Gene expression changes induced by space flight in single-cells of the fern <Emphasis Type="Italic">Ceratopteris richardii</Emphasis>

This work describes a rare high-throughput evaluation of gene expression changes induced by space flight in a single plant cell. The cell evaluated is the spore of the fern Ceratopteris richardii, which exhibits both perception and response to gravity. cDNA microarray and Q RT-PCR analysis of spores germinating in microgravity onboard NASA space shuttle flight STS-93 revealed changes in the mRNA expression of roughly 5% of genes analyzed. These gene expression changes were compared with gene expression changes that occur during gravity perception and response in animal cells and multicellular plants. Our data contribute to a better understanding of the impact of space flight conditions, including microgravity, on cellular growth and development, and provide insights into the adaptive strategies of individual cells in response to these conditions.     

Changes in gene expression and signal transduction in microgravity

Studies from space flights over the past three decades have demonstrated that basic physiological changes occur in humans during space flight. These changes include cephalic fluid shifts, loss of fluid and electrolytes, loss of muscle mass, space motion sickness, anemia, reduced immune response, and loss of calcium and mineralized bone. The cause of most of these manifestations is not known and until recently, the general approach was to investigate general systemic changes, not basic cellular responses to microgravity. This laboratory has recently studied gene growth and activation of normal osteoblasts (MC3T3-El) during spaceflight. Osteoblast cells were grown on glass coverslips and loaded in the Biorack plunger boxes. The osteoblasts were launched in a serum deprived state, activated in microgravity and collected in microgravity. The osteoblasts were examined for changes in gene expression and signal transduction. Approximately one day after growth activation significant changes were observed in gene expression in 0-G flight samples. Immediate early growth genes/growth factors cox-2, c-myc, bcl2, TGF beta1, bFGF and PCNA showed a significant diminished mRNA induction in microgravity FCS activated cells when compared to ground and 1-G flight controls. Cox-1 was not detected in any of the samples. There were no significant differences in the expression of reference gene mRNA between the ground, 0-G and 1-G samples. The data suggest that quiescent osteoblasts are slower to enter the cell cycle in microgravity and that the lack of gravity itself may be a significant factor in bone loss in spaceflight. Preliminary data from our STS 76 flight experiment support our hypothesis that a basic biological response occurs at the tissue, cellular, and molecular level in 0-G. Here we examine ground-based and space flown data to help us understand the mechanism of bone loss in microgravity.     

Development of a whole blood staining device for use during space shuttle flights.

BACKGROUND: Exposure to microgravity during space flight results in profound physiologic changes. Numerous studies have shown changes in circulating populations of peripheral blood immune cells immediately after space flight. It is currently unknown if these changes result from exposure to microgravity or are caused by the stress of reentry and readaptation to gravity. METHODS: We have developed the whole blood staining device (WBSD) as a system for the staining of whole blood collected during space flight for subsequent flow cytometric analysis. This device contains all liquids to address safety issues concerned with space flight and also moves the cells through the staining, lyse/fixation, and dilution steps. RESULTS: Data from flow cytometric analysis of samples stained in the WBSD was found to be comparable to data from samples stained by the conventional methods. Cells stained with the WBSD remain stable in the device for up to 14 days. The necessary manipulations required to use the device were tested on the KC-135 aircraft during the reduced gravity segment of parabolic flight. CONCLUSIONS: With the WBSD immunophenotype analysis can be performed at various time points for the duration of an entire Shuttle flight. In addition, this device has significant terrestrial applications for rapid and easy immunofluorescence labeling of whole blood in remote and isolated locations where immediate access to specialized equipment and skilled laboratory personnel may not be available. The WBSD provides a simple mechanism to design specific immunophenotyping tests for use by nontechnical personnel at bedside or in field locations. Cytometry 37:74-80, 1999. Published 1999 Wiley-Liss, Inc.     

Effect of culture in a rotating wall bioreactor on the physiology of differentiated neuron-like PC12 and SH-SY5Y cells.

A variety of evidence suggests that nervous system function is altered during microgravity, however, assessing changes in neuronal physiology during space flight is a non-trivial task. We have used a rotating wall bioreactor with a high aspect ratio vessel (HARV), which simulates the microgravity environment, to investigate the how the viability, neurite extension, and signaling of differentiated neuron-like cells changes in different culture environments. We show that culture of differentiated PC12 and SH-SY5Y cells in the simulated microgravity HARV bioreactor resulted in high cell viability, moderate neurite extension, and cell aggregation accompanied by NO production. Neurite extension was less than that seen in static cultures, suggesting that less than optimal differentiation occurs in simulated microgravity relative to normal gravity. Cells grown in a mixed vessel under normal gravity (a spinner flask) had low viability, low neurite extension, and high glutamate release. This work demonstrates the feasibility of using a rotating wall bioreactor to explore the effects of simulated microgravity on differentiation and physiology of neuron-like cells.     

Caveolae and caveolae constituents in mechanosensing

Studies in modeled microgravity or during orbital space flights have clearly demonstrated that endothelial cell physiology is strongly affected by the reduction of gravity. Nevertheless, the molecular mechanisms by which endothelial cells may sense gravity force remain unclear. We previously hypothesized that endothelial cell caveolae could be a mechanosensing system involved in hypergravity adaptation of human endothelial cells. In this study, we analyzed the effect on the physiology of human umbilical vein endothelial cell monolayers of short exposure to modeled microgravity (24–48h) obtained by clinorotation. For this purpose, we evaluated the levels of compounds, such as nitric oxide and prostacyclin, involved in vascular tone regulation and synthesized starting from caveolae-related enzymes. Furthermore, we examined posttranslational modifications of Caveolin (Cav)-1 induced by simulated microgravity. The results we collected clearly indicated that short microgravity exposure strongly affected endothelial nitrix oxide synthase activity associated with Cav-1 (Tyr 14) phosphorylation, without modifying the angiogenic response of human umbilical vein endothelial cells. We propose here that one of the early molecular mechanisms responsible for gravity sensing of endothelium involves endothelial cell caveolae and Cav-1 phosphorylation.     

Shift in arm-pointing movements during gravity changes produced by aircraft parabolic flight.

It has been shown that target-pointing arm movements without visual feedback shift downward in space microgravity and upward in centrifuge hypergravity. Under gravity changes in aircraft parabolic flight, however, arm movements have been reported shifting upward in hypergravity as well, but a downward shift under microgravity is contradicted. In order to explain this discrepancy, we reexamined the pointing movements using an experimental design which was different from prior ones. Arm-pointing movements were measured by goniometry around the shoulder joint of subjects with and without eyes closed or with a weight in the hand, during hyper- and microgravity in parabolic flight. Subjects were fastened securely to the seat with the neck fixed and the elbow maintained in an extended position, and the eyes were kept closed for a period of time before each episode of parabolic flight. Under these new conditions, the arm consistently shifted downward during microgravity and mostly upward during hypergravity, as expected. We concluded that arm-pointing deviation induced by parabolic flight could be also be valid for studying the mechanism underlying disorientation under varying gravity conditions.     

Effect of simulated microgravity on human lymphocytes

During space flight the function of the immune system changes significantly. Several papers reported that postflight the number and the proportion of circulating leukocytes in astronauts are modified (Leach, 1992), the in vitro mitogen induced T cell activation is depressed (Cogoli et al., 1985; Konstantinova et al. 1993) and there are detectable differences in cytokine production of leukocytes as well (Talas et al. 1983; Batkai et al. 1988; Chapes et al. 1992). One of the possible modifying forces is the microgravity condition itself. Our aim was to analyse mechanisms responsible for changing leukocyte functions in low gravity environment. For terrestrial simulation of microgravity we used a Rotary Cell Culture System (RCCS) developed by NASA. We investigated the effect of simulated microgravity on separated human peripheral blood mononuclear cells (PBMCs). We detected the populations of different cells by antibodies conjugated to fluorofors using a Flow Cytometer. Since space flight reduces the number of peripheral blood lymphocytes (Stowe et al., 1999) we supposed that apoptotic (programmed cell death) processes might be involved. This hypothesis was supported by the result of our earlier experiment demonstrating that simulated microgravity increased the level of secreted Tumor Necrosis Factor-alpha (TNFalpha, a known apoptotic signal molecule) significantly (Batkai et al. 1999).     

Immune suppression of human lymphoid tissues and cells in rotating suspension culture and onboard the International Space Station

The immune responses of human lymphoid tissue explants or cells isolated from this tissue were studied quantitatively under normal gravity and microgravity. Microgravity was either modeled by solid body suspension in a rotating, oxygenated culture vessel or was actually achieved on the International Space Station (ISS). Our experiments demonstrate that tissues or cells challenged by recall antigen or by polyclonal activator in modeled microgravity lose all their ability to produce antibodies and cytokines and to increase their metabolic activity. In contrast, if the cells were challenged before being exposed to modeled microgravity suspension culture, they maintained their responses. Similarly, in microgravity in the ISS, lymphoid cells did not respond to antigenic or polyclonal challenge, whereas cells challenged prior to the space flight maintained their antibody and cytokine responses in space. Thus, immune activation of cells of lymphoid tissue is severely blunted both in modeled and true microgravity. This suggests that suspension culture via solid body rotation is sufficient to induce the changes in cellular physiology seen in true microgravity. This phenomenon may reflect immune dysfunction observed in astronauts during space flights. If so, the ex vivo system described above can be used to understand cellular and molecular mechanisms of this dysfunction.     

Effects of microgravity on cell cytoskeleton and embryogenesis

The aim of this review is to compile, summarize and discuss the effects of microgravity on embryos, cell structure and function that have been demonstrated from data obtained during experiments performed in space or in altered gravity induced by clinostats. In cells and tissues cellular structure and genetic expression may be changed in microgravity and this has a variety of effects on embryogenesis which include death of the embryo, failure of neural tube closure, or final deformities to the overall morphology of the newborn or hatchling. Many species and protocols have been used for microgravity space experiments making it difficult to compare results. Experiments on the ways in which embryonic development and cell interactions occur in microgravity could also be performed. Experiments that have been done with cells in microgravity show changes in morphology, cytoskeleton and function. Changes in cytoskeleton have been noted and studies on microtubules in gravity have shown that they are gravity sensitive. Further study of basic chemical reactions that occur in cells should be done to shed some light on the underling processes leading to the changes that are observed in cells and embryos in microgravity.     

Impact of modeled microgravity on microvascular endothelial cells

Microvascular endothelial cells are protagonists in inflammation and angiogenesis. They contribute to the integrity of microvasculature by synthesizing a large array of cytokines, growth factors and mediators active on the endothelium itself, on smooth muscle cells and circulating leukocytes. Because space flight (i) associates with vascular impairment and (ii) modulates the cytokine network, we evaluated the effect of modeled microgravity on microvascular 1G11 cells. We found that modeled microgravity reversibly inhibits endothelial growth and this correlates with an upregulation of p21, a cyclin-dependent kinases inhibitor. By protein array, we found that microgravity inhibits the synthesis of interleukin 6, an event that may contribute to growth retardation. We also detected increased amounts of nitric oxide, a mediator of inflammatory responses, a potent vasodilator and a player in angiogenesis. The increased synthesis of nitric oxide is due, at least in part, to an upregulation of endothelial nitric oxide synthase. Because low levels of IL-6 might contribute to endothelial growth retardation as well as to the enhancement of nitric oxide synthesis, we hypothesize a central role of IL-6 in modulating microvascular endothelial cell behaviour in modeled microgravity.     

Modification of proteins secreted by endothelial cells during modeled low gravity exposure

The exposure of the human body to microgravity, conditions that occurs during space flights, causes significant changes in the cardiovascular system. Many cell types have been involved in these changes, and the endothelium seems to play a major role. In endothelial cells (EC), it has been shown that modeled low gravity impairs nitric oxide synthesis, cell adhesion, extracellular matrix composition, cytoskeleton organization, cytokines, and growth factors secretion. Nevertheless, detailed analysis of EC physiological changes induced by microgravity exposure is still lacking. Secretome analysis is one of the most promising approaches for the identification of biomarkers directly related to the physiopathological cellular state. In this study, we analyzed in details the modifications of EC secretome by using umbilical vein endothelial (HUVE) cells exposed to modeled low gravity conditions. By adopting a two‐dimensional (2‐D) proteomic approach, in conjunction with a technique for the compression of the dynamic range of proteins, we observed that modeled low gravity exposure of HUVE cells affected the secretion of proteins involved in the regulation of cytoskeleton assembly. Moreover, by using Luminex® suspension array systems, we found that the low gravity condition decreased in ECs the secretion of some key pro‐inflammatory cytokines, including IL‐1α and IL‐8, and of the pro‐angiogenic factor bFGF. On the contrary, microgravity increase the secretion of two chemokines (Rantes and Eotaxin), involved in leukocytes recruitment. J. Cell. Biochem. 112: 265–272, 2011. © 2010 Wiley‐Liss, Inc.     

Problems of the gravitational physiology of a cell

The article is an overview of the results of experimental studies of cell gravisensitivity per se. The first experiments on simple biological models in short-term space flights under uncontrolled conditions yielded conflicting data and yet implied no lethal or irreversible consequences of exposure to microgravity. The use of special devices and partial environmental control in the absence of adequate analytical procedures of assessing a cell’s functional state led some researchers to the supposition that cell cultures are insensitive to gravity. Recent advances in molecular and cell technologies provided the way of revealing numerous and often reversible morphofunctional changes in cells during space flight and laboratory simulation of microgravity effects. These are remodeling of cytoskeleton elements, alteration of gene expression, and mosaic rearrangement of intracellular regulation systems. Bioengineering and stem cell technologies gave another impetus to these investigations. A better understanding of intercellular interactions, the role of cytokines, and creation of 3D structures in the absence of the force of gravity made possible evidence-based substantiation of the differentiation lag, as well as the dependence of these changes on cell mechanic responses, and other phenomena. Academician O.G. Gazenko, who was the director of the Institute for Biomedical Problems for many years, was at the head of the Soviet biosatellite research program demonstrating his devotion to and awareness of the importance of the fundamental knowledge about the influence of gravity on every level of organization of a living system.     

Strategies of cell biology experimentation in space

The purpose of this article is to inform newcomers on the most important aspects of experimentation with living cells and tissues in space laboratories and platforms. There are strong arguments that justify the efforts and investments in such activity. Experimentation in space is subject to safety and technological constraints that require considerable attention to the development of the flight protocols and of the flight instrumentation. Nevertheless to fly an experiment in space is a unique opportunity to study living systems under conditions not reproducible on Earth and it is also a contribution to human exploration of space. Thereby important progress in basic and applied science can be expected. Parallel investigations on ground with devices averaging the exposure to the gravity vector but not reproducing microgravity shall always be part of a space flight project.     

How Effectively Does a Clinostat Mimic the Ultrastructural Effects of Microgravity on Plant Cells?

Columella cells of seedlings of Zea mays L. cv. Bear Hybridgrown in the microgravity of orbital flight allocate significantlylarger relative-volumes to hyaloplasm and lipid bodies, andsignificantly smaller relative-volumes to dictyosomes, plastids,and starch than do columella cells of seedlings grown at I g.The ultrastructure of columella cells of seedlings grown atI g and on a rotating clinostat is not significantly different.However, the ultrastructure of cells exposed to these treatmentsdiffers significantly from that of seedlings grown in microgravity.These results indicate that the actions of a rotating clinostatdo not mimic the ultrastructural effects of microgravity incolumella cells of Z. mays. Zea mays L., gravity, microgravity, ultrastructure, clinostat, space shuttle, space biology     

Reduced receptor aggregation and altered cytoskeleton in cultured myocytes after space-flight.

We carried out parallel experiments first on the slow clinostat and then in space-flight to examine the effects of altered gravity on the aggregation of the nicotinic acetylcholine receptors and the structure of the cytoskeleton in cultured Xenopus embryonic muscle cells. By examining the concordance between results from space flight and the clinostat, we tested whether the slow clinostat is a relevant simulation paradigm. Space-flown cells showed marked changes in the distribution and organization of actin filaments and had a reduced incidence of acetylcholine receptor aggregates at the site of contact with polystyrene beads. Similar effects were found after clinostat rotation. The sensitivity of synaptic receptor aggregation and cytoskeletal morphology suggests that in the microgravity of space cell behavior may be importantly altered.     

Effects of microgravity and clinostating on plants

Abstract

Results of many space flight and clinostate experiments performed with growing and developing lower and higher plants, tissue and protoplast cultures are presented. Biological effects of gravity changes on organism, cellular, subcellular and membrane levels are described. Regularities of rearrangements of organelle structural-functional organizations and cell metabolism as well as possible cell mechanisms of the adaptation to microgravity are discussed.     

The sea urchin larva, a suitable model for biomineralisation studies in space (IML-2 ESA Biorack experiment '24-F urchin')

By the ESA Biorack 'F-24 urchin' experiment of the IML-2 mission, for the first time the biomineralisation process in developing sea urchin larvae could be studied under real microgravity conditions. The main objectives were to determine whether in microgravity the process of skeleton formation does occur correctly compared to normal gravity conditions and whether larvae with differentiated skeletons do 'de-mineralise'. These objectives have been essentially achieved. Postflight studies on the recovered 'sub-normal' skeletons focused on qualitative, statistical and quantitative aspects. Clear evidence is obtained that the basic biomineralisation process does actually occur normally in microgravity. No significant differences are observed between flight and ground samples. The sub-normal skeleton architectures indicate, however, that the process of positioning of the skeletogenic cells (determining primarily shape and size of the skeleton) is particularly sensitive to modifications of environmental factors, potentially including gravity. The anatomical heterogeneity of the recovered skeletons, interpreted as long term effect of an accidental thermal shock during artificial egg fertilisation (break of climatisation at LSSF), masks possible effects of microgravity. No pronounced demineralisation appears to occur in microgravity; the magnesium component of the skeleton seems yet less stable than the calcium. On the basis of these results, a continuation of biomineralisation studies in space, with the sea urchin larva as model system, appears well justified and desirable.     

Development of a noninvasive technique for the measurement of intracranial pressure.

Intracranial pressure (ICP) dynamics are important for understanding adjustments to altered gravity. Previous flight observations document significant facial edema during exposure to microgravity, which suggests that ICP is elevated during microgravity. However, there are no experimental results obtained during space flight, primarily due to the invasiveness of currently available techniques. We have developed and refined a noninvasive technique to measure intracranial pressure noninvasively. The technique is based upon detecting skull movements of a few micrometers in association with altered intracranial pressure. We reported that the PPLL technique has enough sensitivity to detect changes in cranial distance associated with the pulsation of ICP in cadavera. In normal operations, however, we place a transducer on the scalp. Thus, we cannot rule out the possibility that the PPLL technique picks up cutaneous pulsation. The purpose of the present study was therefore to show that the PPLL technique has enough sensitivity to detect changes in cranial distance associated with cardiac cycles in vivo.