A comprehensive molecular and morphological study of the effects of space flight on human capillary endothelial cells: sample quality assessment and preliminary results.
-
1
University of Pisa, Italy
-
2
Scuola Sant'Anna di Studi Avanzati, Institute of Life Sciences, Italy
-
3
Università degli Studi di Modena e Reggio Emilia, Italy
-
4
University of Hertfordshire, United Kingdom
-
5
Brunel University London, United Kingdom
Human subjects and experimental animal models returning from Space show health issues related to the endothelium, a diffuse organ fundamental in maintaining body homeostasis. There are striking similarities between the effects of Space Flight (SF) on astronauts, and the consequences of old age, degenerative diseases and leading a sedentary life on Earth. Therefore, understanding the pathophysiological processes caused by SF might significantly advance prevention and treatment of frequent pathologies associated with ageing and lack of exercise.
Up to now, several cell types were characterized, although partially, for their response to SF, except endothelial cells (ECs) of capillary origin, a fundamental part of the endothelium. Therefore, we launched a human EC line from dermal capillary (HMEC-1, human Microvascular Endothelial Cells 1, CDC, USA) to the International Space Station (ISS): 'Endothelial cells' project, http://blogs.esa.int/iriss/2015/08/25/how-do-blood-vessel-cells-react-to-weightlessness/ (Soyuz 44S-42S launch campaign, September 2015) and undertook a cell and molecular biology study that included analysis of protein markers, of the transcriptome (RNASeq) and the methylome. Cells were seeded onto Cyclic Olefin Copolymer coverslips (IBIDI, Germany) coated with 2% pork skin gelatine, in MCDB131 medium supplemented with 20 mM HEPES (all reagents: Sigma-Aldrich, USA). Cell cultures were placed into micro-incubators (Experimental Units, EUs, Kayser Italia, Livorno, Italy) that, when connected to power, are able to fulfill under electronic control and without human intervention a scientific protocol of cell culture and fixation 2. We prepared 40 EUs: 24, were integrated on the Soyuz rocket and reached the ISS, 16 others remained on Earth to serve as ground controls (GC), (Figure 1, Table 1). The study required six-days of culturing in space, within KUBIK onboard ISS, with two cell growth medium changes, and a final fixation step. The short-duration mission foresaw a 6-hours rendez-vous flight to the ISS that instead, for unpredicted reasons, lasted 48 hours. Only afterwards, the samples were integrated into KUBIK. In parallel, we activated the GC EUs within the KISS facility in Pisa. Due to the delay, the medium change within KUBIK and KISS was still activated twice but after two instead of three days each time. At the end of space mission, the science material was returned via a commercial flight to Pisa, traveling in the ESA Thermocase B5 (Yellow Box). Cell culturing procedures, fixation methods and feasibility of planned experiments had been extensively tested during the experiment definition and experiment sequence test phases of the project 2,3. However, due to the operational complexity of the whole study, when the samples arrived at Pisa, before undertaking the molecular analyses, we performed a quality assessment of fixed samples whose results we present here with some preliminary data.
Initially, we evaluated the cell general morphology with light-microscopy observation. All coverslips (except GM03, GM08, FM05, FM18, that were somewhat less satisfactory) were found covered with cells (about 100% confluent) adherent and elongated (examples in Figure 2A). DNA (Figure 2B) and RNA were extracted and quantified. Each sample type, i.e. microgravity exposed (μ−g), centrifuge exposed (space-1g), and GCs, yielded in average about 500 ng total RNA, and about 1 μg genomic DNA.
Eight samples (Table 1) were used for whole-transcriptome RNASeq analysis (Applied Genomics Institute, IGA, Udine, Italy). Libraries were prepared with TruSeq Stranded Total RNA with Ribo-Zero Library Prep Kit (Illumina, San Diego, CA) and sequenced with Illumina HiSeq 2500 platform; CASAVA v1.8.2 was used for base calling. Quality of 2X125bp reads was assessed with FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Raw reads were aligned using STAR v2.5.3 4 to build version hg38 of the GENCODE human genome release 25. All samples had at least 44 millions PE reads uniquely aligned to the genome (Figure 2C). Counts for GENCODE basic annotated genes were calculated from the aligned reads using featureCounts function of the Rsubread R package 5. Normalization was carried out using edgeR R package 6. Raw counts were normalized according to library size to obtain counts per millions (CPM). Only genes with CPM greater than 1 in at least two samples were retained for subsequent analysis. Differential analysis was performed with voomWithQualityWeights function of limma R package 7, to down-weight outlier samples. Gene Set Enrichment Analysis (GSEA) 8 is in progress to determine whether the set of genes transcriptionally affected by microgravity or cosmic radiation was enriched in molecular pathways.
Eight samples (Table 1) were reserved for methylome analysis, currently in progress.
To evaluate cell morphology we began with F-actin staining because several reports describe the effect of microgravity 9–14, either real or simulated, on this protein in several cell types, so as to compare our own original results regarding microvascular ECs in real SF.
GCs showed well-defined F-actin stress and peripheral fibers whereas μ−g and space-1g cells showed collapsed F-actin networks (Figure 2D), with fluorescence intensity peaks considerably decreased (Figure 2D'). Also, stress granules (cytoplasmic structures that appear upon different stress stimuli 15,16) of different size were found in all space samples (Figure 2D, yellow arrow). These similarities in both space sample types were possibly caused by the long rendez-vous flight, and/or other space-related factors, e.g. cosmic radiation, that were not counterbalanced by four days at 1g. The distribution of beta-catenin appeared less cytoplasmic and more in structured and oriented adherens junctions of μ-g cells compared to GCs (Figure 2E). HMEC-1 cells showed statistically significant change of morphology, measured with descriptors as circularity, roundness and solidity (Figure 2F) 17.
Altogether, these preliminary observations confirm that SF stresses ECs, damaging basic assets of cytoskeleton and transcriptome activity, whose dynamics are known to be strongly intertwined. The disorganization of F-actin seems counterbalanced by well-structured adherens junctions that preserve the endothelial sheet in SF.
For the first time, this work attempts at an integrated study of morphological, protein and transcriptome markers on a same biological system, specifically microvascular ECs to describe as integrally as possible their response to SF. On-going analysis will uncover further molecular details regarding the adaptive response of this fundamental component of physiological homeostasis.
Acknowledgements
The Endothelial cells experiment was supported by the ESA (ESA ILSRA-2009-1026); ASI (contract no. 5681); Regione Toscana (POR FSE 2007-2013-FORTEC); Kayser Italia; Lions Club International, District 108LA, Toscana, Italy.
The authors thank all personnel at Biotesc, especially B. Rattenbacher, J. Winkler, F. Wyss and S. Richard; key officials at ESA, especially P. Manieri, P. Provasi, J. Krause and A. Koehler (HSO-PIL); V. Zolesi, A. Donati and colleagues at Kayser Italia.
References
1. Ades, E. W. et al. HMEC-1: establishment of an immortalized human microvascular endothelial cell line. J. Invest. Dermatol. 99, 683–690 (1992).
2. Balsamo, M. et al. Molecular and Cellular Characterization of Space Flight Effects on Microvascular Endothelial Cell Function – PreparatoryWork for the SFEF Project. Microgravity Sci. Technol. 26, 351–363 (2014).
3. Barravecchia, I. et al. Pitting Corrosion Within Bioreactors for Space Cell-Culture Contaminated by Paenibacillus glucanolyticus, a Case Report. Microgravity Sci. Technol. 30, 309–319 (2018).
4. Dobin, A. et al. STAR: Ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).
5. Liao, Y., Smyth, G. K. & Shi, W. FeatureCounts: An efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30, 923–930 (2014).
6. Robinson, M. D., McCarthy, D. J. & Smyth, G. K. edgeR: A Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2009).
7. Liu, R. et al. Why weight? Modelling sample and observational level variability improves power in RNA-seq analyses. Nucleic Acids Res. 43, e97 (2015).
8. Subramanian, A. et al. Gene set enrichment analysis: A knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl. Acad. Sci. 102, 15545–15550 (2005).
9. Versari, S., Villa, A., Bradamante, S. & Maier, J. A. M. Alterations of the actin cytoskeleton and increased nitric oxide synthesis are common features in human primary endothelial cell response to changes in gravity. Biochim. Biophys. Acta 1773, 1645–52 (2007).
10. Carlsson, S. I. M., Bertilaccio, M. T. S., Ballabio, E. & Maier, J. A. M. Endothelial stress by gravitational unloading: effects on cell growth and cytoskeletal organization. Biochim. Biophys. Acta 1642, 173–9 (2003).
11. Morbidelli, L. et al. Simulated hypogravity impairs the angiogenic response of endothelium by up-regulating apoptotic signals. Biochem. Biophys. Res. Commun. 334, 491–499 (2005).
12. Infanger, M. et al. Modeled gravitational unloading induced downregulation of endothelin-1 in human endothelial cells. J. Cell. Biochem. 101, 1439–1455 (2007).
13. Pietsch, J. et al. Three-dimensional growth of human endothelial cells in an automated cell culture experiment container during the SpaceX CRS-8 ISS space mission – The SPHEROIDS project. Biomaterials 124, 126–156 (2017).
14. Monici, M. et al. An in Vitro Study on Tissue Repair: Impact of Unloading on Cells Involved in the Remodelling Phase. Microgravity Sci. Technol. 23, 391–401 (2011).
15. Berlin, S.-V. et al. NATO ASI Series Advanced Science Institutes Series Volume 284-Advances in Morphometries edited Volume 285-Molecular, Cellular, and Clinical Aspects of Angiogenesis edited. (1996). doi:10.1007/978-1-4613-0389-3
16. Costa-Almeida, R. et al. Effects of hypergravity on the angiogenic potential of endothelial cells. J. R. Soc. Interface 13, 20160688 (2016).
17. Martino, F., Perestrelo, A. R., Vinarský, V., Pagliari, S. & Forte, G. Cellular Mechanotransduction: From Tension to Function. Front. Physiol. 9, 824 (2018).
18. Ewels, P., Magnusson, M., Lundin, S. & Käller, M. MultiQC: summarize analysis results for multiple tools and samples in a single report. Bioinformatics 32, 3047–8 (2016).
19. Marino, A. et al. The Osteoprint: A bioinspired two-photon polymerized 3-D structure for the enhancement of bone-like cell differentiation. Acta Biomater. 10, 4304–4313 (2014).
Keywords:
Capillary endothelium,
F-actin = filamentous actin,
Human Microvascular Endothelial cells-1 HMEC-1,
International Space Station (ISS),
microgravity,
RNA sequencing (RNAseq),
Illumina HiSeq 2500,
Soyuz
Conference:
39th ISGP Meeting & ESA Life Sciences Meeting, Noordwijk, Netherlands, 18 Jun - 22 Jun, 2018.
Presentation Type:
Extended abstract
Topic:
Cardiovascular, Fluid Shift and Respiration
Citation:
BARRAVECCHIA
I,
De Cesari
C,
Pyankova
OV,
Scebba
F,
Pè
ME,
Forcato
M,
Bicciato
S,
Foster
HA,
Bridger
JM and
Angeloni
D
(2019). A comprehensive molecular and morphological study of the effects of space flight on human capillary endothelial cells: sample quality assessment and preliminary results..
Front. Physiol.
Conference Abstract:
39th ISGP Meeting & ESA Life Sciences Meeting.
doi: 10.3389/conf.fphys.2018.26.00050
Copyright:
The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers.
They are made available through the Frontiers publishing platform as a service to conference organizers and presenters.
The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated.
Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed.
For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions.
Received:
02 Dec 2018;
Published Online:
16 Jan 2019.
*
Correspondence:
PhD. Debora Angeloni, Scuola Sant'Anna di Studi Avanzati, Institute of Life Sciences, Pisa, Italy, angeloni@ifc.cnr.it