Exposure to microgravity generates alterations that are similar to those involved in age-related diseases, such as cardiovascular deconditioning, bone loss, muscle atrophy, and immune response impairment. Endothelial dysfunction is the common denominator.
The objective of this study is to determine how HUVECs (Human Umbilical Vein Endothelial Cell - cells that line the interior of blood vessel) modify their behaviour when exposed to real microgravity. This could provide better knowledge of endothelial function, which could be useful for clinical application.
1. To evaluate endothelial protein profile by protein arrays.
2. To evaluate endothelial gene expression by cDNA arrays.
3. To evaluate endothelial synthesis of nitric oxide.
It has been previously demonstrated that simulated μg reversibly stimulates the growth of macrovascular ECs (Endothelial Cell). In order to investigate the effects of microgravity on HUVEC genomics, proteomics and Nitrogen monoxide synthesis, cell cultures will be flown and incubated in microgravity for several days. At the end of this period, the culture medium will be separated from the cells and both will be fixed for postflight analysis.
The specific benefits of the proposed space experiment range from a better understanding of the molecular mechanisms influencing endothelial behaviour to the possibility of outlining new countermeasures against the astronaut cardiovascular deconditioning and bone demineralisation.
Figure 1: SPHINX Experiment Concept Diagram.
This 10-day study which consisted of 12 in-flight and 12 ground-based control modules is important to maintaining crew health during long-duration space exploration.
Biological Samples and Fixatives
- HUVEC (Human Umbilical Vein Endothelial Cell);
- Medium composition: Medium 199 supplemented with 10% FCS, 10 μg/mL ECGF (Endothelial Cell Growth Factor), 1% antibiotics, 1% L-glutamine, 1% sodium pyruvate and 10 U/mL heparin, 12.5 mM HEPES;
- PBS (phosphate buffered saline);
- RNAlater for fixation of cells;
- Sigma P8340 Protease inhibitor cocktail.
Number of samples and culture chamber requirements
Preferred 12 samples (minimum 8 samples). No in-flight centrifuge control.
This number of samples is required to ensure sufficient number of experimental replicates & sample material for analysis.
- The culture chambers shall have a volume of at least 0.8 - 0.9 mL.
- Each refresh volume (fixation included) shall be at least 1 - 1.2 mL.
- The culture chamber shall be completely filled with fresh medium (or fixative) at the end of the refreshment (or fixation) step.
General description of experiment protocol
During the SPHINX experiment, HUVECs are incubated in microgravity for several days with regular medium refresh. At the end of the active phase of the experiment, medium and cells are suitably fixed for post-flight analyses.
GENERAL EXPERIMENT PROCEDURE
Pre-flight sample preparation
To assure HUVEC availability at the launch site, HUVECs will be carried:
1) in warm condition (seeded in Opticells in medium M199) and
2) frozen (in liquid nitrogen). During the ground transportation phase, the temperature for cells and medium will be as defined in F.O. (Functional Objective) Step 0 (see Fig. 3). For HUVECs in Opticells, it will be necessary to refresh the medium depending upon the duration of the transport.
At the launch site
1) medium of HUVECs in Opticells will refreshed;
2) frozen extra cells will be thawed, seeded in culture dishes and the medium will be refreshed after one day.
In both cases, HUVECs will be seeded on SPHINX cell culture supports to be inserted in SPHINX EH (Experiment Hardware composed of Experiment Units and Experiment Container) filled with media and fixatives.
Samples may be subjected to X-rays without risk. HUVECs will be prepared on ground.
Cells are launched and then incubated on board ISS. There is a 1st culture medium exchange which marks the starting point of the experiment. Then there is a second and a third medium exchange (PBS wash). The fourth medium exchange will be done witha fixative medium containing RNAlater.The exchanged culture media will be fixed with a non toxic protease inhibitor cocktail.
In-flight sample preservation
In order to preserve samples for post flight analysis, at the end of the experiment HUVEC will be stored in RNAlater solution until the return to Earth and ground analyses.
In order to avoid protein degradation, the culture medium, added of a nontoxic protease inhibitor cocktail (Sigma P8340 containing leupeptin, aprotinin, pestatin A, AEBSF and bestatin), will remain fixed until the return to earth and ground analyses. (Refer to F.O. Step11 - see Fig. 3).
Frozen samples may thaw without risk during the download phase.
Post-flight sample analyses
Back on Earth, samples will be analysed as following:
a. the conditioned media will be utilized to measure the amounts of nitric oxide;
b. the conditioned media will be utilized for protein array to gain insights into the profile of secreted cytokines and angiogenic factors in EC (Endothelial Cells) in space;
c. Cell lysates will be processed by PCR array to obtain EC (Endothelial Cells) gene expression profile in space.
Ground reference experiment(s)
The experiment proposes to validate in space the results on HUVECs obtained in laboratories in simulated microgravity. The ground reference experiment should be performed with identical protocol and hardware to flight experiment. During microgravity culture in space, shear and turbulence approximate zero, whereas co-spatial relations of cells and three-dimensionality are almost perfect. It is expected that these event would greatly affect endothelial physiology.
Permissible Cross contamination
Maximum permissible concentration of reagents with cell culture medium during culture of experiment, prior to addition of activator/medium exchange/fixation.
Maximum allowable concentration
Culture medium for refresh
Maximum permissible concentration of culture medium that can remain following addition of fixative. Contaminant Maximum allowable concentration Phosphate buffered saline in RNAlater fixative 21 % Culture medium in RNAlater fixative 9 %
Maximum allowable concentration
Phosphate buffered saline in RNAlater fixative
Culture medium in RNAlater fixative
- All flown samples fixed in RNAlater.
- Total spent culture medium fixed with proteinase inhibitor cocktail.
- Mission temperature log and timing of experimental steps.
On fixed medium: Protein array and NO (Nitrogen Monoxide) quantification.
On fixed cells: cDNA array
Figure 2: SPHINX Overview Experiment Timeline and associated Functional Objectives.
Figure 3: Flow Diagram with Functional Objectives indicated.
1. We expect to validate in space the results obtained in endothelial cells by simulating microgravity on Earth with different devices, namely the RPM (Random Positioning Machine) and the RWV (Rotating Wall Vessel). The proposed experiments will allow to determine whether true microgravity promotes alterations in the cytokine network: this issue is relevant since different cytokines and chemokines are involved in promoting endothelial dysfunction, which is known to promote cardiovascular diseases. In space-flown cells, we will also study gene expression and the results will be compared with our studies showing the modulation of about 7% of the genes in simulated microgravity. In addition, we described an increased synthesis of nitric oxide in the RWV (Rotating Wall Vessel) and RPM (Random Positioning Machine). We therefore plan to determine the levels of nitric oxide also in space-flown cells.
2. Because endothelial cells are responsible for the integrity of the vascular wall, a better understanding of the modulation of endothelial functions in space might direct future studies to individuate potential countermeasures to prevent cardiovascular deconditioning in astronauts. In addition, vascularization being crucial for bone metabolism, we argue that our results might offer new insights also in elucidating novel strategies to prevent bone loss in space.The teams Bradamante-Maier, supported by ASI, participated to the FOTON M3 mission (14.09.2007) with the experiment named SCORE (Saccharomyces Cerevisiae Oxidative-stress Response Evaluation).
S. Versari, A. Villa, S. Bradamante, J.A.M. Maier, (2007), "Cytoskeletal disorganization and increased nitric oxide synthesis are common features in human primary endothelial cell response to hyper- and microgravity", Biochimica et Biophysica Acta - Molecular Cell Research, 1773, 11, pp. 1645-1652.
S. Bradamante, L. Barenghi, S. Versari, A. Villa, (2006), "From hypergravity to microgravity: choosing the suitable simulator", Microgravity Science and Technology, 18, pp. 250-253.
A. Villa, S. Versari, L. Barenghi, J.A.M. Maier, S. Bradamante, (2005), "Effects of space-flight simulation on human cells", European Space Agency, Special Publication, SP-585, Villa / 1-2, pp. 11.
A. Villa, S. Versari, J.A.M. Maier, S. Bradamante, (2005), "Cell behavior in simulated microgravity: a comparison of results obtained with RWV and RPM", Gravitational and Space Biology Bulletin, 18, 2, pp. 89-90.
S. Bradamante, L. Barenghi, F. Piccinini, A.A.E. Bertelli, R. de Jonge, P. Beemster et al., (2003), "Resveratrol provides late-phase cardioprotection by means of a nitric oxide- and adenosine-mediated mechanism", European Journal of Pharmacology, 465, pp. 115-123.
S.I. Carlsson, M.T. Bertilaccio, I. Ascari, S. Bradamante, J.A.M. Maier, (2002), "Modulation of human endothelial cell behaviour in simulated microgravity", Journal of Gravitational Physiology, 9, pp. P273-274.
D. Bernardini, E. Ballabio, M. Mariotti, J.A.M. Maier, (2005), "Differential expression of EDF-1 and endothelial nitric oxide synthase by proliferating, quiescent and senescent microvascular endothelial cells", Biochimica et Biophysica Acta - Molecular Cell Research, 1745, 2, pp. 265-272.
S. Cotrupi, D. Ranzani, J.A.M. Maier, (2005), "Impact of modeled microgravity on microvascular endothelial cells", Biochimica et Biophysica Acta - Molecular Cell Research, 1746, 2, pp. 163-168.
A. Villa, S. Versari, L. Barenghi, J.A.M. Maier, S. Bradamante, (2005), "Effects of space flight simulation on human cells", Journal of Gravitational Physiology, 12, pp. 277-278.
S. Cotrupi, J.A.M. Maier, (2004), "HSP70 upregulation is crucial for cellular proliferative response in simulated microgravity", Journal of Gravitational Physiology, 11, 2, pp. P173-176.
S. Bradamante, J.A.M. Maier, S. Versari, (2012), "SPaceflight of Huvec: an Integrated eXperiment - SPHINX onboard the ISS", Conference Paper for Life in Space for Life on Earth Symposium, Aberdeen, United Kingdom, 18-22 June 2012.
S. Versari, G. Longinotti, L. Barenghi, J.A.M. Maier, S. Bradamante, (2013), "The challenging environment on board the International Space Station affects endothelial cell function by triggering oxidative stress through thioredoxin interacting protein overexpression: the ESA-SPHINX experiment", FASEB Journal, 27, 11, DOI: 10.1096/fj.13-229195, pp. 4466-4475.