was significantly lower in the melatonin-deficient C57Bl mice than in the melatonin-proficient C3H mice during the second part of the day and during the night. In each strain, the percentage of FSH-immunoreactive cells was lowest at the early morning and gradually increases until the maximum at late night. In wild type (MelAABB) and MelAAbb mice the percentage of lactotroph cells with nuclear pCREB immunoreactions varied significantly over 24 h period, whereas in MelaaBB and Melaabb mice no significant differences were found between the five time points analyzed. The number of Prl mRNA expressing cells was significantly higher in MelaaBB and MelAAbb than in their wild type (MelAABB) littermates. pCREB levels in the pars intermedia did not show rhythmic variation in wild type or Melaabb animals, but wild type mice had higher pCREB levels than Melaabb. The observation that, during darkness, the percentage of lactotroph cells with nuclear pCREB immunoreaction is significantly higher in C3H than in C57BL mice suggests the existence of a distinct cell population that is under the control of melatonin-dependent intrapituitary signaling. Results with melatonin receptor knockout mice indicate that Mel1a and Mel1b melatonin receptors are involved in the control of the activity state of lactotroph cells, but to a differing degree. Analysis of cells expressing Prl mRNA showed that inhibitory action on the Prl expression is mostly mediated through the Mel1a receptor. The significant difference between pCREB immunoreaction in gonadotroph cells of C3H and C57BL mice might suggest that, like lactotrophes, FSH cells represent a heterogeneous population and only a subpopulation is under control of melatonin signaling. The present study is first to show that melatonin signaling also affects pCREB levels in pars intermedia of mice.
To determine the effects of inhaled IL-10 at different doses and different time points on the pulmonary and systemic inflammatory response during endotoxemia, 48 ventilated, anaesthetized rats (mean body weight ± standard deviation, 500 ± 33g) were randomly assigned to six groups (n = 8, each). Interleukin-10 was nebulised either prior to or following the intravenous injection of LPS (5mg/kg) at two doses (5.0 mycro-g or 0.5 mycro-g) in our groups. Eight rats received the same insult with no further treatment (LPS-only group). Another eight rats served as controls without endotoxemia but with aerosolized phosphate-buffered saline, the solvent of IL-10 (Sham group). Concentrations of TNF-alpha, IL-1beta, IL-6, and IFN-gamma were analyzed in plasma and bronchoalveolar lavage fluid (BALF). In addition, the nitrite release from ex-vivo cultured alveolar macrophages was determined. As compared to the LPS-only group, the concentrations of the proinflammatory cytokines TNF-alpha, IL-1beta, IL-6, and IFN-gamma in plasma were significantly reduced in the group, which inhaled 5 mycro-g IL-10 before LPS injection (p< 0.0125). Spontaneous nitrite release from exvivo cultured alveolar macrophages was suppressed in this group (p< 0.0125). Inhalation of 0.5 mycro-g IL-10 before LPS injection and both dosages of IL-10 inhalation (5 mycro-g or 0.5 mycro-g) after LPS injection did not significantly influence either inflammatory cytokine concentrations in BALF, in plasma or the nitrite release from ex-vivo cultured alveolar macrophages. In this study, inhaled IL-10 only demonstrated anti-inflammatory effects when it was administered at 5 mycro-g prior to the induction of experimental endotoxemia. Interleukin-10 aerosol had no effect when it was given either following induction of endotoxemia or given at a lower dosage (which here was 0.5 mycro-g) either before or following injection of lipopolysaccharide.
HELEX Septal Occluder for transcatheter closure of patent foramen ovale : multicenter experience
(2006)
Aims Patients with cryptogenic embolic events and a patent foramen ovale (PFO) are at risk of paradoxical embolism causing recurrent cerebral events; however, transcatheter PFO closure remains controversial. The aim of this multicenter trial was to demonstrate the feasibility and safety of transcatheter closure of PFO with the HELEX Septal Occluder. Methods and results The study enrolled 128 patients (66 female; mean age, 50 years). Mean (± SD) PFO size was 10±3.7 mm; 38 patients also had an atrial septal aneurysm. Device implantation was successful in 127 patients. Device-related events during implantation or follow-up were device embolisation, wire-frame fracture, and retrieval cord breaks (two cases each; no sequelae). Other adverse events included atrial arrhythmia (two patients), migraine, convulsion, and transient ischaemic attack (one case each). There were no recurrent strokes, deaths, perforations, or accumulations of thrombi on the device. Within a mean follow-up period of 21±11 months, complete PFO closure using one device was achieved in 114 patients (90%). Five patients with a moderate to large residual shunt received a second device. Conclusion The HELEX Occluder can be used for PFO closure. Device- and procedure-related complications are rare. The closure procedure appears to reduce recurrence rates of stroke and transient ischaemic attack.
The substantia nigra is not the induction site in the brain of the neurodegenerative process that underlies Parkinson’s disease. Instead, the results of this semiquantitative study of 30 autopsy cases with incidental Lewy body pathology indicate that Parkinson’s disease in the brain commences with the formation of the very first immunoreactive Lewy neurites and Lewy bodies in noncatecholaminergic visceromotor neurons of the dorsal glossopharyngeusvagus complex, in projection neurons of the intermediate reticular zone, and in specific nerve cell types of the gain setting system (coeruleussubcoeruleus complex, caudal raphe nuclei, gigantocellular reticular nucleus), olfactory bulb, olfactory tract, and/or anterior olfactory nucleus in the absence of nigral involvement. The topographical parcellation of the nuclear grays described here is based upon known architectonic analyses of the human brain stem and takes into consideration the pigmentation properties of a few highly susceptible nerve cell types involved in Parkinson’s disease. In this sample and in all 58 ageand gendermatched controls, Lewy bodies and Lewy neurites do not occur in any of the known telencephalic Parkinson’s disease predilection sites (hippocampal formation, temporal mesocortex, proneocortical cingulate areas, amygdala, basal nucleus of Meynert, interstitial nucleus of the diagonal band of Broca, hypothalamic tuberomamillary nucleus).
Removal of apoptotic cells by macrophages or resident semi-professional phagocytes is a prominent principle with important implications for the pathophysiology of chronic inflammatory diseases, viral infections, or cancer. To characterize mechanisms which may determine the fate of apoptotic cells, I investigated chemokine expression in apoptotic promonocytic U-937 cells or PBMC. Exposure of U-937 cells to the anti-cancer drug etoposide (VP-16), an inducer of apoptosis in these cells, was associated with increased expression of the chemokines IL-8 and macrophage inflammatory protein 1alpha (MIP-1alpha). Upregulation of IL-8 mRNA expression by VP-16 was observed as early as 4 h after onset of treatment and was still detectable after 19h of exposure. A serine protease inhibitor prevented both VP-16-induced apoptosis and release of IL-8, whereas inhibition of p38 MAP-kinases reduced IL-8 secretion only. Moreover, I observed that incubation with 2-chlorodeoxyadenosine (CdA) upregulated release of IL-8 from adherent PBMC in parallel to induction of apoptosis. In these cells a modest but significant induction of TNF-alpha release by CdA was also detected. In addition, CdA augmented release of IL-8 from whole blood cultures. By facilitating adequate recruitment of phagocytes to sites of cell death, stress-induced upregulation of chemokines associated with apoptosis may contribute to mechanisms aiming at efficient removal of apoptotic cells.
Hepatitis E virus (HEV) is a positive-stranded RNA virus with a 7.2 kb genome that is capped and polyadenylated. The virus is currently unclassified : the organisation of the genome resembles that of the Caliciviridae but sequence analyses suggest that it is more closely related to the Togaviridae. HEV is an enterically transmitted virus that causes both epidemics and sporadic cases of acute hepatitis in many countries of Asia and Africa but only rarely causes disease in more industrialised countries. Initially the virus was believed to have a limited geographical distribution. However, serological studies suggest that that HEV may be endemic also in the United states and Europe even though it infrequently causes overt disease in these countries. Many different animal species worldwide recently have been shown to have antibodies to HEV suggesting that hepatitis E may be zoonotic. Although two related strains have been experimentally transmitted between species, direct transmission from animal to a human has not been documented. Our main objective in this study is to evaluate the suitability of current available HEV antibody assays for use in low-endemicity areas such as in Germany. Methods: We selected sera on the basis of at least borderline reactivity in the routinely used Abbot EIA. Most were tested as part of routine screening of long-term expatriates in endemic countries. The following assays (recombinant antigens : ORF2 and ORF3) were used: Abbot EIA, Genelabs ELISA, Mikrogen recomBlot and a 'Prototype' DSL-ELISA. We observed a wide range of sensitivity ( average of 56.8%) and specificity ( an average of 61.4%) in these used assays. These results implies that , these assays might be unreliable for detection of HEV infection in areas where hepatitis E is not endemic. However, most anti- HEV assays have not been correlated with the HEV RNA determined by reverse transcription. Many of these unexpected results and discrepancies can be alluded to the following reasons: I. The choice and the size of the HEV antigen. II. Duration of the antibody persistence III. A cross reactivity with different agent IV. Due to geographic species V. A low sensitivity of the available assays. VI. And infection with non-pathogenic HEV strain. (zoonotic strain?). We therefore suggest that, further studies will be required to improve the sensitivity and specificity of the available commercial assays on the market.
Die vorliegende Dissertation berichtet über eine Serie von Verhaltens- und funktionellen Bildgebungsstudien zu experimentalpsychologischen Paradigmata, die eine räumliche Analyse und Koordinatentransformation von Material der visuellen Wahrnehmung oder Vorstellung beinhalten. Nach einer Einführung in die Prinzipien und Techniken der funktionellen Kernspintomographie, der hier benutzten Methode für die Messung von Gehirnaktivität, werden die Versuche einer Replikation des berühmten Stratton'schen Umkehrbrillen-Experiments dargestellt (Kapitel 1). Unsere vier Probanden zeigten zwar eine zügige Anpassung der visuomotorischen Funktionen an die neue visuelle Umwelt, berichteten aber, anders als Stratton, nicht, daß sie die Welt nach einigen Tagen mit der Umkehrbrille wieder normal sähen. Diese Persistenz des umgekehrten Bildes wurde durch eine psychphysische Testbatterie bestätigt. Des weiteren ergaben die funktionellen Kernspinmessungen, daß sich die kortikale retinotope Organisation im Verlaufe des Experiments nicht geändert hat. Da sich also Strattons Haupteffekt, das Aufrechtsehen durch die Umkehrbrille nach einwöchiger Adaptation, nicht replizieren ließ, werden andere Möglichkeiten der Interpretation der verschiedenen Umkehrexperimente der letzten hundert Jahre vorgeschlagen. Dieses Ergebnis einer funktionellen Anpassung ohne größere Änderungen der visuellen Wahrnehmung (und ohne Veränderungen der Repräsentation der Netzhautareale in der Sehrinde) führte zu der Hypothese, daß die erforderlichen Transformationen auf einer höheren Stufe der kortikalen Hierarchie der visuellen Verarbeitung erfolgen. Zur Testung dieser Hypothese wurde eine funktionelle Kernspinstudie des Umkehrlesens durchgeführt (Kapitel 2). Hierbei lasen die Probanden Wörter und Sätze in Spiegelschrift oder auf dem Kopf. Der neuronale Mechanismus der räumlichen Transformationen, die zur Bewältigung dieser Aufgabe nötig sind, konnte in bestimmten Regionen des Parietallappens, die zwischen den Probanden sehr konstant waren, lokalisiert werden. Weiterhin fand sich eine Koaktivierung okzipitootemporaler Objekterkennungs-Areale. Die Spezifizität der parietalen Aktivierungsfoci wurde durch ein Kontrollexperiment bestätigt, in welchem das kortikale System für räumliche Transformationen von den Netzwerken der allgemeinen visuellen Aufmerksamkeit und der Augenbewegungskontrolle unterschieden werden konnte. In einem weiteren Experiment wurden die räumlichen Funktionen des Parietallappens unter dem Vorzeichen der visuellen Vorstellung untersucht. Als Paradigma wurde der "mental clock" - Test verwendet, bei welchem die Probanden die Winkel der Zeiger zweier Uhren vergleichen müssen, deren Zeiten nur akustisch vorgegeben werden (Kapitel 3). Diese Aufgabe erfordert die Generierung eines entsprechenden Vorstellungsbildes und dessen räumliche Analyse, stellt also sowohl ein kontrolliertes Vorstellungs-Paradigma als auch einen Test räumlicher Funktionen dar, der nicht auf visuell präsentiertem Material beruht. Das parietale Aktivierungsmuster, das der Analyse der Winkel dieser vorgestellten Uhren zugeschrieben werden konnte, entsprach weitgehend demjenigen, das mit der räumlichen Transformation von Buchstaben verbunden war. Es handelt sich also wahrscheinlich um ein kortikales System für räumliche Analyse und Koordinatentransformationen, das nicht auf eine visuelle Stimulation angewiesen ist, sondern auch bei bloßer visueller Vorstellung aktiv werden kann. Die vorgelegten Resultate werden im Kontext neuerer neuropsychologischer Befunde zu Defiziten räumlicher Analyse und Vorstellung bei Läsionen des Parietallappens diskutiert (Kapitel 4). Auch die methodologischen Probleme der kognitiven Subtraktion, die in unseren Studien teilweise benutzt wurde, werden behandelt. Dabei wird erläutert, inwiefern diese für die Beurteilung der vorgelegten Studien nur von untergeordneter Bedeutung sind. Nichtsdestoweniger schlagen wir Modifikationen der experimentellen Paradigmata im Sinne des parametrischen Designs und des "event-related functional magnetic resonance imaging" vor, die bei zukünftigen Studien einen vollständigen Verzicht auf die kognitive Subtraktion ermöglichen dürften. Schließlich wird die Bedeutung der vorgelegten Ergebnisse für die Erforschung der Anpassungsfähigkeit des menschlichen Gehirns und des Verhältnisses von Vorstellung und visueller Wahrnehmung dargelegt.