The effect of the lathyrogens beta-aminopropionitril and homocysteine on osteoblastic differentiation in regard to collagen matrix formation and epigenetic regulation of gene expression

Kollagen Typ I, der Hauptbestandteil der extrazellulären Matrix des Knochens, weist eine super-molekulare Organisation auf. Die einzelnen Kollagenmoleküle werden einer Reihe von intra- und extrazellulären Modifikationen unterzogen, die es ihnen ermöglicht extrazellulär Kollagenfibrillen auszubilden. Ein wesentlicher Schritt dabei ist die Ausbildung von Kollagenquervernetzungen. Dieser Prozess ist gewebespezifisch und wird von vielen zellulären und matrix-abhängigen Signalen gesteuert. Hemmung der Lysyloxidase (Lox), ein Schlüsselenzym der Kollagenvernetzung, führt zu veränderten Quervernetzungen und gestörter Fibrillogenese. Ergebnisse aus in-vitro als auch in-vivo durchgeführten Experimenten bestätigten, dass solche Veränderungen zu Knochenmineralverlust, reduzierter Knochenfestigkeit und einer veränderten Mineralisation führen. Ein weiterer Faktor der eine wesentliche Rolle in der Organ und Gewebsentwicklung spielt ist die Regulierung der Expression von Genen durch epigenetische DNA-Methylierung. Bis zum jetzigen Zeitpunkt ist die Bedeutung von diesem Mechanismus in der Knochenentwicklung und Pathogenese nur wenig erforscht worden. In dieser Dissertation wurde daher auf die Rolle epigenetischer Genregulationen im Osteoblasten intensiv eingegangen. Nach Behandlung der pre-osteoblastären MC3T3-E1 Maus Zelllinie mit den zwei Inhibitoren der Lox, beta aminopropionitrile (bAPN) und Homocysteine (hcys), haben wir den Effekt von diesen zwei Lathyrogenen auf die Zelllinie analysiert und verglichen. Die Expression von osteoblastären Genen wurde mittels „real time polymerase chain reaction“ (qPCR) und „gene expression microarrays“ untersucht. Marker der osteoblastischen Aktivität und Zellproliferation wurden durch Messung der Aktivität der Alkalischen Phosphatase sowie Viabilitätstest bestimmt. Die Effekte der Substanzen auf die Kollagenquervernetzung wurden durch „Fourier-Transform-Infrarot-Spektrometrie“ (FTIR) gemessen. Die ersten Ergebnisse zeigten dass sowohl bAPN als auch hcys Lox nicht nur enzymatisch hemmen sondern auch dessen mRNA Expression vermindern. Unter Berücksichtigung der klinischen Bedeutung von hcys, haben wir den zellulären Signalweg für die hcys-abhängige Verminderung der Lox Expression erforscht. Dabei nutzten wir Techniken wie „Enzyme-linked immunosorbent assay“ (ELISA), Immuno Blotting und Chromatin-Immunopräzipitation (ChIP). Auswertungen ergaben das Interleukin 6 (IL-6), der Transkriptionsfaktor „Friend leukemia integration 1“ (FLI1) und die DNA-Methyltransferse 1 (DNMT1) in der Repression von Lox durch hcys involviert sind. Untersuchungen an der Promotorregion des Lox-Gens ergaben eine DNA-methylierungsabhängige Regulation der Lox Expression durch hcys. Im letzten Teil der Arbeit wurde ein neuer Signal–Transduktionsweg aufgeklärt, durch den die extrazelluläre Matrix (ECM) die Proliferation und Differenzierung von Osteoblasten fördern kann. Beim Aussähen von MC3T3-E1 Zellen auf mit Kollagen Typ I beschichtete Platten haben wir eine erhöhte Expression an osteoblastären Genen und eine verminderte Expression des pro-apoptotischen Gens Fas beobachtet. Durch die Verwendung verschiedener Inhibitoren, qPCR und DNA-Methylierungsanalysen konnten wir zeigen, dass das extrazelluläre Kollagen Typ I via FAK, MAPK und den Transkriptionsfaktor AP1 direkt die Expression des Gens Dnmt1 stimuliert was in weiterer Folge für die Stilllegung des Gens Fas durch epigenetischer DNA-Methylierung verantwortlich ist.Collagen, the main component of bone extracellular matrix (ECM) represents a protein with a defined super-molecular organization. For stable fibril formation, the basic element of collagen, the fiber undergoes several intra- and extracellular modifications. One major process of collagen / matrix maturation is collagen cross-link formation. This process is tissue-specific, and involves a variety of cellular and matrix derived signals. Inhibition of lysyl oxidase (Lox), an enzymatic key player in cross-link formation, leads to improper collagen cross-linking and altered fibrillogenesis. In vitro and in vivo experiments confirmed that these aberrations in matrix formation are accompanied by loss of bone mineral, reduced bone strength and altered mineralization. A further factor involved in tissue development is regulation of gene expressions by epigenetic DNA methylation. To date, sparse research has been performed regarding the significance of this mechanism for bone development and pathogenesis. Therefore, in this thesis the role of epigenetic mechanisms in osteoblastic development was examined. By treatment of the murine pre-osteoblastic MC3T3-E1 cell line with two lysyl oxidase inhibitors, namely beta aminopropionitrile (bAPN) and homocysteine (hcys), we monitored and compared the effects of these two lathyrogens on the osteoblastic cells. The expression levels of osteoblastic genes were analyzed employing quantitative real time PCR (qPCR) and gene expression microarrays. Markers for osteoblastic activity and cell proliferation were analyzed by viability tests and alkaline phosphatase activity tests and effects on collagen cross-link formation were measured by FTIR. First results indicated that bAPN as well as hcys, besides the enzymatic inhibition down regulate the mRNA expression of Lox. Due to the greater clinical relevance of hcys, using ELISA, immuno blotting and chromatin immuno precipitation techniques we identified the cellular pathway responsible for the effect of hcys on the expression of Lox. This involves IL-6, the transcription factor FLI1 and the DNA methyltransferase DNMT1. DNA methylation analysis of the Lox promoter revealed that hcys suppresses Lox expression by DNA methylation. Finally, a new mechanism controlling ECM mediated proliferation and differentiation of osteoblasts was elucidated. By seeding MC3T3-E1 cells on collagen type I we observed an up-regulation of osteoblastic genes and a down regulation of the pro-apoptotic gene Fas when compared to control. Using several inhibitors, qPCR and DNA methylation analysis we found that extra-cellular collagen type I via FAK, MAPK and the transcription factor AP1 directly up-regulates Dnmt1 which in turn maintains promoter methylation of the gene Fas thus repressing its expression

Inhibition of the mevalonate pathway affects epigenetic regulation in cancer cells

The mevalonate pathway provides metabolites for post-translational modifications such as farnesylation, which are critical for the activity of RAS downstream signaling. Subsequently occurring regulatory processes can induce an aberrant stimulation of DNA methyltransferase (DNMT1) as well as changes in histone deacetylases (HDACs) and microRNAs in many cancer cell lines. Inhibitors of the mevalonate pathway are increasingly recognized as anticancer drugs. Extensive evidence indicates an intense cross-talk between signaling pathways, which affect growth, differentiation, and apoptosis either directly or indirectly via epigenetic mechanisms. Herein, we show data obtained by novel transcriptomic and corresponding methylomic or proteomic analyses from cell lines treated with pharmacologic doses of respective inhibitors (i.e., simvastatin, ibandronate). Metabolic pathways and their epigenetic consequences appear to be affected by a changed concentration of NADPH. Moreover, since the mevalonate metabolism is part of a signaling network, including vitamin D metabolism or fatty acid synthesis, the epigenetic activity of associated pathways is also presented. This emphasizes the far-reaching epigenetic impact of metabolic therapies on cancer cells and provides some explanation for clinical observations, which indicate the anticancer activity of statins and bisphosphonates

Bovine Colostrum Supplementation and Bone Health: a Pilot Study

Research has shown the positive effects of some bovine colostrum components in bone cells; for instance, lactoferrin is reported to stimulate osteoblast proliferation and inhibit osteoclast activity in cell cultures. However, whether bovine colostrum as a whole can induce bone mass gains in osteoporotic bones is relatively unclear. The aim of this study was to investigate the effects of bovine colostrum supplementation in ovariectomized-induced bone loss (OVX) rats. Methods: Twenty-seven-month-old female Wister rats (n=16) were randomly assigned to the following two groups: 1) a healthy control (non-OVX) with no supplementation, and 2) a OVX with bovine colostrum supplementation (0.5g/day; oral consumption). After 5 months supplementation, bone microstructure was scanned using micro-CT (right tibia). Bone formation markers (serum: pre-and post supplementation) were analysed (alkaline phosphatase and osteocalcin) by ECLIA. The study was approved by the National Ethics Committee for the Use of Animals in Research (ORBEA). Results: No significant differences were found between groups in serum alkaline phosphatase either before or after supplementation (p>0.05). Serum osteocalcin significantly increased post-supple-mentation in the OVX compared to pre-supplementation (pre: 11.32+/-1.61; post: 12.45+/-1.21μg/L, p0.05). Trabecular bone mineral content (BMC), trabecular thickness, cortical bone mineral density (BMD) and cortical BMC were similar between groups after supplementation (p>0.05). However, OVX group revealed significantly higher trabecular porosity (5.6%, p<0.01), trabecular separation (36.3%, p<0.01), and cortical porosity (8.0%, p<0.01) compared to the healthy control post-supplementation. Conclusion: Bovine colostrum seems to preserve bone mass of OVX by stimulating bone formation. However, these positive effects seem not to be sufficient to restore bone micro-architecture in the OVX group, possibly because the administrated dose of bovine colostrum was not sufficient for OVX to catch-up healthy rats in terms of trabecular and cortical porosity. The potential therapeutic use of bovine colostrum for osteoporosis deserves further investigation

Prior Stroke in PFO Patients Is Associated With Both PFO-Related and -Unrelated Factors.

Background and Purpose: To identify factors associated with prior stroke at presentation in patients with cryptogenic stroke (CS) and patent foramen ovale (PFO). Methods: We studied cross-sectional data from the International PFO Consortium Study (NCT00859885). Patients with first-ever stroke and those with prior stroke at baseline were analyzed for an association with PFO-related (right-to-left shunt at rest, atrial septal aneurysm, deep venous thrombosis, pulmonary embolism, and Valsalva maneuver) and PFO-unrelated factors (age, gender, BMI, hypertension, diabetes mellitus, hypercholesterolemia, smoking, migraine, coronary artery disease, aortic plaque). A multivariable analysis was used to adjust effect estimation for confounding, e.g., owing to the age-dependent definition of study groups in this cross-sectional study design. Results: We identified 635 patients with first-ever and 53 patients with prior stroke. Age, BMI, hypertension, diabetes mellitus, hypercholesterolemia, coronary artery disease, and right-to-left shunt (RLS) at rest were significantly associated with prior stroke. Using a pre-specified multivariable logistic regression model, age (Odds Ratio 1.06), BMI (OR 1.06), hypercholesterolemia (OR 1.90) and RLS at rest (OR 1.88) were strongly associated with prior stroke.Based on these factors, we developed a nomogram to illustrate the strength of the relation of individual factors to prior stroke. Conclusion: In patients with CS and PFO, the likelihood of prior stroke is associated with both, PFO-related and PFO-unrelated factors

Effects of halogens on European air-quality

Halogens (Cl, Br) have a profound influence on stratospheric ozone (O3). They (Cl, Br and I) have recently also been shown to impact the troposphere, notably by reducing the mixing ratios of O3 and OH. Their potential for impacting regional air-quality is less well understood. We explore the impact of halogens on regional pollutants (focussing on O3) with the European grid of the GEOS-Chem model (0.25° × 0.3125°). It has recently been updated to include a representation of halogen chemistry. We focus on the summer of 2015 during the ICOZA campaign at the Weybourne Atmospheric Observatory on the North Sea coast of the UK. Comparisons between these observations together with those from the UK air-quality network show that the model has some skill in representing the mixing ratios/concentration of pollutants during this period. Although the model has some success in simulating the Weybourne ClNO2 observations, it significantly underestimates ClNO2 observations reported at inland locations. It also underestimates mixing ratios of IO, OIO, I2 and BrO, but this may reflect the coastal nature of these observations. Model simulations, with and without halogens, highlight the processes by which halogens can impact O3. Throughout the domain O3 mixing ratios are reduced by halogens. In northern Europe this is due to a change in the background O3 advected into the region, whereas in southern Europe this is due to local chemistry driven by Mediterranean emissions. The proportion of hourly O3 above 50 nmol mol-1 in Europe is reduced from 46% to 18% by halogens. ClNO2 from N2O5 uptake onto sea-salt leads to increases in O3 mixing ratio, but these are smaller than the decreases caused by the bromine and iodine. 12% of ethane and 16% of acetone within the boundary layer is oxidised by Cl. Aerosol response to halogens is complex with small (∼10%) reductions in PM2.5 in most locations. A lack of observational constraints coupled to large uncertainties in emissions and chemical processing of halogens make these conclusions tentative at best. However, the results here point to the potential for halogen chemistry to influence air quality policy in Europe and other parts of the world

EuReCa ONE—27 Nations, ONE Europe, ONE Registry A prospective one month analysis of out-of-hospital cardiac arrest outcomes in 27 countries in Europe

AbstractIntroductionThe aim of the EuReCa ONE study was to determine the incidence, process, and outcome for out of hospital cardiac arrest (OHCA) throughout Europe.MethodsThis was an international, prospective, multi-centre one-month study. Patients who suffered an OHCA during October 2014 who were attended and/or treated by an Emergency Medical Service (EMS) were eligible for inclusion in the study. Data were extracted from national, regional or local registries.ResultsData on 10,682 confirmed OHCAs from 248 regions in 27 countries, covering an estimated population of 174 million. In 7146 (66%) cases, CPR was started by a bystander or by the EMS. The incidence of CPR attempts ranged from 19.0 to 104.0 per 100,000 population per year. 1735 had ROSC on arrival at hospital (25.2%), Overall, 662/6414 (10.3%) in all cases with CPR attempted survived for at least 30 days or to hospital discharge.ConclusionThe results of EuReCa ONE highlight that OHCA is still a major public health problem accounting for a substantial number of deaths in Europe.EuReCa ONE very clearly demonstrates marked differences in the processes for data collection and reported outcomes following OHCA all over Europe. Using these data and analyses, different countries, regions, systems, and concepts can benchmark themselves and may learn from each other to further improve survival following one of our major health care events

Active silver nanoparticles for wound healing

In this preliminary study, the silver nanoparticle (Ag NP)-based dressing, ActicoatTM Flex 3, has been applied to a 3D fibroblast cell culture in vitro and to a real partial thickness burn patient. The in vitro results show that Ag NPs greatly reduce mitochondrial activity, while cellular staining techniques show that nuclear integrity is maintained, with no signs of cell death. For the first time, transmission electron microscopy (TEM) and inductively coupled plasma mass spectrometry (ICP-MS) analyses were carried out on skin biopsies taken from a single patient during treatment. The results show that Ag NPs are released as aggregates and are localized in the cytoplasm of fibroblasts. No signs of cell death were observed, and the nanoparticles had different distributions within the cells of the upper and lower dermis. Depth profiles of the Ag concentrations were determined along the skin biopsies. In the healed sample, most of the silver remained in the surface layers, whereas in the unhealed sample, the silver penetrated more deeply. The Ag concentrations in the cell cultures were also determined. Clinical observations and experimental data collected here are consistent with previously published articles and support the safety of Ag NP-based dressing in wound treatment