Rolle der Telomerase und zellulärer Seneszenz beim myokardialen Remodeling nach experimenteller Myokardischämie in Mäusen

Die Zellalterung könnte für das so genannte myokardiale Remodeling, also einen Anpassungsprozess des Herzmuskels nach kardialer Schädigung, eine wichtige Rolle spielen. Die komplexe Regulation von Vorgängen wie Zellüberleben / Apoptose, Zellproliferation, Fibrose und Seneszenz betreffen nicht nur die primär von dem kardialen Schaden betroffenen Zellen des Myokards, sondern involvieren auch Einwanderung und Überleben von extrakardialen Zellen wie Entzündungszellen mit dem Ziel der Heilung, Narbenbildung und Regeneration. Telomere und telomer-regulierende Proteine wie Telomerase und telomeric repeat-binding factor 2 (TRF2) sind nicht nur für die Steuerung und Kontrolle der Zellalterung wichtig, sondern interagieren bekanntermaßen mit allen o.g. Vorgängen. Durch das Modell der experimentellen Myokardischämie (MI) mittels Ligatur der proximalen left anterior descending (LAD) artery in Mäusen ist es möglich, diese bislang wenig erforschten Mechanismen der Zellalterung nach einem Herzinfarkt besser zu verstehen. Dies war das Hauptziel der vorliegenden Promotionsarbeit, in der, neben der Erforschung der kardialen Regulation der Telomerparameter bei genetisch unveränderten Tieren, die kausale Rolle der Telomerase sowohl für das myokardiale Remodeling als auch für die Regulation in extrakardialen Organen in Knockout-Mäusen untersucht wurde. Zunächst wurde der Frage nachgegangen, ob es eine zeitlich (Tag 3, 7 und 28) und regional (Infarkt- (IZ) und Randzone (RZ)) unterschiedliche Regulation der Telomerlänge, Telomerase und telomer-assoziierter Proteine nach Induktion eines MI durch proximale LAD-Ligatur gibt. In männlichen, 10-14 Wochen alten C57Bl/6N-Wildtypmäusen (WT) konnten wir mit Hilfe des Telomere-Repeat Amplification Protocol (TRAP)-Assays zeigen, dass initial bloß in der IZ und nicht in der RZ eine signifikante Steigerung der Telomeraseaktivität vorlag. An Tag 7 nach Operation fand sich dann eine weitere Zunahme, wobei dieser Effekt an Tag 28 nur in der IZ noch stärker ausgeprägt war. Die Telomerlänge wurde nach 4 Wochen bestimmt und war in der RZ mehr als in der IZ verlängert. Parallel zur Telomeraseaktivität beobachteten wir gleichzeitig eine Aktivierung anti-seneszenter / anti-apoptotischer (TRF2, pAKT) und pro-seneszenter / pro-apoptotischer (p53, Cell-Cycle Checkpoint Kinase 2 (Chk2), Bax, p16) Signalwege. Diese schien zeitlich begrenzt zu sein mit einem Maximum an Tag 7 nach MI, und sich nach 4 Wochen eine Gegenregulation unter den Ausgangswert (TRF2, p53, Chk2, pAkT) bzw. Rückkehr zur Baseline (Bax) fand. Sirtuin-1 (SIRT1) und p16 hingegen waren beide an Tag 28 nach MI noch deutlich hochreguliert. Des Weiteren wurden drei extrakardiale Organsysteme untersucht: 1.) die Aorta ascendens, weil sich im Rahmen der kardialen Funktionsverschlechterung nach MI durch geänderte Scherkräfte in den zentralen Gefäßen eine zelluläre Regulation ergeben könnte. Die Assays lieferten insgesamt kein valides Ergebnis. 2.) Die Leber, weil dieses Organ zum einen empfindlich auf einen kardialen Rückstau ist, zum anderen eines der hämatopoetischen Organe der Maus und somit Quelle für inflammatorische Zellen und Stammzellen, welche vom Herzen nach einer Ischämie durch Chemokine angelockt werden, ist. Die wichtigsten Befunde waren eine reduzierte Telomeraseaktivität und eine gleichzeitig erhöhte Expression von Bax und SIRT1 in den Infarkttieren. 3.) Die mononukleären Zellen (MNC) der Milz, da sie ebenfalls ein hämatopoetisches Organ in der Maus ist und über die zirkulierenden Zellen an der Infarktheilung teilnimmt. Die Ergebnisse in der heterogenen Population der MNC streuten, ähnlich wie in der Aorta, stark. Es zeigte sich allerdings konsistent eine gesteigerte Telomerlänge nach 4 Wochen, hinweisend auf eine Ausschwemmung von Stammzellen mit langen Telomeren aus dem blutbildenden System. Zusätzlich wurden weibliche, 11-34 Wochen alte Mäuse der 2. Generation (d.h. mit noch nicht verkürzten Telomeren) mit globaler genetischer Defizienz der Telomerase-Reverse-Transkriptase (TERT), der katalytischen Untereinheit der Telomerase, an Tag 28 nach Scheinoperation bzw. MI untersucht. Im Vergleich zu den C57Bl/6N- und den TERT+/+-Tieren zeigten die Infarktmäuse ohne Telomeraseaktivität zum Teil deutliche Unterschiede in der Regulation. Die Telomerlänge und TRF2-Expression waren z.b. in TERT-/--Infarktmäusen nicht verändert, jedoch waren bereits in der TERT-/-- im Vgl. zur TERT+/+-Shamgruppe die pro-apoptotischen Proteine p53 und Bax vermindert und der Seneszenzmarker p16 erhöht, was durch den Infarkt nicht weiter beeinflusst wurde. In histologischen Untersuchungen zeigte sich ebenfalls ein entscheidender Unterschied bei den TERT-defizienten MI-Tieren gegenüber den TERT-positiven Kontrollen: auffällig war eine deutlich vermehrte Myokardfibrose in der Sirius Red-Färbung. Dieser wichtige Befund konnte durch den Nachweis einer gesteigerten Genexpression von Collagen-1α und Connective Tissue Growth Factor (CTGF) molekularbiologisch noch untermauern werden. Die oben genannten Veränderungen der Proteinexpression könnten eine Erklärung für den möglichen Einfluss der TERT-Defizienz auf die Kollagenakkumulation sein. Die Untersuchungen in extrakardialen Geweben führten, ähnlich wie bei den C57Bl/6N-Tieren, zu heterogenen Ergebnissen im Myokard, wiesen aber insgesamt auf eine Störung der extrakardialen Regulation des kardialen Remodeling, z.B. durch fehlenden Nachweis von MNC mit längeren Telomeren bei TERT-/--Infarkttieren hin, während andere Regulationen vergleichbar waren. Eine weitere Fragestellung der Arbeit betraf die funktionellen Konsequenzen der TERT-Defizienz im Kontext des MI. Die Messung der Indices von Herzgewicht / Tibialänge bzw. / Körpergewicht (KG), Lungenfeuchtgewicht / KG und Leberfeuchtgewicht / KG zeigte, dass 4 Wochen nach LAD-Ligatur weder eine Myokardhypertrophie noch eine Herzinsuffizienz vorlagen. Die Kardio-Magnetresonanztomografie (MRT) und der Millar-Katheter verifizierten zwar die kardiale Funktionsminderung mit erniedrigter Pump- bzw. Druckleistung durch den operativ induzierten MI, eindeutige genotypische Unterschiede waren jedoch nicht zu verzeichnen. Dies ist auf den kurzen Zeitpunkt der MRT-Untersuchung 4 Wochen nach dem MI zurückzuführen, welcher bewusst gewählt wurde, um die mittelfristige Regulation nach Herzinfarkt zu beurteilen. Die vorliegende Arbeit führte zu einer Reihe neuer Erkenntnisse mit Fokus auf die Rolle der Telomerase und zellulärer Seneszenzmarker bei Ischämie-induziertem Myokardremodeling in Mäusen. Die Ergebnisse könnten eine Grundlage für die zukünftige Entwicklung neuer therapeutischer Strategien darstellen. Über eine Beeinflussung der Zellalterung wäre es vielleicht möglich, die Entwicklung einer ischämischen Kardiomyopathie als gefürchtete Folgeerscheinung nach MI, die unter anderem durch maladaptives Remodeling verursacht wird, günstig zu beeinflussen.Role of telomerase and cellular senescence in myocardial remodeling after experimental myocardial ischemia in mice Cell aging may play an important role in myocardial remodeling, which is a process of adaptation of the cardiac muscle following cardiac damage. The complex regulation of mechanisms such as cell survival / apoptosis, cell proliferation, fibrosis and senescence involve not only the cardiac cells affected by the cardiac damage primarily, but also the immigration and survival of extracardiac cells with the aim of healing, scarring and regeneration. Telomeres and telomere-regulating proteins such as telomerase and telomeric repeat-binding factor 2 are not only important for the regulation and control of cell aging, but are also known for interacting with all of the above-mentioned mechanisms. Using the model of experimental myocardial ischemia by ligating the proximal left anterior descending artery in mice makes it possible to better understand these mechanisms of cell aging following a heart infarction, which have been poorly understood so far. This was the main goal of the present doctoral thesis, which investigated, apart from the study of the cardiac regulation of telomeric parameters in genetically unmodified mice, the causal role of telomerase for both the myocardial remodeling and the regulation in extracardiac organs in knockout mice. First, we pursued the question whether there was a temporal (day 3, 7 and 28) and regional (infarct and remote zone) different regulation of telomere length, telomerase and telomere-associated proteins after induction of MI by ligation of the proximal LAD. In male 10- to 14-week-old C57Bl/6N wild-type mice, we were able to demonstrate, using the Telomere Repeat Amplification Protocol assay, that initially there was a significant increase in telomerase activity only in the IZ and not in the RZ. On day 7 after surgery there was a further increase, whereby this effect was even more pronounced on day 28 only in the IZ. The telomere length was determined after 4 weeks and was more extended in the RZ than in the IZ. In parallel to the telomerase activity, we simultaneously observed an activation of anti-senescent / anti-apoptotic (TRF2, pAKT) and pro-senescent / pro-apoptotic (p53, cell-cycle checkpoint kinase 2, Bax, p16) signaling pathways. This seemed to be temporary with a maximum on day 7 after MI, and after 4 weeks a counterregulation was found below baseline (TRF2, p53, Chk2, pAkT) or return to baseline (Bax), respectively. Sirtuin-1 and p16, however, were both significantly upregulated at day 28 after MI. In addition, three extracardiac organ systems were investigated: 1.) the ascending aorta, since cardiac dysfunction after MI with altered shear forces in central vessels could result in a cellular regulation. The assays did not give a valid result. 2.) The liver, because this organ is sensitive to congestion on the one hand, and is one of the hematopoietic organs of the mouse on the other hand, and thus the source of inflammatory and stem cells that are attracted to the heart by chemokines after ischemia. The most important findings were a reduced telomerase activity and a simultaneously increased expression of Bax and SIRT1 in the animals with MI. 3.) The mononuclear cells in the spleen, as it is also a hematopoietic organ in the mouse, participating in infarct healing via the circulating cells. The results in the heterogeneous population of MNC scattered strongly, similar to those in the aorta. However, there was consistently an increased telomere length after 4 weeks, indicating a recruitment of stem cells with long telomeres from the hematopoietic system. Furthermore, female 11- to 34-week-old second generation mice (i.e. with not yet shortened telomeres) with global genetic deficiency of telomerase reverse transcriptase, the catalytic subunit of telomerase, were analyzed on day 28 after sham surgery and MI, respectively. In comparison to the C57Bl/6N and the TERT+/+ animals, mice with MI without telomerase activity showed partially marked differences in the regulation. For example, the telomere length and TRF2 expression were not altered in TERT-/- mice with MI, but in the TERT-/-- in comparison to the TERT+/+- sham group the pro-apoptotic proteins p53 and Bax were decreased and the senescence marker p16 was increased, which was not further influenced by the infarction. In histologic examinations, there was also a crucial difference in TERT-deficient animals with MI versus TERT-positive controls: a significant increase of myocardial fibrosis with the Sirius Red staining. This important finding could be substantiated molecular biologically by demonstrating an increased gene expression of Collagen-1α and Connective Tissue Growth Factor. The above-mentioned alterations of protein expression may be an explanation for the possible influence of TERT deficiency on collagen accumulation. Examinations in extracardiac tissues, similar to the C57Bl/6N animals, resulted in heterogeneous results in the myocardium, but overall indicated an impairment of extracardiac regulation of cardiac remodeling, e.g. lack of MNC with longer telomeres in TERT-/- animals with MI, while other regulations were comparable. Another scientific question of this work concerned with the functional consequences of TERT deficiency in the context of MI. The measurement of the indices of heart weight / tibial length and / body weight, lung wet weight / body weight and liver wet weight / body weight indicated that 4 weeks after LAD ligation neither myocardial hypertrophy nor heart failure was present. Cardiac magnetic resonance imaging and Millar catheter verified cardiac dysfunction with reduced pumping performance and pressure load, respectively, due to surgically induced MI, but there were no clear genotypic differences. This is due to the short time of MRI examination 4 weeks after MI, which was deliberately chosen to assess the mid-term regulation after heart infarction. The present work provided a bunch of new findings focusing on the role of telomerase and cellular senescence markers in ischemia-induced myocardial remodeling in mice. The results could provide a basis for the future development of new therapeutic strategies. By influencing cell aging, it might be possible to favorably influence the development of ischemic cardiomyopathy as a dreaded sequelae of MI caused by maladaptive remodeling, among other things

Global prevalence and genotype distribution of hepatitis C virus infection in 2015 : A modelling study

Publisher Copyright: © 2017 Elsevier LtdBackground The 69th World Health Assembly approved the Global Health Sector Strategy to eliminate hepatitis C virus (HCV) infection by 2030, which can become a reality with the recent launch of direct acting antiviral therapies. Reliable disease burden estimates are required for national strategies. This analysis estimates the global prevalence of viraemic HCV at the end of 2015, an update of—and expansion on—the 2014 analysis, which reported 80 million (95% CI 64–103) viraemic infections in 2013. Methods We developed country-level disease burden models following a systematic review of HCV prevalence (number of studies, n=6754) and genotype (n=11 342) studies published after 2013. A Delphi process was used to gain country expert consensus and validate inputs. Published estimates alone were used for countries where expert panel meetings could not be scheduled. Global prevalence was estimated using regional averages for countries without data. Findings Models were built for 100 countries, 59 of which were approved by country experts, with the remaining 41 estimated using published data alone. The remaining countries had insufficient data to create a model. The global prevalence of viraemic HCV is estimated to be 1·0% (95% uncertainty interval 0·8–1·1) in 2015, corresponding to 71·1 million (62·5–79·4) viraemic infections. Genotypes 1 and 3 were the most common cause of infections (44% and 25%, respectively). Interpretation The global estimate of viraemic infections is lower than previous estimates, largely due to more recent (lower) prevalence estimates in Africa. Additionally, increased mortality due to liver-related causes and an ageing population may have contributed to a reduction in infections. Funding John C Martin Foundation.publishersversionPeer reviewe

Identification of Genomic Regions Associated with Phenotypic Variation between Dog Breeds using Selection Mapping

Peer reviewe

Influence of germination time and temperature on the properties of rye malt and rye malt based worts

The effects of germination time and temperature on the quality of rye malt and worts derived thereof were investigated using Response Surface Methodology. Amylolytic and proteolytic enzyme activities were increased by long germination periods, while beta-glucanase activity was not influenced. Total and Soluble Nitrogen content were also not significantly affected by the variations in germination conditions. Free Amino Nitrogen (FAN) was found in higher amounts in worts prepared from rye malts with long germination times. Extract contents were higher in rye malt than in the control barley malt and could be increased by a favourable germination regime, while no such impact on wort fermentability was found. High wort viscosities could be significantly reduced by a long germination period at low temperatures, but were still unacceptably high. The same conditions favoured the development of endoxylanase activity. Arabinoxylan (AX) accumulated during the germination process and their extractability increased. The results suggest that longer germination periods resulted in an increased number of AX molecules with lower molecular mass. Optimal rye malt qualities within the limits of this study were found for a germination time of 144 h at 10 degrees C, which resulted in an acceptable FAN content and the lowest measured viscosity. (C) 2010 Elsevier Ltd. All rights reserved.status: publishe

Evaluation of an adaptive detector collimation for prospectively ECG-triggered coronary CT angiography with third-generation dual-source CT

OBJECTIVES: To investigate the impact of an adaptive detector collimation on the dose parameters and accurateness of scan length adaption at prospectively ECG-triggered sequential cardiac CT with a wide-detector third-generation dual-source CT. METHODS: Ideal scan lengths for human hearts were retrospectively derived from 103 triple-rule-out examinations. These measures were entered into the new scanner operated in prospectively ECG-triggered sequential cardiac scan mode with three different detector settings: (1) adaptive collimation, (2) fixed 64 × 0.6-mm collimation, and (3) fixed 96 × 0.6-mm collimation. Differences in effective scan length and deviation from the ideal scan length and dose parameters (CTDIvol, DLP) were documented. RESULTS: The ideal cardiac scan length could be matched by the adaptive collimation in every case while the mean scanned length was longer by 15.4% with the 64 × 0.6 mm and by 27.2% with the fixed 96 × 0.6-mm collimation. While the DLP was almost identical between the adaptive and the 64 × 0.6-mm collimation (83 vs. 89 mGycm at 120 kV), it was 62.7% higher with the 96 × 0.6-mm collimation (135 mGycm), p < 0.001. CONCLUSION: The adaptive detector collimation for prospectively ECG-triggered sequential acquisition allows for adjusting the scan length as accurate as this can only be achieved with a spiral acquisition. This technique allows keeping patient exposure low where patient dose would significantly increase with the traditional step-and-shoot mode. KEY POINTS: • Adaptive detector collimation allows keeping patient exposure low in cardiac CT. • With novel detectors the desired scan length can be accurately matched. • Differences in detector settings may cause 62.7% of excessive dose

Quantitative perfusion-CMR is significantly influenced by the placement of the arterial input function

The aim of this study is to provide a systematic assessment of the influence of the position on the arterial input function (AIF) for perfusion quantification. In 39 patients with a wide range of left ventricular function the AIF was determined using a diluted contrast bolus of a cardiac magnetic resonance imaging in three left ventricular levels (basal, mid, apex) as well as aortic sinus (AoS). Time to peak signal intensities, baseline corrected peak signal intensity and upslopes were determined and compared to those obtained in the AoS. The error induced by sampling the AIF in a position different to the AoS was determined by Fermi deconvolution. The time to peak signal intensity was strongly correlated (r2 > 0.9) for all positions with a systematic earlier arrival in the basal (− 2153 ± 818 ms), the mid (− 1429 ± 928 ms) and the apical slice (− 450 ± 739 ms) relative to the AoS (all p  0.9 for both) for all positions with a systematic overestimation in all positions relative to the AoS (all p < 0.001 and all p < 0.05). Differences between the positions were more pronounced for patients with reduced ejection fraction. The error of averaged MBF quantification was 8%, 13% and 27% for the base, mid and apex. The location of the AIF significantly influences core parameters for perfusion quantification with a systematic and ejection fraction dependent error. Full quantification should be based on obtaining the AIF as close as possible to the myocardium to minimize these errors

Head and neck single- and dual-energy CT: differences in radiation dose and image quality of 2nd and 3rd generation dual-source CT

Objectives: To compare radiation dose and image quality of single-energy (SECT) and dual-energy (DECT) head and neck CT examinations performed with second- and third-generation dual-source CT (DSCT) in matched patient cohorts. Methods: 200 patients (mean age 55.1 ± 16.9 years) who underwent venous phase head and neck CT with a vendor-preset protocol were retrospectively divided into four equal groups (n = 50) matched by gender and BMI: second (Group A, SECT, 100-kV; Group B, DECT, 80/Sn140-kV), and third-generation DSCT (Group C, SECT, 100-kV; Group D, DECT, 90/Sn150-kV). Assess- ment of radiation dose was performed for an average scan length of 27 cm. Contrast-to-noise ratio measure- ments and dose-independent figure-of-merit calcu- lations of the submandibular gland, thyroid, internal jugular vein, and common carotid artery were analyzed quantitatively. Qualitative image parameters were evalu- ated regarding overall image quality, artifacts and reader confidence using 5-point Likert scales. Results: Effective radiation dose (ED) was not signifi- cantly different between SECT and DECT acquisition for each scanner generation (p = 0.10). Significantly lower effective radiation dose (p 0.06). Conclusion: Contrast-enhanced head and neck DECT can be performed with second- and third-generation DSCT systems without radiation penalty or impaired image quality compared with SECT, while third-generation DSCT is the most dose efficient acquisition method. Advances in knowledge: Differences in radiation dose between SECT and DECT of the dose-vulnerable head and neck region using DSCT systems have not been evaluated so far. Therefore, this study directly compares radiation dose and image quality of standard SECT and DECT protocols of second- and third-generation DSCT platforms