Die nicht-apoptotische Funktion von Caspase-8 in endothelialen Vorläuferzellen

Vasculogenesis as well as angiogenesis are important for postnatal development of blood vessels. Peripheral blood or bone marrow-derived endothelial precursor cells are used in clinical trials for therapeutic enhancement of postnatal neovascularization in patients suffering from coronary artery diseases. The vasculogenic potential of the precursor cell population depends on the appropriate retention of the infused cells to the ischemic tissue. However, cell-autonomous mechanisms regulating the attraction and retention of circulating cells in inflammatory tissue are not well understood. Caspases belong to a family of pro-apoptotic enzymes. Beyond cell death signals, caspase proteases additionally regulate non-apoptotic processes like cell morphology and migration in many cell types. The isoform Caspase-8 is essential for embryonal vasculogenesis in conditional knockout mice. In this study, we identified a novel apoptosis-unrelated role of Caspase-8 in circulating and bone marrow-derived cells for vascular repair. Caspase-8-specific inhibition abrogated the ex vivo formation of EPC from human peripheral blood. Moreover, Caspase-8 inhibition disables EPC migration and adhesion to different matrices and decreases the cell surface expression of the fibronectin receptor subunit integrin alpha 5 and the chemokine receptor CXCR4. In vitro and in vivo studies using bone marrow mononuclear cells derived from inducible Caspase-8- deficient mice revealed an essential role of Caspase-8 for EPC formation and neovascularization enhancing capacities of progenitor cells. Caspase-8 activity appears to be required for maintaining responses to matrix interaction and chemoattractants of EPC. Additional studies showed that the E3 ubiquitin ligase Cbl-b, a negative regulator of cell adhesion molecules including integrin alpha 5, is present in EPC at low protein levels under basal conditions, but markedly increases upon Caspase-8 inhibition. In vitro assays and overexpression studies in intact cells confirmed Caspase-8-dependent degradation of Cbl-b, providing a potential requirement for Caspase-8-regulated adhesion. Indeed, neovascularization of matrigel plugs was enhanced in mice lacking Cbl-b. Moreover, Cbl-b degradation in the presence of active Caspase-8 prevents the down-regulation of integrin alpha 5 and is associated with an enhanced vasculogenic activity of progenitor cells in hind limb ischemia. The identified upstream regulation of caspase-8 by cytokine IL-6 is only one possibility for fine-tuning the non-apoptotic enzymatic activity. In summary, this study shows a novel essential role of Caspase-8 for proper EPC adhesion-related signaling. Caspase-8 is involved in the function of adhesion molecules by regulation the E3 ubiquitin ligase Cbl-b. Strategies to improve survival of therapeutic injected progenitor cells by using caspase inhibitors should be addressed with caution. Because of the broad spectrum of activity of caspase-8, downstream targets of this caspase isoform and Cbl-b should be in more focus for therapeutic pretreatment to improve neovascularization of myocardial and ischemic tissue.Sowohl Vaskulogenese als auch Angiogenese spielen eine bedeutende Rolle in der nachgeburtlichen Entwicklung von Blutgefäßen. Im Blutstrom zirkulierende oder direkt aus dem Knochenmark isolierte endotheliale Vorläuferzellen werden in klinischen Studien zur therapeutischen Unterstützung der nachgeburtlichen Gefäßneubildung bei Patienten mit koronaren Herzkrankheiten eingesetzt. Das Potential zur Unterstützung der Gefäßneubildung durch Vorläuferzellen hängt von dem entsprechenden Verbleiben der applizierten Zellen im ischämischen Gewebe ab. Dennoch sind die zelleigenen Mechanismen, die zur Regulation der Anziehung und dem Verbleiben von zirkulierenden Zellen im entzündlichen Gewebe beitragen, noch nicht weit genug untersucht und verstanden. Caspasen gehören zu einer Familie von Enzymen die den Zelltod programmieren. Neben Signalen für den Zelltod kann diese Art der Proteasen zusätzlich Prozesse wie Zellform und Fortbewegung in verschiedenen Zelltypen regulieren. Die Isoform Caspase-8 ist wichtig für die embryonale Vaskulogenese, was in Knockout-Mäusen nachgewissen wurde. In dieser vorliegenden Doktorarbeit identifizierten wir eine neue Zelltod-unabhängige Rolle von Caspase-8 für die im Blut zirkulierenden oder aus dem Knochenmark stammmenden Vorläuferzellen und der vermittelten Gefäßreparatur. Die spezifische Caspase-8 Inhibition verhindert die ex vivo Bildung von EPC aus mononukleären Zellen des humanen Peripherbluts. Weiterhin blockiert Caspase-8 die Fortbewegung und Anhaftung von EPC an verschiedene Matrizen und verringert die Oberflächenexpression der Fibronektin-Rezeptoruntereinheit Integrin alpha 5 und des Chemokine-Rezeptors CXCR4 auf EPC. Sowohl in vitro als auch in vivo Untersuchungen mit Knochenmarksmononukleären Zellen von induzierten Mäusen mangelhaft an Caspase-8 zeigten, dass Caspase-8 eine wichtige Rolle für die Ausbildung von EPC und dessen unterstützende Wirkung zur Gefäßneubildung spielt. Die Aktivität von Caspase-8 erscheint erforderlich für die Interaktionen zwischen EPC und Matrizen und die Anziehung durch Chemokine. Weitere Untersuchungen zeigten, dass die E3 Ubiquitin Ligase Cbl-b als ein negativer Regulator von Adhäsionsmolekülen wie Integrin alpha 5, unter basalen Bedingungen in sehr niedrigen Proteinkonzentrationen in EPC vorhanden ist. Dieser Proteinanteil steigt mit der Inhibierung von Caspase-8 in endothelialen Vorläuferzellen. In vitro Untersuchungen und Überexpressionen in humanen Zellen bestätigten den Caspase-8 abhängigen Abbau von Cbl-b. Dies wiederum lässt eine mögliche Notwenigkeit der Caspase-8 regulierten Adhäsion vermuten. Tatsächlich war die Gefäßneubildung in Matrigelplugs in Mäusen ohne Cbl-b verbessert. Desweiteren verhindert der Cbl-b Abbau in Anwesenheit aktiver Caspase-8 die Runterregulation von Integrin alpha 5 und ist zugleich verbunden mit einer erhöhten Aktivität der EPC bei der Gefäßneubildung in der Hinterlaufischämie. Interleukin-6 ist vermutlich nur eine der vielen Möglichkeiten zur Regulation der nicht-apoptotischen enzymatischen Aktivität von Caspase-8 und muss hier weiter untersucht werden. Zusammenfassend lässt sich sagen, dass diese Arbeit eine neue wichtige Rolle von Caspase-8 für eine funktionierende Signalübertragung in der Adhäsion in EPC zeigt. Caspase-8 ist beteiligt an der korrekten Funktion von Adhäsionsmolekülen durch die Regulation der E3 Ubiquitin Ligase Cbl-b. Mögliche Strategien zur Steigerung des Überlebens von therapeutisch injizierten Vorläuferzellen durch die Vorbehandlung mit Caspase Inhibitoren sollte mit Vorsicht betrachtet werden. Aufgrund des vielfältigen Aktivitätsspektrums von Caspase-8 sollten Zielobjekte von Caspase-8 und Cbl-b in nähere Betrachtung für therapeutische Ansätze gezogen werden, um die Gefäßneubildung im myokardialen und ischämischen Gewebe zu verbessern

Animal Models for Anorexia Nervosa—A Systematic Review

Anorexia nervosa is an eating disorder characterized by intense fear of gaining weight and a distorted body image which usually leads to low caloric intake and hyperactivity. The underlying mechanism and pathogenesis of anorexia nervosa is still poorly understood. In order to learn more about the underlying pathophysiology of anorexia nervosa and to find further possible treatment options, several animal models mimicking anorexia nervosa have been developed. The aim of this review is to systematically search different databases and provide an overview of existing animal models and to discuss the current knowledge gained from animal models of anorexia nervosa. For the systematic data search, the Pubmed—Medline database, Embase database, and Web of Science database were searched. After removal of duplicates and the systematic process of selection, 108 original research papers were included in this systematic review. One hundred and six studies were performed with rodents and 2 on monkeys. Eighteen different animal models for anorexia nervosa were used in these studies. Parameters assessed in many studies were body weight, food intake, physical activity, cessation of the estrous cycle in female animals, behavioral changes, metabolic and hormonal alterations. The most commonly used animal model (75 of the studies) is the activity-based anorexia model in which typically young rodents are exposed to time-reduced access to food (a certain number of hours a day) with unrestricted access to a running wheel. Of the genetic animal models, one that is of particular interest is the anx/anx mice model. Animal models have so far contributed many findings to the understanding of mechanisms of hunger and satiety, physical activity and cognition in an underweight state and other mechanisms relevant for anorexia nervosa in humans

The muscle satellite cell at 50: the formative years

In February 1961, Alexander Mauro described a cell 'wedged' between the plasma membrane of the muscle fibre and the surrounding basement membrane. He postulated that it could be a dormant myoblast, poised to repair muscle when needed. In the same month, Bernard Katz also reported a cell in a similar location on muscle spindles, suggesting that it was associated with development and growth of intrafusal muscle fibres. Both Mauro and Katz used the term 'satellite cell' in relation to their discoveries. Today, the muscle satellite cell is widely accepted as the resident stem cell of skeletal muscle, supplying myoblasts for growth, homeostasis and repair

Control of Ground Movements for a Multi-Level-Anchored, Diaphragm Wall During Excavation

An excavation up to 23m deep for the Dana Farber research tower in the Longwood medical area of Boston, was supported by a permanent perimeter diaphragm wall extending into the underlying conglomerate and up to 6 levels of prestressed tiebacks anchored in the rock. The lateral earth support system was very successful in limiting wall deflections to less than ±15mm on each of the four sides of the excavation. However, ground surface settlements up to 65mm occurred on two sides and were attributed to ground losses that occurred when tiebacks were installed through overpressured sand layers at depths of 15-18m. Finite element simulations are able to describe consistently the effects of the different excavation and support sequences on each side of the project using backfigured soil properties, while surface settlements can be explained by including local ground losses within the analyses

Activity-Based Anorexia Reduces Body Weight without Inducing a Separate Food Intake Microstructure or Activity Phenotype in Female Rats—Mediation via an Activation of Distinct Brain Nuclei

Anorexia nervosa (AN) is accompanied by severe somatic and psychosocial complications. However, the underlying pathogenesis is poorly understood, treatment is challenging and often hampered by high relapse. Therefore, more basic research is needed to better understand the disease. Since hyperactivity often plays a role in AN, we characterized an animal model to mimic AN using restricted feeding and hyperactivity. Female Sprague-Dawley rats were divided into four groups: no activity/ad libitum feeding (ad libitum, AL, n=9), activity/ad libitum feeding (activity, AC, n=9), no activity/restricted feeding (RF, n=12) and activity/restricted feeding (activity-based anorexia, ABA, n=11). During the first week all rats were fed ad libitum, ABA and AC had access to a running wheel for 24h/d. From week two ABA and RF only had access to food from 9:00-10:30 am. Body weight was assessed daily, activity and food intake monitored electronically, brain activation assessed using Fos immunohistochemistry at the end of the experiment. While during the first week no body weight differences were observed (p>0.05), after food restriction RF rats showed a body weight decrease: -13% vs. day eight (p0.05). Similarly, the daily physical activity was not different between AC and ABA (p>0.05). The investigation of Fos expression in the brain showed neuronal activation in several brain nuclei such as the supraoptic nucleus, arcuate nucleus, locus coeruleus and nucleus of the solitary tract of ABA compared to AL rats. In conclusion, ABA combining physical activity and restricted feeding likely represents a suited animal model for AN to study pathophysiological alterations and pharmacological treatment options. Nonetheless, cautious interpretation of the data is necessary since rats do not voluntarily reduce their body weight as observed in human AN

Delivery of GalNAc-Conjugated Splice-Switching ASOs to Non-hepatic Cells through Ectopic Expression of Asialoglycoprotein Receptor

Splice-switching antisense oligonucleotides (ASOs) are promising therapeutic tools to target various genetic diseases, including cancer. However, in vivo delivery of ASOs to orthotopic tumors in cancer mouse models or to certain target tissues remains challenging. A viable solution already in use is receptor-mediated uptake of ASOs via tissue-specific receptors. For example, the asialoglycoprotein receptor (ASGP-R) is exclusively expressed in hepatocytes. Triantennary N-acetylgalactosamine (GalNAc) (GN3)-conjugated ASOs bind to the receptor and are efficiently internalized by endocytosis, enhancing ASO potency in the liver. Here we explore the use of GalNAc-mediated targeting to deliver therapeutic splice-switching ASOs to cancer cells that ectopically express ASGP-R, both in vitro and in tumor mouse models. We found that ectopic expression of the major isoform ASGP-R1 H1a is sufficient to promote uptake and increase GN3-ASO potency to various degrees in four of five tested cancer cells. We show that cell-type-specific glycosylation of the receptor does not affect its activity. In vivo, GN3-conjugated ASOs specifically target subcutaneous xenograft tumors that ectopically express ASGP-R1, and modulate splicing significantly more strongly than unconjugated ASOs. Our work shows that GN3-targeting is a useful tool for proof-of-principle studies in orthotopic cancer models, until endogenous receptors are identified and exploited for efficiently targeting cancer cells

The autonomous acoustic buoy

Treball desenvolupat dins el marc del programa 'European Project Semester'.The purpose of this project is to design an Autonomous Acoustic Buoy (AAB) for the Laboratori d’Aplicacions Bioacústiques (LAB) and the Universitat Politècnica de Catalunya (UPC). The project is composed of five sub-projects: the Marketing Project, the Mechanical Design Project, the Electronics Project, the Computer Science Project, and the Wireless Communication and Power Supply Project. The Marketing Project created a leaflet for the AAB. The Mechanical Design Project has modelled the buoy using a computer aided design program. The Electronic Project has designed a programmable filtering circuit. The Computer Science Project has developed software for a single board computer called: the Hercules Board. The Wireless Communication and Power Supply Project has examined wireless communication and power supply solutions. All of the work done for the AAB Project is presented in this report and is addressed to both the LAB and the UPC

Culturing muscle fibres in hanging drop: A novel approach to solve an old problem

Background Information. The satellite cells (SCs) associated with muscle fibres play a key role in postnatal growth and regeneration of skeletal muscle. Commonly used methods of isolation and in vitro culture of SCs lead to the mixture of their subpopulations that exist within muscle. To solve this problem, we used the well established technique, the hanging drop system, to culture SCs in a three-dimensional environment and thus, to monitor them in their original niche. Results. Using hanging drop technique, we were able to culture SCs associated with the fibre at least for 9 days with one transfer of fibres to the fresh drops. In comparison, in the classical method of myofibres culture, that is, on the dishes coated with Matrigel, SCs leave the fibres within 3 days after the isolation. Cells cultured in both systems differed in expression of Pax7 and MyoD. While almost all cells cultured in adhesion system expressed MyoD before the fifth day of the culture, the majority of SCs cultured in hanging drop still maintained expression of Pax7 and were not characterised by the presence of MyoD. Among the cells cultured with single myofibre for up to 9 days, we identified two different subclones of SCs: low proliferative clone and high proliferative clone, which differed in proliferation rate and membrane potential. Conclusions. The hanging drop enables the myofibres to be kept in suspension for at least 9 days, and thus, allows SCs and their niche to interact each other for prolonged time. In a consequence, SCs cultured in hanging drop maintain expression of Pax7 while those cultured in a traditional adhesion culture, that is, devoid of signals from the original niche, activate and preferentially undergo differentiation as manifested by expression of MyoD. Thus, the innovative method of SCs culturing in the hanging drop system may serve as a useful tool to study the fate of different subpopulations of these cells in their anatomical location and to determine reciprocal interactions between them and their niche

Uncoordinated Transcription and Compromised Muscle Function in the Lmna-Null Mouse Model of Emery-Dreifuss Muscular Dystrophy

LMNA encodes both lamin A and C: major components of the nuclear lamina. Mutations in LMNA underlie a range of tissue-specific degenerative diseases, including those that affect skeletal muscle, such as autosomal-Emery-Dreifuss muscular dystrophy (A-EDMD) and limb girdle muscular dystrophy 1B. Here, we examine the morphology and transcriptional activity of myonuclei, the structure of the myotendinous junction and the muscle contraction dynamics in the lmna-null mouse model of A-EDMD. We found that there were fewer myonuclei in lmna-null mice, of which ∼50% had morphological abnormalities. Assaying transcriptional activity by examining acetylated histone H3 and PABPN1 levels indicated that there was a lack of coordinated transcription between myonuclei lacking lamin A/C. Myonuclei with abnormal morphology and transcriptional activity were distributed along the length of the myofibre, but accumulated at the myotendinous junction. Indeed, in addition to the presence of abnormal myonuclei, the structure of the myotendinous junction was perturbed, with disorganised sarcomeres and reduced interdigitation with the tendon, together with lipid and collagen deposition. Functionally, muscle contraction became severely affected within weeks of birth, with specific force generation dropping as low as ∼65% and ∼27% of control values in the extensor digitorum longus and soleus muscles respectively. These observations illustrate the importance of lamin A/C for correct myonuclear function, which likely acts synergistically with myotendinous junction disorganisation in the development of A-EDMD, and the consequential reduction in force generation and muscle wasting

Comparison of incisional complications between skin closures using a simple continuous or intradermal pattern: a pilot study in horses undergoing ventral median celiotomy

Background Development of incisional complications following ventral median celiotomy might depend on suture pattern for skin closure. Methods In this prospective study, 21 healthy male horses underwent celiotomy. Skin closure was either performed via a continuous percutaneous pattern (CO group; 5 warmbloods/5 ponies) or an intradermal pattern (ID group; 5 warmbloods/6 ponies). Follow-up examination of the incisional site included daily monitoring for edema, dehiscence, and drainage. Transcutaneous ultrasound was performed at Days 3, 6, and 10 as well as on Week 8 and 12 to evaluate size of edema and presence or absence of sinus formation, and hernia formation. Prevalence of incisional infection on base of positive microbiological analysis at any time up to Day 10 was evaluated and compared between ID and CO group. Furthermore, edema size was analysed by a linear mixed-effect model for group and time dependency. Results Observed incisional complications included edema (9/10 in CO, 10/11 in ID), suture sinus formation (2/10 in CO, 1/11 in ID), surgical site infection (2/10 in CO, 0/11 in ID), and incisional hernia (1/10 in CO, 0/11 in ID). The overall prevalence of incisional infection was 9.5% without significant differences between both groups (20% in CO, 0% in ID; p = 0.214). Edema size was not dependent on time or group (p = 0.545 and p = 0.627, respectively). Discussion CO and ID suture pattern are appropriate for skin closure following ventral median celiotomy in horses. None of the animals in the continuous ID group developed surgical site infections, even without the use of antibiotics