Cell-to-cell interactions in changed gravity: ground-based and flight experiments

Cell-to-cell interactions play an important role in all physiological processes and are mediated by humoral and mechanical factors. Mechanosensitive cells (e.g., osteocytes, chondrocytes, and fibroblasts) can be studied ex vivo to understand the effects of an altered gravity environment. In particular, cultured endothelial cells (EC) are very sensitive to a broad spectrum of mechanical and biochemical stimuli. Earlier, we demonstrated that clinorotation leads to cytoskeletal remodeling in cultured ECs. Long-term gravity vector changes also modulate the expression of surface adhesion molecules (ICAM-1, E-selectin, VCAM-1) on cultured ECs. To study the interactions of geterological cells, we cocultured endothelial monolayers and human lymphocytes, immune cells and myeloleucemic (K-560) cells. It was found that, although clinorotation did not alter the basal adhesion level of non-activated immune cells on endothelial monolayers, the adhesion of PMA-activated lymphocytes was increased. During flight experiments onboard the Russian segment of the International Space Station, we measured the cytotoxic activity of natural killer (NK) cells incubated with labeled target cells. It was found that immune cells in microgravity retained their ability to contact, recognize, and destroy oncogenic cells in vitro. Together, our data concerning the effects of simulated and real microgravity suggest that, despite changes in the cytoskeleton, cell motility, and expression of adhesion molecules, cell-cell interactions are not compromised, thus preserving the critical physiological functions of immune and endothelial cells.     

Spaceflight and clinorotation cause cytoskeleton and mitochondria changes and increases in apoptosis in cultured cells.

The cytoskeleton is a complex network of fibers that is sensitive to environmental factors including microgravity and altered gravitational forces. Cellular functions such as transport of cell organelles depend on cytoskeletal integrity; regulation of cytoskeletal activity plays a role in cell maintenance, cell division, and apoptosis. Here we report cytoskeletal and mitochondria alterations in cultured human lymphocyte (Jurkat) cells after exposure to spaceflight and in insect cells of Drosophila melanogaster (Schneider S-1) after exposure to conditions created by clinostat rotation. Jurkat cells were flown on the space shuttle in Biorack cassettes while Schneider S-1 cells were exposed to altered gravity forces as produced by clinostat rotation. The effects of both treatments were similar in the different cell types. Fifty percent of cells displayed effects on the microtubule network in both cell lines. Under these experimental conditions mitochondria clustering and morphological alterations of mitochondrial cristae was observed to various degrees after 4 and 48 hours of culture. Jurkat cells underwent cell divisions during exposure to spaceflight but a large number of apoptotic cells was also observed. Similar results were obtained in Schneider S-1 cells cultured under clinostat rotation. Both cell lines displayed mitochondria abnormalities and mitochondria clustering toward one side of the cells which is interpreted to be the result of microtubule disruption and failure of mitochondria transport along microtubules. The number of mitochondria was increased in cells exposed to altered gravity while cristae morphology was severely affected indicating altered mitochondria function. These results show that spaceflight as well as altered gravity produced by clinostat rotation affects microtubule and mitochondria organization and results in increases in apoptosis. Grant numbers: NAG 10-0224, NAG2-985.     

Induction of vascular endothelial phenotype and cellular proliferation from human cord blood stem cells cultured in simulated microgravity

Recent studies have demonstrated that stem cells derived from adult hematopoietic tissues are capable of trans-differentiation into non-hematopoietic cells, and that the culture in microgravity (microg) may modulate the proliferation and differentiation. We investigated the application of microg to human umbilical cord blood stem cells (CBSC) in the induction of vascular endothelial phenotype expression and cellular proliferation. CD34+ mononuclear cells were isolated from waste human umbilical cord blood samples and cultured in simulated microg for 14 days. The cells were seeded in rotary wall vessels (RWV) with or without microcarrier beads (MCB) and vascular endothelial growth factor was added during culture. Controls consisted of culture in 1 G. The cell cultures in RWV were examined by inverted microscopy. Cell counts, endothelial cell and leukocyte markers performed by flow cytometry and FACS scan were assayed at days 1, 4, 7 and at the termination of the experiments. Culture in RWV revealed significantly increased cellular proliferation with three-dimensional (3D) tissue-like aggregates. At day 4, CD34+ cells cultured in RWV bioreactor without MCB developed vascular tubular assemblies and exhibited endothelial phenotypic markers. These data suggest that CD34+ human umbilical cord blood progenitors are capable of trans-differentiation into vascular endothelial cell phenotype and assemble into 3D tissue structures. Culture of CBSC in simulated microg may be potentially beneficial in the fields of stem cell biology and somatic cell therapy.     

Response to hypogravity of normal in vitro cultured follicular cells from thyroid

Aim of this investigation is the study of molecular modifications occurring in differentiated mammalian cells exposed to gravitational changes. The test system chosen is a well characterized clone of differentiated, normal thyroid follicular cells (FRTL5) in long-term culture. As a follow-up to our recent experiment performed during the MASER-7 sounding rocket mission, flown for European Space Agency by Swedish Space Corporation in May 1996, we evaluated FRTL5 cells responses to Thyroid Stimulating Hormone dependent cAMP production under acute hypogravity conditions obtained in a fast rotating clinostat. Following this approach, we evaluated the FRTL5 cells response to TSH under microgravity conditions in order to optimize experimental tools and strategies in preparation to, and in between real flight missions.     

Can ultrasound counteract bone loss? Effect of low-intensity ultrasound stimulation on a model of osteoclastic precursor

The aim of the present work is to determine whether mechanical stress caused by ultrasound (US) exposure affects osteoclastic precursor cells, thus addressing the hypothesis that mechanical strain-induced perturbation of preosteoclastic cell machinery can contribute to the occurrence of bone turnover alterations. Moreover, cell cytoskeleton was studied because of its supposed involvement in cell mechanotransduction.Our experimental model was the FLG 29.1 human cell line, previously characterized as an osteoclastic precursor model. Cell proliferation was quantified by trypan blue exclusion assay. Cell morpho-functional state was monitored by multispectral imaging autofluorescence microscopy. The expression of cytoskeletal components and markers of proliferation (Ki67) and osteoclastic differentiation (RANK) was analysed by immunocytochemistry.The findings demonstrated that US stimulation affects FLG 29.1 cell growth, depresses the expression of cytoskeletal components and markers of proliferation and differentiation, induces cell damage, thus supporting the hypothesis that US exposure inhibits osteoclastogenesis.These results have been compared with those obtained previously by exposure of FLG 29.1 cells to modelled hypogravity conditions. Finally, the possibility to utilize US stimulation for counteracting osteoporosis has been discussed.     

Taxane recovery from cells of Taxus in micro- and hypergravity

Cell suspension cultures of Taxus cuspidata produce taxanes that are released from the outer surface of cells into the culture medium as free and bound alkaloids. Paclitaxel (Taxol (TM)), is an anti-cancer drug in short supply. It has a taxane ring derived from baccatin III and a C-13 phenylisoserine side-chain. This drug is produced over a wide range of gravitational forces. Monoclonal and polyclonal antibodies to paclitaxel, baccatin III, and the C-13 phenylisoserine side chain were combined in multiple-labeling studies to localize taxanes and paclitaxel on cell surfaces or on particles released into the culture medium. Bioreactor vessel design altered the composition of taxanes recovered from cells in simulated microgravity. At 10(-2) and 2x10(-4)g, taxane recovery was reduced but biomass growth and percent paclitaxel was significantly increased. At 1 to 24g, growth was reduced with a significant recovery of total taxanes with low percent paclitaxel. Bound paclitaxel was also localized in endonuclease-rich fragmenting nuclei of individual apoptotic cells. A model is presented comprising TCH (touch) genes encoding enzymes that modify taxane-bearing xylan residues in cell walls, the calcium-sensing of gravitational forces by the cytoplasm, and the predisposition of nuclei to apoptosis. This integrates the adaptive physiological and biochemical responses of drug-producing genomes with gravitational forces.     

Prospects of autotrophic link functioning in biological life-support systems based on cell biology studies

On the basis of using of modern methods of cytological analysis the cell growth peculiarities, reproduction and structure of plant cells of different level organisation (unicellular algae, mosses, angiosperms, cell cultures of higher factors--weightlessness, hypogravity, magnetic fields of various intensity, vibration and acceleration were cleared. It is shown that many discovered biological effects of space flight are connected with the degree of complexity of the object organisation and intensified with the increasing duration of influence.     

T cell regulation in microgravity – The current knowledge from in vitro experiments conducted in space,parabolic flights and ground-based facilities

Dating back to the Apollo and Skylab missions, it has been reported that astronauts suffered from bacterial and viral infections during space flight or after returning to Earth. Blood analyses revealed strongly reduced capability of human lymphocytes to become active upon mitogenic stimulation. Since then, a large number of in vitro studies on human immune cells have been conducted in space, in parabolic flights, and in ground-based facilities. It became obvious that microgravity affects cell morphology and important cellular functions. Observed changes include cell proliferation, the cytoskeleton, signal transduction and gene expression. This review gives an overview of the current knowledge of T cell regulation under altered gravity conditions obtained by in vitro studies with special emphasis on the cell culture conditions used. We propose that future in vitro experiments should follow rigorous standardized cell culture conditions, which allows better comparison of the results obtained in different flight- and ground-based experiment platforms.     

Invariant aspects of human locomotion in different gravitational environments.

Previous literature showed that walking gait follows the same mechanical paradigm, i.e. the straight/inverted pendulum, regardless the body size, the number of legs, and the amount of gravity acceleration. The Froude number, a dimensionless parameter originally designed to normalize the same (pendulum-like) motion in differently sized subjects, proved to be useful also in the comparison, within the same subject, of walking in heterogravity. In this paper the theory of dynamic similarity is tested by comparing the predictive power of the Froude number in terms of walking speed to previously published data on walking in hypogravity simulators. It is concluded that the Froude number is a good first predictor of the optimal walking speed and of the transition speed between walking and running in different gravitational conditions. According to the Froude number a dynamically similar walking speed on another planet can be calculated as [formula: see text] where V(Earth) is the reference speed on Earth.     

Mechanisms of spinal motoneurons survival in rats under simulated hypogravity on earth

It was previously shown that different cell types in vivo and in vitro may die via apoptosis under weightlessness conditions in space as well as in simulated hypogravity on the Earth. We assessed survivability of spinal motoneurons of rats after 35-day antiorthostatic hind limb suspension. Following weight bearing, unloading the total protein content in lumbar spinal cord is dropped by 21%. The electrophysiological studies of m. gastrocnemius revealed an elevated motoneurons’ reflex excitability and conduction disturbances in the sciatic nerve axons. The number of myelinated fibers in the ventral root of experimental animals was insignificantly increased by 35-day of antiorthostatic hind limb suspension, although the retrograde axonal transport was significantly decreased during the first week of simulated hypogravity. The results of the immunohistochemical assay with antibodies against proapoptotic protein caspase 9 and cytotoxicity marker neuron specific nitric oxide synthase (nNOS) and the TUNEL staining did not reveal any signs of apoptosis in motoneurons of suspended and control animals. To examine the possible adaptation mechanisms activated in motoneurons in response to simulated hypogravity we investigated immunoexpression of Hsp25 and Hsp70 in lumbar spinal cord of the rats after 35-day antiorthostatic hind limb suspension. Comparative analysis of the immunohistochemical reaction with anti-Hsp25 antibodies revealed differential staining of motoneurons in intact and experimental animals. The density of immunoprecipitate with anti-Hsp25 antibodies was substantially higher in motoneurons of the 35-day suspended than control rats and the more intensive precipitate in this reaction was observed in motoneuron neuritis. Quantitative analysis of Hsp25 expression demonstrated an increase in the Hsp25 level by 95% in experimental rats compared to the control. The immunoexpression of Hsp70 found no qualitative and quantitative differences in control and experimental lumbar spinal cords. Taken together our results show that (1) rat motoneurons survived after 35-day antiorthostatic hind limb suspension and the changes in neurons had a mostly functional character, and (2) the increased immunoexpression of Hsp25 can be considered as the anti-apoptotic factor.     

Parametric excitation of gyroscopic waves in Thomson-Delaney cells of the ocean of Jovian moon Europa

A possible mechanism (of parametric resonance type) of excitation of planetary gyroscopic waves in Thomson-Delaney cells of the ocean of Jovian moon Europa is considered. It is assumed that the basis of this mechanism is a variation of liquid depth in a cell caused by tidal oscillations under the action of gravitational perturbing influence of Galilean satellites of Jupiter. Such a model leads to a system of linear differential equations with periodic coefficients of the Hill’s type. Under some additional assumptions it changes over into a system of independent Mathieu equations. The regions of parametric resonance of this system are constructed.     

Thyroid cell growth: sphingomyelin metabolism as non-invasive marker for cell damage acquired during spaceflight

Prolonged spaceflights are known to elicit changes in human cardiovascular, musculoskeletal, and nervous systems, whose functions are regulated by the thyroid gland. It is known that sphingomyelin metabolism is involved in apoptosis (programmed cell death) of thyroid cells induced by UVC radiation, but at present no data exists with regard to this phenomenon, which occurs during space missions. The aim of this study was to analyze, for the first time, the effect of spaceflight on the enzymes of sphingomyelin metabolism, sphingomyelinase, and sphingomyelin synthase, and to determine whether the ratio between the two enzymes might be used as a possible marker for thyroid activity during space missions. Both quiescent thyroid cells and thyroid cells stimulated to proliferate with thyrotropin (TSH) were cultured during the Eneide and Esperia missions on the International Space Station. The results show that during space missions the cells treated with TSH grew only 1.5?±?0.65-fold and, thus, behave similarly to quiescent cells, while on the ground the same cells, maintained in experimental conditions that reproduced those of the flight, grew 7.71?±?0.67-fold. Comparison of the sphingomyelinase/sphingomyelin-synthase ratio and the levels of Bax, STAT3, and RNA polymerase II in proliferating, quiescent, pro-apoptotic, or apoptotic cells demonstrated that thyroid cells during space missions were induced into a pro-apoptotic state. Given its specificity and the small amount of cells needed for analysis, we propose the use of the sphingomyelinase/sphingomyelin-synthase ratio as a marker of functional status of thyroid cells during space missions. Further studies could lead to its use in real time during prolonged spaceflights.     

Application of GFP technique for cytoskeleton visualization onboard the International Space Station

Cytoskeleton recently attracted wide attention of cell and molecular biologists due to its crucial role in gravity sensing and trunsduction. Most of cytoskeletal research is conducted by the means of immunohistochemical reactions, different modifications of which are beneficial for the ground-based experiments. But for the performance onboard the space vehicles, they represent quite complicated technique which requires time and special skills for astronauts. In addition, immunocytochemistry provides only static images of the cytoskeleton arrangement in fixed cells while its localization in living cells is needed for the better understanding of cytoskeletal function. In this connection, we propose a new approach for cytoskeletal visualization onboard the ISS, namely, application of green fluorescent protein (GFP) from Aequorea victoria, which has the unique properties as a marker for protein localization in vivo. The creation of chimerical protein-GFP gene constructs, obtaining the transformed plant cells possessed protein-GFP in their cytoskeletal composition will allow receiving a simple and efficient model for screening of the cytoskeleton functional status in microgravity.     

The physical price of a ticket into space

As a direct consequence of exposure to microgravity astronauts experience a number of physiological changes, which can have serious medical implications when they return to Earth. Most immediate and significant are the head-ward shift of body fluids and the removal of gravitational loading from bone and muscles, which lead to progressive changes in the cardiovascular and musculoskeletal systems. Cardiovascular adaptations result in an increased incidence of orthostatic intolerance (fainting) post-flight, decreased cardiac output and reduced exercise capacity. Changes in the musculoskeletal system contribute significantly to the impaired functions experienced in the post-flight period. The underlying factor producing these changes is the absence of gravity. Countermeasures, therefore, are designed primarily to simulate Earth-like movements, stresses and system interactions. Exercise is one approach that has received wide operational use and acceptance in both the US and Russian space programmes, and has enabled humans to stay relatively healthy in space for well over a year. Although it remains the most effective countermeasure currently available, significant physiological degradation still occurs. The development of other countermeasures will therefore be necessary for longer duration missions, such as the human exploration of Mars.     

Hematological and immunological changes during space flight.

This paper gives a summary of the principal hematological and immunological changes observed in crews after space flight. Reduction of red blood cell mass (2-21%) and of hemoglobin mass (12-33%) is generally observed after the US and Soviet space missions. The changes are accompanied with a loss of plasma volume (4-16%). Erythrocyte and hemoglobin concentrations in the blood remain constant, suggesting that the changes are driven by a feed-back mechanism. Immunological changes consist mainly of reduced T-lymphocyte reactivity. The results of the 96-day and 140-day Salyut-6 missions suggest that the adaptation of the immune system to spaceflight occurs in two stages: the first takes place during the first 2-3 months in space, the second follows and consists of further weakening of the immune response. Our experiments with human lymphocytes in vitro indicate that high-g enhance, whereas low-g depress lymphocyte activity. Finally, our investigations to be performed on Spacelab are described.     

Separation of bacterial cells by free flow electrophoresis under microgravity: a result of the SpaceLab-Japan project on Space Shuttle flight STS-47

We demonstrated free flow electrophoresis (FFE) of charged cells under microgravity, where gravitational effects are almost eliminated. Separation of a mixture of three bacterial strains (mutants of Salmonella typhimurium LT2) by FFE was conducted on NASA Space Shuttle flight STS-47 (September 1992). The experiment was designed to differentiate three strains having different lipopolysaccharide core structures in the cell membrane. The results were compared to those of ground experiments, in order to examine whether or not FFE in a weightless environment provides distinct advantages. Smooth strain SL1027 and rough strain SL3749 migrated to two separated fractions. The quality (viability) and the yields of the separated samples were sufficient to show the advantage of microgravity. Another rough strain, SL1102, exhibited unexpected electrophoretic behavior, which prevented the complete resolution of the three strains. All the strains were recovered as viable cells after 8 days of flight. The present study suggests that electrophoretic separation of bacterial cells is much more effective under microgravity conditions with relatively good resolution in comparison with the ground operation.