Marcel Egli

Circulating relaxin acts on subfornical organ neurons to stimulate water drinking in the rat

Relaxin, a peptide hormone secreted by the corpus luteum during pregnancy, exerts actions on reproductive tissues such as the pubic symphysis, uterus, and cervix. It may also influence body fluid balance by actions on the brain to stimulate thirst and vasopressin secretion. We mapped the sites in the brain that are activated by i.v. infusion of a dipsogenic dose of relaxin (25 μg/h) by immunohistochemically detecting Fos expression. Relaxin administration resulted in increased Fos expression in the subfornical organ (SFO), organum vasculosum of the lamina terminalis (OVLT), median preoptic nucleus, and magnocellular neurons in the supraoptic and paraventricular nuclei. Ablation of the SFO abolished relaxin-induced water drinking, but did not prevent increased Fos expression in the OVLT, supraoptic or paraventricular nuclei. Although ablation of the OVLT did not inhibit relaxin-induced drinking, it did cause a large reduction in Fos expression in the supraoptic nucleus and posterior magnocellular subdivision of the paraventricular nucleus. In vitro single-unit recording of electrical activity of neurons in isolated slices of the SFO showed that relaxin (10−7 M) added to the perfusion medium caused marked and prolonged increase in neuronal activity. Most of these neurons also responded to 10−7 M angiotensin II. The data indicate that blood-borne relaxin can directly stimulate neurons in the SFO to initiate water drinking. It is likely that circulating relaxin also stimulates neurons in the OVLT that influence vasopressin secretion. These two circumventricular organs that lack a blood–brain barrier may have regulatory influences on fluid balance during pregnancy in rats.

The Effects of Weightlessness on the Human Organism and Mammalian Cells

It has always been a desire of mankind to conquest Space. A major step in realizing this dream was the completion of the International Space Station (ISS). Living there for several months confirmed early observations of short-term spaceflights that a loss of gravity affects the health of astronauts. Space medicine tries to understand the mechanism of microgravity-induced health problems and to conceive potent countermeasures. There are four different aspects which make space medicine appealing: i) finding better strategies for adapting astronauts to weightlessness; ii) identification of microgravity-induced diseases (e.g. osteoporosis, muscle atrophy, cardiac problems and others); iii) defining new therapies to conquer these diseases which will benefit astronauts as well as people on Earth in the end; and iv) on top of that, unveiling the mechanisms of weightlessness-dependent molecular and cellular changes is a requirement for improving space medicine. In mammalian cells, microgravity induces apoptosis and alters the cytoskeleton and affects signal transduction pathways, cell differentiation, growth, proliferation, migration and adhesion. This review focused on gravi-sensitive signal transduction elements and pathways as well as molecular mechanisms in human cells, aiming to understand the cellular changes in altered gravity. Moreover, the latest information on how these changes lead to clinically relevant health problems and current strategies of countermeasures are reviewed.

Prolactin secretion patterns: basic mechanisms and clinical implications for reproduction

Prolactin (PRL) is one of the most versatile hormones in the mammalian body affecting reproductive, sexual, metabolic, immune, and other functions. It is therefore not surprising that the neural control of PRL secretion is complex, involving the coordinated actions of several hypothalamic nuclei. A plethora of experimental data exists on the hypothalamic control of hormone secretion under various physiological stimuli. There have been even mathematical models and computer studies published, which help to understand the complex hypothalamic-pituitary network. Nevertheless, the putative role of PRL for human reproduction still has to be clarified. Here, we review data on the underlying mechanisms controlling PRL secretion using both experimental and mathematical approaches. These investigations primarily focus on rhythmic secretion in rats during early pregnancy or pseudopregnancy, and they point to the important role of oxytocin as a crucial PRL-releasing factor. Recent data on human studies and their theoretical and clinical implications are reviewed as well. In particular, studies demonstrating a sustained PRL surge after sexual climax in males and females are presented, indicating possible implications for both sexual satiation and reproductive functions. Taking these data together, there is evidence for the hypothesis that the PRL surge induced by sexual activity, together with the altered PRL rhythmic pattern, is important for successful initialization of pregnancy not only in rodents but also possibly in humans. However, further investigations are needed to clarify such a role in humans.

Short-term weightlessness produced by parabolic flight maneuvers altered gene expression patterns in human endothelial cells

This study focused on the effects of short‐term microgravity (22 s) on the gene expression and morphology of endothelial cells (ECs) and evaluated gravisensitive signaling elements. ECs were investigated during four German Space Agency (Deutsches Zentrum für Luft‐ und Raumfahrt) parabolic flight campaigns. Hoechst 33342 and acridine orange/ethidium bromide staining showed no signs of cell death in ECs after 31 parabolas (P31). Gene array analysis revealed 320 significantly regulated genes after the first parabola (P1) and P31. COL4A5, COL8A1, ITGA6, ITGA10, and ITGB3 mRNAs were down‐regulated after P1. EDN1 and TNFRSF12A mRNAs were up‐regulated. ADAM19, CARD8, CD40, GSN, PRKCA (all down‐regulated after P1), and PRKAA1 (AMPKα1) mRNAs (up‐regulated) provide a very early protective mechanism of cell survival induced by 22 s microgravity. The ABL2 gene was significantly up‐regulated after P1 and P31, TUBB was slightly induced, but ACTA2 and VIM mRNAs were not changed. β‐Tubulin immunofluorescence revealed a cytoplasmic rearrangement. Vibration had no effect. Hypergravity reduced CARD8, NOS3, VASH1, SERPINH1 (all P1), CAV2, ADAM19, TNFRSF12A, CD40, and ITGA6 (P31) mRNAs. These data suggest that microgravity alters the gene expression patterns and the cytoskeleton of ECs very early. Several gravisensitive signaling elements, such as AMPKα1 and integrins, are involved in the reaction of ECs to altered gravity.—Grosse, J., Wehland, M., Pietsch, J., Ma, X., Ulbrich, C., Schulz, H., Saar, K., Hübner, N., Hauslage, J., Hemmersbach, R., Braun, M., van Loon, J., Vagt, N., Infanger, M., Eilles, C., Egli, M., Richter, P., Baltz, T., Einspanier, R., Sharbati, S., Grimm, D. Short‐term weightlessness produced by parabolic flight maneuvers altered gene expression patterns in human endothelial cells. FASEB J. 26, 639–655 (2012). www.fasebj.org

Differential gene regulation under altered gravity conditions in follicular thyroid cancer cells: relationship between the extracellular matrix and the cytoskeleton

Extracellular matrix proteins, adhesion molecules, and cytoskeletal proteins form a dynamic network interacting with signalling molecules as an adaptive response to altered gravity. An important issue is the exact differentiation between real microgravity responses of the cells or cellular reactions to hypergravity and/or vibrations. To determine the effects of real microgravity on human cells, we used four DLR parabolic flight campaigns and focused on the effects of short-term microgravity (22 s), hypergravity (1.8 g), and vibrations on ML-1 thyroid cancer cells. No signs of apoptosis or necrosis were detectable. Gene array analysis revealed 2430 significantly changed transcripts. After 22 s microgravity, the F-actin and cytokeratin cytoskeleton was altered, and ACTB and KRT80 mRNAs were significantly upregulated after the first and thirty-first parabolas. The COL4A5 mRNA was downregulated under microgravity, whereas OPN and FN were significantly upregulated. Hypergravity and vibrations did not change ACTB, KRT-80 or COL4A5 mRNA. MTSS1 and LIMA1 mRNAs were downregulated/slightly upregulated under microgravity, upregulated in hypergravity and unchanged by vibrations. These data indicate that the graviresponse of ML-1 cells occurred very early, within the first few seconds. Downregulated MTSS1 and upregulated LIMA1 may be an adaptive mechanism of human cells for stabilizing the cytoskeleton under microgravity conditions.

Effects of basic fibroblast growth factor on endothelial cells under conditions of simulated microgravity

Fibroblast growth factors interact with appropriate endothelial cell (EC) surface receptors and initiate intracellular signal cascades, which participate in modulating blood vessel growth. EC, upon exposure to basic fibroblast growth factors (bFGFs) undergo profound functional alterations, which depend on their actual sensitivity and involve gene expression and de novo protein synthesis. We investigated the effects of bFGF on signaling pathways of EA.hy926 cells in different environments. EC were cultured under normal gravity (1 g) and simulated microgravity (µg) using a three‐dimensional (3D) clinostat. Microgravity induced early and late apoptosis, extracellular matrix proteins, endothelin‐1 (ET‐1) and TGF‐β1 expression. Microgravity reduced eNOS mRNA within 24 h. Moreover, a six‐ to eightfold higher amount of IL‐6 and IL‐8 was secreted within 24 h µg. In addition, microgravity induced a duplication of NF‐kappaB p50, while p65 was quadrupled. At 1 g, bFGF application (4 h) reduced ET‐1, TGF‐β1 and eNOS gene expression. After 24 h, bFGF enhanced fibronectin, VEGF, Flk‐1, Flt‐1, the release of IL‐6, IL‐8, and TGF‐β1. Furthermore, bFGF promoted apoptosis, reduced NFkB p50, but enhanced NFkB p65. After 4 h µg, bFGF decreased TGF‐β1, eNOS, and ET‐1 gene expression. After 24 h µg, bFGF elevated fibronectin, Flk‐1 and Flt‐1 protein, and reduced IL‐6 and IL‐8 compared with vehicle treated µg cultures. In µg, bFGF enhanced NF‐KappaB p50 by 50%, Bax by 25% and attenuated p65, activation of caspase‐3 and annexin V‐positive cells. bFGF differently changes intracellular signals in ECs depending whether it is applied under microgravity or normal gravity conditions. In microgravity, bFGF contributes to protect the EC from apoptosis. J. Cell. Biochem. 104: 1324–1341, 2008.

A proteomic approach to analysing spheroid formation of two human thyroid cell lines cultured on a random positioning machine

The human cell lines FTC‐133 and CGTH W‐1, both derived from patients with thyroid cancer, assemble to form different types of spheroids when cultured on a random positioning machine. In order to obtain a possible explanation for their distinguishable aggregation behaviour under equal culturing conditions, we evaluated a proteomic analysis emphasising cytoskeletal and membrane‐associated proteins. For this analysis, we treated the cells by ultrasound, which freed up some of the proteins into the supernatant but left some attached to the cell fragments. Both types of proteins were further separated by free‐flow IEF and SDS gel electrophoresis until their identity was determined by MS. The MS data revealed differences between the two cell lines with regard to various structural proteins such as vimentin, tubulins and actin. Interestingly, integrin α‐5 chains, myosin‐10 and filamin B were only found in FTC‐133 cells, while collagen was only detected in CGTH W‐1 cells. These analyses suggest that FTC‐133 cells express surface proteins that bind fibronectin, strengthening the three‐dimensional cell cohesion.

Low Intensity and Frequency Pulsed Electromagnetic Fields Selectively Impair Breast Cancer Cell Viability

Introduction A common drawback of many anticancer therapies is non-specificity in action of killing. We investigated the potential of ultra-low intensity and frequency pulsed electromagnetic fields (PEMFs) to kill breast cancer cells. Our criteria to accept this technology as a potentially valid therapeutic approach were: 1) cytotoxicity to breast cancer cells and; 2) that the designed fields proved innocuous to healthy cell classes that would be exposed to the PEMFs during clinical treatment. Methods MCF7 breast cancer cells and their normal counterparts, MCF10 cells, were exposed to PEMFs and cytotoxic indices measured in order to design PEMF paradigms that best kill breast cancer cells. The PEMF parameters tested were: 1) frequencies ranging from 20 to 50 Hz; 2) intensities ranging from 2 mT to 5 mT and; 3) exposure durations ranging from 30 to 90 minutes per day for up to three days to determine the optimum parameters for selective cancer cell killing. Results We observed a discrete window of vulnerability of MCF7 cells to PEMFs of 20 Hz frequency, 3 mT magnitude and exposure duration of 60 minutes per day. The cell damage accrued in response to PEMFs increased with time and gained significance after three days of consecutive daily exposure. By contrast, the PEMFs parameters determined to be most cytotoxic to breast cancer MCF-7 cells were not damaging to normal MCF-10 cells. Conclusion Based on our data it appears that PEMF-based anticancer strategies may represent a new therapeutic approach to treat breast cancer without affecting normal tissues in a manner that is non-invasive and can be potentially combined with existing anti-cancer treatments.

Calcium's Role in Mechanotransduction during Muscle Development

Mechanotransduction is a process where cells sense their surroundings and convert the physical forces in their environment into an appropriate response. Calcium plays a crucial role in the translation of such forces to biochemical signals that control various biological processes fundamental in muscle development. The mechanical stimulation of muscle cells may for example result from stretch, electric and magnetic stimulation, shear stress, and altered gravity exposure. The response, mainly involving changes in intracellular calcium concentration then leads to a cascade of events by the activation of downstream signaling pathways. The key calcium-dependent pathways described here include the nuclear factor of activated T cells (NFAT) and mitogen-activated protein kinase (MAPK) activation. The subsequent effects in cellular homeostasis consist of cytoskeletal remodeling, cell cycle progression, growth, differentiation, and apoptosis, all necessary for healthy muscle development, repair, and regeneration. A deregulation from the normal process due to disuse, trauma, or disease can result in a clinical condition such as muscle atrophy, which entails a significant loss of muscle mass. In order to develop therapies against such diseased states, we need to better understand the relevance of calcium signaling and the downstream responses to mechanical forces in skeletal muscle. The purpose of this review is to discuss in detail how diverse mechanical stimuli cause changes in calcium homeostasis by affecting membrane channels and the intracellular stores, which in turn regulate multiple pathways that impart these effects and control the fate of muscle tissue.

Application of free-flow IEF to identify protein candidates changing under microgravity conditions.

Using antibody‐related methods, we recently found that human thyroid cells express various proteins differently depending on whether they are cultured under normal gravity (1g) or simulated microgravity (s‐μg). In this study, we performed proteome analysis in order to identify more gravity‐sensitive thyroid proteins. Cells cultured under 1g or s‐μg conditions were sonicated. Proteins released into the supernatant and those remaining in the cell fragments were fractionated by free‐flow IEF. The fractions obtained were further separated by SDS‐gel electrophoresis. Selected gel pieces were excised and their proteins were determined by MS. A total of 235 different proteins were found. Out of 235 proteins, 37 appeared to be first identifications in human thyroid cells. Comparing SDS gel lanes of equally numbered free‐flow IEF fractions revealed similar patterns with a number of identical bands if proteins of a distinct cell line had been applied, irrespective of whether the cells had been cultured under 1g or s‐μg. Most of the identical band pairs contained identical proteins. However, the concentrations of some types of proteins were different within the two pieces of gel. Proteins that concentrated differently in such pieces of gel are considered as candidates for further investigations of gravitational sensitivity.