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Microgravity and space radiation inhibit autophagy in human capillary endothelial cells, through either opposite or synergistic effects on specific molecular pathways

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Abstract

Microgravity and space radiation (SR) are two highly influential factors affecting humans in space flight (SF). Many health problems reported by astronauts derive from endothelial dysfunction and impaired homeostasis. Here, we describe the adaptive response of human, capillary endothelial cells to SF. Reference samples on the ground and at 1g onboard permitted discrimination between the contribution of microgravity and SR within the combined responses to SF. Cell softening and reduced motility occurred in SF cells, with a loss of actin stress fibers and a broader distribution of microtubules and intermediate filaments within the cytoplasm than in control cells. Furthermore, in space the number of primary cilia per cell increased and DNA repair mechanisms were found to be activated. Transcriptomics revealed the opposing effects of microgravity from SR for specific molecular pathways: SR, unlike microgravity, stimulated pathways for endothelial activation, such as hypoxia and inflammation, DNA repair and apoptosis, inhibiting autophagic flux and promoting an aged-like phenotype. Conversely, microgravity, unlike SR, activated pathways for metabolism and a pro-proliferative phenotype. Therefore, we suggest microgravity and SR should be considered separately to tailor effective countermeasures to protect astronauts’ health.

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Availability of data and materials

All transcriptome analysis raw data were deposited in GEO as GSE157937.

Code availability

Not applicable.

Abbreviations

AcTUBA:

Acetylated tubulin alpha

ATP5H:

Mitochondrial ATP synthase, subunit d

CC:

Culture chamber

COC:

Cyclic olefin copolymer

CTNNB:

Catenin-beta

DEG:

Differentially expressed genes

EC:

Endothelial cell

ESA:

European Space Agency

EU:

Experimental Unit

FISH:

Fluorescent in situ hybridization

GC:

Ground controls, samples prepared in parallel with samples sent to space, but grown on Earth

GSEA:

Gene Set Enrichment Analysis

Hh:

Hedgehog

HMEC-1:

Human microvascular endothelial cells-1

IF:

Immunofluorescence

ISS:

International Space Station

KRT7:

Cytokeratin 7

LC3B:

Light chain 3 isoform B

LINC:

Linker of nucleoskeleton and cytoskeleton

MYL2:

Myosin light chain 2

PBS:

Phosphate buffer saline

γH2AX:

Phosphorylated histone H2AX

PML NB:

Promyelocytic leukemia nuclear bodies

SF:

Space flight

SF-1g:

Space-flown samples cultured at 1g within the centrifuge onboard

SF-μg:

Space-flown samples exposed to real microgravity

SR:

Space radiation

TMA:

Transport modified anthropometric

TUBA:

Tubulin alpha

VIM:

Vimentin

WCP:

Whole chromosome probes

YAP1:

Yes-associated protein 1

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Acknowledgements

All space biology experiments are supported by complex logistics and require by definition a large range of interventions. The authors wish to acknowledge the contribution of several professionals whose roles were critical for completing this work: Valfredo Zolesi, Alessandro Donati, Aleandro Norfini, Michele Balsamo (Kayser Italia, srl, Livorno, Italy); Jason Hatton, Jutta Krause, Paolo Provasi, Andrea Koheler, Pierfilippo Manieri, Sergio Mugnai (ESA); Sara Piccirillo, Gabriele Mascetti (ASI); Andreas Mogensen (Astronaut Corps, ESA) and Kimiya Yui (Astronaut Corps, Jaxa), Paolo Nespoli (Astronaut Corps, ESA, Ret.); Raimondo Fortezza (Telespazio, Napoli, Italy); Fabienne Wyss, Jeannine Winkler, Bernd Rattenbacher (USOC Hergiswil, CH); Alexander Sverev and all his team of PAO RCS Energia (Russia); Rosa Sapone, Paolo Cergna (Altech spa, Torino, Italy); Thomas Berger (DLR); Sonke Burmeister (CAU); Lucia Giorgetti (IBBA CNR, Pisa, Italy); Edoardo Bertolini (Scuola Superiore Sant’Anna, Pisa, Italy); Daniele Panetta (IFC CNR, Pisa, Italy); Gianni Ciofani, Attilio Marino (IIT, Pontedera, Italy); Gianni Costa (Pierburg Italy); Benedetto Grimaldi (IIT, Genova); Jonny Martini (DHL); Mr. and Mrs. Vittorio E. Angeloni. Last but not least, Riccardo M. Andreazzoli and Matteo and Giulia Meccheri for their cheerful comprehension and forbearance.

Funding

The study was supported by European Space Agency (ESA ILSRA-2009-1026), Agenzia Spaziale Italiana (contract number 5681).

Author information

Author notes

  1. Ivana Barravecchia

    Present address: Department of Pharmacy, University of Pisa, 56126, Pisa, Italy

  2. Chiara De Cesari & Massimiliano Andreazzoli

    Present address: Department of Biology, University of Pisa, 56123, Pisa, Italy

Authors and Affiliations

  1. Institute of Life Sciences, Scuola Superiore Sant’Anna, Via G. Moruzzi, 1, 56124, Pisa, Italy

    Ivana Barravecchia, Chiara De Cesari, Francesca Scebba, Olga V. Pyankova, Mario Enrico Pè & Debora Angeloni

  2. Center for Genome Research, Department of Life Science, University of Modena and Reggio Emilia, 41125, Modena, Italy

    Mattia Forcato & Silvio Bicciato

  3. Laboratory of Nuclear and Genomic Health, Centre of Genome Engineering and Maintenance, Division of Biosciences, Department of Life Sciences, College of Health and Life Sciences, Brunel University London, Uxbridge, UB8 3PH, UK

    Joanna M. Bridger

  4. Department of Biological and Environmental Sciences, School of Life and Medical Sciences, University of Hertfordshire, Hatfield, UK

    Helen A. Foster

  5. Fondazione Pisana Per La Scienza, 56124, Pisa, Italy

    Giovanni Signore

  6. Institute of Clinical Physiology, National Research Council, 56124, Pisa, Italy

    Andrea Borghini & Mariagrazia Andreassi

Authors
  1. Ivana Barravecchia
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Contributions

Conceptualization and overall supervision: DA; methodology: DA, IB, MF, SB, JMB; investigation and validation, data collection, image and statistical analysis: IB, CDC, OVP, MF, SB, JMB, HAF, FS, AB, MA, GS; writing—original draft preparation: DA, IB; writing editing: MF, SB, JMB, GS; project administration: DA; funding acquisition: DA, MA, MEP. All authors have read and agreed to the published version of the manuscript.

Corresponding author

Correspondence to Debora Angeloni.

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Supplementary Information

Below is the link to the electronic supplementary material.

18_2021_4025_MOESM1_ESM.docx

Supplementary file 1 Supplementary Table 1 List of antibodies used for IF, with indication of biological source, commercial provider and dilution used (DOCX 14 KB)

18_2021_4025_MOESM2_ESM.tif

Supplementary file 2 Supplementary Fig. 1 Schematic representation of the ENDO EU. A Mission patch of the ENDO study, supported by ESA and ASI. B 3D rendering of the microincubator (Kayser Italia, Livorno, Italy). Reservoirs containing cell culture media is in red, fixative in green, washing solution in grey. The cell culture support is in red on the incubator proximal side. Further details are in [21–23]. C Launch of Soyuz TMA-18M (manned Mission 44S) from Baikonur Cosmodrome (Kazahstan), at 4:37 GMT, September 2nd 2015. D ESA thermal case Yellow Box, used for maintaining samples while traveling after landing from retrieval site to the PI home laboratory (TIF 114298 KB)

18_2021_4025_MOESM3_ESM.tif

Supplementary file 3 Supplementary Fig. 2 Results of IF preparatory work. HMEC-1 cells were variously stimulated on ground to activate autophagy (A–E) and DNA damage response mechanisms (F–K): culture in serum-free medium (Starvation: 48 h); exposure to oxygen reactive species (50 mM H2O2, 1.5 h prior to fixing); DNA synthesis inhibition (10mM Hydroxyurea, HDU); induction of double strand breaks (exposure to 1 Gray X-rays). L–N labeling with chromosome markers 1, 10 and 17 in ground samples. For each experimental group, three independent operators counted at least 60 cells (six cells per at least ten fields). All tests were repeated at least three times (TIF 104213 KB)

18_2021_4025_MOESM4_ESM.tif

Supplementary file 4 Supplementary Fig. 3 Nuclear and cell shape. Shape descriptors indicated absence of differences in terms of Circularity (A), Roundness (B) and Solidity (C) among the three experimental groups. Statistical analysis was performed with One way ANOVA and Bonferroni multiple comparison post-hoc tests (TIF 90558 KB)

18_2021_4025_MOESM5_ESM.tif

Supplementary file 5 Supplementary Fig. 4 PCA analysis. Unsupervised principal component analysis of the RNA-seq samples (TIF 36294 KB)

18_2021_4025_MOESM6_ESM.tif

Supplementary file6 Supplementary Fig. 5 Integration of genomics and transcriptomics data. Circular plots integrated the gene expression information with physical localization of genes differentially affected by SF (A), showing a different contribution of Microgravity (B) and Radiation (C). All chromosomes were  affected. Red and green bars indicate up- and down-regulated genes respectively (TIF 91434 KB)

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Barravecchia, I., De Cesari, C., Forcato, M. et al. Microgravity and space radiation inhibit autophagy in human capillary endothelial cells, through either opposite or synergistic effects on specific molecular pathways. Cell. Mol. Life Sci. 79, 28 (2022). https://doi.org/10.1007/s00018-021-04025-z

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  • DOI: https://doi.org/10.1007/s00018-021-04025-z

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