Thèse Adaptation de l'Endothélium Lymphatique aux Contraintes de l'Environnement Spatial Effets de la Microgravité et des Rayonnements Cosmiques Galactiques H/F Doctorat.Gouv.Fr

Établissement : Université de Toulouse École doctorale : BSB - Biologie, Santé, Biotechnologies Laboratoire de recherche : I2MC - Institut des Maladies Métaboliques et Cardiovasculaires Direction de la thèse : Florent MORFOISSE ORCID 0000000315323665 Début de la thèse : 2027-09-01 Date limite de candidature : 2026-11-23T23:59:59 Le système lymphatique joue un rôle essentiel dans l'homéostasie des fluides interstitiels, le transport des lipides et la surveillance immunitaire. Son fonctionnement repose sur des gradients de pression et des contraintes mécaniques influencés par la gravité. En microgravité, la redistribution des fluides vers le compartiment céphalique entraîne des modifications vasculaires majeures chez les astronautes, mais l'adaptation du système lymphatique à ces conditions reste largement inconnue. Par ailleurs, les missions spatiales exposent les astronautes aux rayonnements cosmiques galactiques (GCR), dont les effets sur l'endothélium lymphatique n'ont pratiquement pas été étudiés.
Ce projet vise à caractériser, pour la première fois, les réponses des cellules endothéliales lymphatiques humaines à la microgravité et aux GCR, et à déterminer comment ces deux contraintes interagissent. Nous faisons l'hypothèse que la microgravité et les GCR induisent des adaptations moléculaires et fonctionnelles distinctes mais interconnectées, et que leur exposition simultanée déclenche une réponse spécifique résultant de leur interaction.
Le projet s'appuiera sur un modèle tridimensionnel de vaisseau lymphatique sur puce (lymphatic-on-chip), développé par le LAAS-CNRS en collaboration avec l'Institute of Industrial Science (IIS) de l'Université de Tokyo, inventeur et développeur de cette technologie. Cette collaboration permettra également d'adapter le dispositif aux contraintes expérimentales spatiales. Les expériences de microgravité simulée seront réalisées au GSBMS, plateforme spécialisée en biologie et médecine spatiales, à l'aide du simulateur martien Mars Simulator (Orius), selon un scénario reproduisant la microgravité de l'ISS.
Trois axes complémentaires seront développés. Le premier étudiera la réponse à la microgravité après 24 h et 96 h, en évaluant la perméabilité vasculaire, l'organisation des jonctions endothéliales et du cytosquelette, ainsi que les modifications transcriptomiques. Le deuxième analysera les effets des GCR, avec une dose initiale de 0,5 Gy, selon les mêmes temps et critères fonctionnels et moléculaires. Le troisième étudiera l'interaction entre les deux contraintes selon un plan factoriel 2 × 2 comparant 1 g ou microgravité, avec ou sans irradiation, afin d'identifier les réponses spécifiquement liées à leur combinaison.
Les analyses transcriptomiques permettront d'identifier les voies et mécanismes candidats, qui seront ensuite validés fonctionnellement par des approches de perte et de gain de fonction, puis dans le modèle 3D de vaisseau lymphatique sur puce. Ce projet devrait ainsi révéler de nouveaux mécanismes d'adaptation de l'endothélium lymphatique aux conditions spatiales et identifier des cibles susceptibles de préserver sa fonction. À terme, ces résultats pourront soutenir le développement d'expériences autonomes en orbite basse et de futures missions scientifiques avec l'ESA et la JAXA.

The lymphatic vascular system is essential for maintaining tissue fluid homeostasis, immune surveillance, and lipid transport. By collecting excess interstitial fluid, macromolecules, and immune cells from peripheral tissues and returning them to the venous circulation, the lymphatic vasculature plays a central role in maintaining tissue homeostasis. Unlike the blood vascular system, however, lymphatic circulation is not driven by a central pump and must continuously transport fluid through a network of lymphatic capillaries, collecting vessels, and lymph nodes. Lymph transport therefore relies on the coordinated contribution of intrinsic lymphatic vessel contractility, intraluminal valves, skeletal muscle activity, respiratory movements, and, importantly, hydrostatic pressure gradients generated by gravity and body posture. Normal lymphatic function is consequently intimately linked to mechanical and gravitational forces [1,2].
This dependence on gravity becomes particularly relevant during spaceflight. Exposure to microgravity causes a profound redistribution of body fluids, with a cephalad fluid shift from the lower extremities toward the thoracic and cranial compartments. This redistribution is associated with characteristic physiological adaptations in astronauts, including facial edema, venous congestion, increased intracranial pressure, and ocular alterations collectively referred to as Spaceflight-Associated Neuro-Ocular Syndrome (SANS) [3,4]. Although these phenomena have predominantly been investigated from a cardiovascular perspective, growing evidence suggests that alterations in lymphatic drainage and interstitial fluid homeostasis may also contribute to their development. The parallels with lymphatic disorders such as secondary lymphedema are particularly striking: both conditions involve abnormal fluid accumulation, altered mechanical and pressure environments, vascular dysfunction, inflammatory remodeling, and impaired tissue drainage [5]. In lymphedema, chronic alterations in lymphatic flow progressively promote endothelial dysfunction, vascular leakage, inflammation, and tissue remodeling. Similarly, the sustained redistribution of fluids during microgravity may expose lymphatic vessels and surrounding tissues to profoundly altered mechanical conditions while challenging the mechanisms that normally maintain efficient fluid drainage. The recent recognition of meningeal lymphatic vessels as important regulators of cerebrospinal fluid clearance further strengthens the potential relevance of lymphatic dysfunction to intracranial fluid regulation and spaceflight-associated neurological and ocular complications [6].
Despite these strong physiological connections, the lymphatic vasculature remains remarkably understudied in space biology. Most studies investigating the effects of microgravity on the vascular endothelium have focused on blood endothelial cells, particularly HUVECs, and have demonstrated extensive alterations in cytoskeletal organization, cell adhesion, proliferation, oxidative stress, apoptosis, and vascular signaling [7,10]. However, endothelial responses to microgravity are highly dependent on cellular identity and function [7], and these observations cannot simply be extrapolated to lymphatic endothelial cells (LECs). LECs possess specialized structural and molecular characteristics adapted to their unique role in fluid uptake, permeability regulation, immune cell trafficking, and lymphatic vessel function. Their physiology is also tightly controlled by mechanosensitive pathways that allow them to respond to changes in fluid flow, pressure, and tissue mechanics. Given the central role of gravitationally dependent hydrostatic gradients in lymphatic drainage, LECs may therefore represent a particularly sensitive cellular population within the vascular system during spaceflight.
A major limitation of current approaches is that most available microgravity studies rely on conventional two-dimensional endothelial cultures, which cannot reproduce the three-dimensional architecture, lumen, cell-cell organization, or fluidic environment of a lymphatic vessel. To overcome this limitation, our team has developed, in collaboration with LAAS-CNRS and the University of Tokyo, a human lymphatic vessel-on-chip model that recreates key structural and functional properties of lymphatic vessels, including a perfusable lumen, three-dimensional endothelial organization, and measurable barrier function [11-13]. This platform provides a unique opportunity to investigate how altered mechanical environments directly affect human lymphatic endothelial function under controlled experimental conditions.
Against this background, a fundamental question remains unresolved: how does the human lymphatic endothelium adapt to the profound mechanical and environmental changes imposed by spaceflight, and how might these adaptations contribute to impaired fluid homeostasis? Addressing this question requires moving beyond the study of microgravity as an isolated mechanical stimulus and considering the broader space exposome, which combines mechanical unloading with other environmental stresses, including chronic exposure to space radiation. Understanding how LECs respond to these individual and combined challenges is therefore essential to determine whether spaceflight induces a specific lymphatic endothelial dysfunction and to identify the molecular mechanisms that could ultimately be targeted to preserve lymphatic function during long-duration space missions.
The overall objective of this project is to determine how lymphatic endothelial cells adapt to the major environmental stresses encountered during spaceflight, and to identify the molecular pathways and therapeutic targets underlying spaceflight-induced lymphatic dysfunction.

Axis 1 - Microgravity: Characterize the functional and molecular response of human lymphatic endothelial cells to simulated microgravity, and identify the signaling pathways and molecular targets responsible for alterations in endothelial barrier function.

Axis 2 - Space radiation: Determine the impact of Galactic Cosmic Ray-like irradiation on lymphatic endothelial function and identify the molecular pathways and candidate targets mediating radiation-induced endothelial dysfunction.

Axis 3 - Combined spaceflight stress: Determine how lymphatic endothelial cells integrate microgravity and radiation exposure, establish whether their combined effects are additive, synergistic, or antagonistic, and identify interaction-specific pathways and therapeutic targets that could be exploited to preserve lymphatic endothelial function during long-duration spaceflight. Axis 1 - Defining the lymphatic endothelial response to microgravity
The first objective will be to determine how exposure to microgravity affects the structure, molecular state and function of human lymphatic endothelial cells (LECs). The project will rely on a human lymphatic vessel-on-chip model developed through a collaboration between the LAAS-CNRS and the University of Tokyo, which reproduces the three-dimensional organization and key barrier functions of lymphatic vessels [11-13]. This platform will provide a physiologically relevant three-dimensional system in which molecular alterations induced by altered gravity can be directly linked to vascular function.
Simulated microgravity experiments will be performed at GSBMS using the Mars Simulator (Orius) configured with the ISS microgravity scenario, which will be used to reproduce the gravitational conditions experienced aboard the International Space Station [14]. The target gravitational level will therefore correspond to the microgravity environment of the ISS. The detailed operational parameters of the device, including the rotation/exposure characteristics and validated experimental configuration, will be defined on the basis of previous experiments performed with the same Mars Simulator and documented using the corresponding experimental references. For each experimental condition, parallel control samples maintained at normal gravity will be processed under otherwise identical conditions.
Two exposure durations will initially be investigated: 24 h and 96 h. These time points are commonly used to investigate endothelial responses to simulated microgravity and will allow the characterization of both early cellular responses and more established adaptive mechanisms [6]. The 24 h time point will primarily capture early molecular and functional alterations following exposure to altered gravity, whereas the 96 h condition will allow the identification of more sustained transcriptional and structural adaptations.
For each exposure duration and experimental condition, functional analyses will be performed on groups of 10 independent lymphatic vessels-on-chip, a number routinely used within the laboratory to account for inter-vessel variability and to provide sufficient statistical power for functional measurements. Three independent biological experiments will be performed for each experimental condition. The complete experimental design will therefore incorporate both technical replication at the vessel level and biological replication across independent experiments.
The first experimental objective will be to determine whether simulated microgravity alters lymphatic endothelial barrier function. Vascular permeability will be quantified using fluorescent FITC-dextran introduced into the endothelial lumen, followed by time-resolved fluorescence imaging using confocal microscopy, as previously established for the lymphatic vessel-on-chip platform [9,10]. Quantification of dextran extravasation into the surrounding extracellular matrix will provide a direct functional measurement of endothelial barrier integrity. This approach will determine whether altered gravity induces a measurable increase in vascular leakage and will establish the functional phenotype that will subsequently be linked to molecular alterations.
Following permeability measurements, the lymphatic vessels-on-chip will be fixed using 4% paraformaldehyde (PFA) and processed for immunofluorescence analysis. Particular emphasis will be placed on proteins involved in endothelial junctional integrity and cytoskeletal organization, including VE-cadherin, ZO-1 and filamentous actin (F-actin). High-resolution imaging will be used to assess junctional continuity, cellular organization and cytoskeletal architecture, allowing functional alterations in permeability to be related to structural changes within the endothelial monolayer. Additional junctional markers, including claudin-5 and occludin, may be incorporated according to the phenotypes identified during the initial experiments.
In parallel with the functional characterization, the molecular response of LECs to simulated microgravity will be investigated by transcriptomic profiling. For each experimental condition, RNA will be extracted from the lymphatic vessels-on-chip and subjected to bulk RNA sequencing (RNA-seq). Because the amount of RNA obtainable from an individual microvessel is limited, each sequencing sample will consist of a pool of RNA collected from vessels generated across three independent biological experiments. This strategy will provide sufficient biological material for transcriptomic analysis while maintaining reproducibility across independent experimental batches.
The RNA-seq analysis will initially follow an unbiased, discovery-driven strategy rather than being restricted to a predefined list of genes. Differential expression analysis and pathway-level analyses will be used to identify biological processes altered by simulated microgravity, including endothelial activation, inflammatory signaling, mechanotransduction, cytoskeletal remodeling, oxidative stress, cell survival and regulation of endothelial junctions. Genes previously implicated in endothelial adaptation to simulated microgravity, including SELE, VCAM1, ICAM1, IL6 and IL8, will be used as reference markers to facilitate comparison with previous studies, while the principal candidate-selection strategy will remain driven by the newly generated transcriptomic data.
Where appropriate, transcriptomic changes will be complemented by analysis of the endothelial secretome, allowing changes in gene expression to be related to the production of inflammatory mediators. This integrated approach will determine whether microgravity induces a predominantly structural, inflammatory, stress-associated or multifactorial response in lymphatic endothelial cells.
The strongest candidate genes and pathways emerging from the RNA-seq analysis will subsequently be prioritized for functional validation. Candidate selection will take into account the magnitude and reproducibility of transcriptional regulation, pathway-level significance, biological relevance to endothelial function and, where possible, the relationship between molecular changes and the observed permeability phenotype. Rather than validating a large predefined panel, the project will focus on the best candidates emerging from the transcriptomic analysis.
Selected candidates will be investigated using complementary loss- and gain-of-function approaches. siRNA and/or shRNA-mediated silencing will be used to determine whether candidate genes are required for the microgravity-induced phenotype, whereas lentiviral overexpression will assess whether modulation of these pathways is sufficient to reproduce, modify or rescue the observed response. Candidates demonstrating a causal role will subsequently be validated in the three-dimensional lymphatic vessel-on-chip model, where permeability and endothelial junctional organization will provide direct functional endpoints.
Axis 1 will therefore establish a quantitative functional and molecular map of lymphatic endothelial adaptation to simulated microgravity, while generating a ranked set of candidate pathways for investigation under combined spaceflight stress.
Axis 2 - Defining the impact of Galactic Cosmic Rays on lymphatic endothelial homeostasis
The second axis will determine how space-relevant radiation affects lymphatic endothelial homeostasis independently of altered gravity. Human LECs will be exposed to simulated Galactic Cosmic Ray (GCR) radiation using the irradiation platform available to the consortium. An initial cumulative dose of 0.5 Gy will be used as the first experimental condition. This dose will provide a standardized starting point for characterizing the biological response of lymphatic endothelial cells to GCR exposure, while the precise irradiation parameters, including the radiation composition, particle characteristics and exposure configuration, will be defined according to the validated experimental protocols of the irradiation platform and the available preliminary data.
For each irradiation condition, sham-irradiated cells will be maintained under otherwise identical experimental conditions and will serve as controls. As in Axis 1, two experimental time points, 24 h and 96 h, will initially be investigated. These time points will allow the characterization of both early responses to radiation-induced cellular stress and more sustained alterations in endothelial homeostasis. Importantly, using the same temporal framework as for the microgravity experiments will facilitate direct comparison of the kinetics and magnitude of the responses induced by the two environmental stressors.
The functional consequences of GCR exposure will first be investigated using the lymphatic vessel-on-chip model. For each condition and time point, functional analyses will be performed on groups of 10 lymphatic vessels-on-chip, with three independent biological experiments performed for each condition. The primary functional endpoint will again be endothelial barrier integrity. FITC-dextran will be introduced into the endothelial lumen and its extravasation into the surrounding matrix will be quantified by fluorescence imaging, providing a direct measurement of radiation-induced changes in vascular permeability [12,13].
Following permeability measurements, vessels will be fixed using 4% PFA and analyzed by immunofluorescence. VE-cadherin, ZO-1 and F-actin will constitute the principal markers used to assess endothelial junctional organization and cytoskeletal architecture. Additional junctional proteins, including claudin-5 and occludin, may be examined depending on the phenotypes identified during the initial experiments. These analyses will determine whether changes in permeability are associated with disruption of endothelial junctions, cytoskeletal remodeling or broader alterations in endothelial architecture.
The functional analysis will be complemented by characterization of radiation-associated cellular stress responses. Depending on the initial phenotype, these analyses will include markers of inflammatory activation, oxidative stress, DNA damage, apoptosis and cellular senescence. This integrated characterization will determine whether GCR exposure primarily induces a barrier defect, a stress-associated endothelial phenotype, or a combination of these responses.
The molecular response to radiation will subsequently be investigated by bulk RNA sequencing. RNA will be extracted from the lymphatic vessels-on-chip for each experimental condition, with sequencing samples generated by pooling RNA obtained from vessels across the three independent biological experiments. This approach will provide sufficient RNA for transcriptomic analysis while maintaining biological reproducibility across experimental batches.
As in Axis 1, transcriptomic analysis will initially be performed using an unbiased discovery-driven strategy. Differential expression and pathway analyses will be used to identify radiation-responsive biological programs, with particular attention to DNA damage and repair, oxidative stress, mitochondrial function, inflammatory signaling, apoptosis and senescence, as well as endothelial barrier regulation and cytoskeletal organization. The analysis will not be restricted to predefined candidate genes, allowing lymphatic-specific responses to GCR exposure to emerge from the dataset.
The resulting radiation transcriptome will then be compared with the microgravity dataset generated in Axis 1. This comparative analysis will identify molecular programs shared between the two environmental stressors as well as stressor-specific responses. Shared pathways will provide candidate mechanisms through which lymphatic endothelial cells may integrate distinct environmental insults, whereas radiation-specific pathways may explain functional alterations that are not reproduced by altered gravity.
The most relevant candidates emerging from the RNA-seq analysis will be selected for functional validation based on their magnitude and reproducibility of regulation, pathway-level significance, biological relevance and association with the observed endothelial phenotype. siRNA and/or shRNA-mediated silencing will be used to determine whether selected candidates are required for the radiation-induced response, while lentiviral overexpression will be used to assess whether modulation of these pathways is sufficient to alter or rescue endothelial dysfunction.
Candidates that are also significantly regulated under microgravity, or that represent plausible molecular convergence points between mechanical unloading and radiation-induced stress, will be prioritized for further investigation in Axis 3. The most compelling mechanisms will ultimately be validated using the three-dimensional lymphatic vessel-on-chip model, thereby establishing a direct link between radiation-responsive molecular pathways and lymphatic endothelial barrier function.
Axis 3 - Deciphering the integrated response to microgravity and Galactic Cosmic Rays
The third axis will investigate how lymphatic endothelial cells integrate simultaneous exposure to microgravity and GCR radiation. This axis will constitute the central mechanistic component of the PhD project and will specifically address whether the combined space environment generates a response that differs from the independent effects of altered gravity and radiation.
Microgravity and radiation will be applied as a combined exposure in the fourth experimental condition, rather than being studied as two sequentially independent stresses. The microgravity experiments will be performed using the Mars Simulator (Orius) with the ISS microgravity scenario at GSBMS, while the radiation exposure will be performed according to the validated irradiation protocol established for the GCR experiments. The precise operational parameters and temporal coordination between the two platforms will be determined from the validated experimental procedures and practical constraints of the respective facilities.
The same 24 h and 96 h time points used in Axes 1 and 2 will initially be investigated. For each condition and time point, functional analyses will be performed on 10 lymphatic vessels-on-chip, with three independent biological experiments conducted for each experimental group. This standardized experimental framework will allow the magnitude and kinetics of the combined response to be directly compared with those induced by each individual stressor.
Endothelial barrier integrity will constitute the primary functional endpoint. FITC-dextran permeability assays will be performed in the lymphatic vessel-on-chip system, followed by quantitative analysis of dextran extravasation from the endothelial lumen into the surrounding matrix. Immunofluorescence analysis after fixation with 4% PFA will assess VE-cadherin, ZO-1, F-actin and, where relevant, claudin-5 and occludin. Additional analyses of inflammatory activation, oxidative stress, DNA damage, cell survival and senescence will be incorporated according to the phenotypes identified in Axes 1 and 2.
The factorial design will be essential for distinguishing simple additive effects from a genuine interaction between microgravity and radiation. Rather than comparing only the combined condition with the 1 g sham control, the statistical analysis will explicitly test the interaction term between gravitational condition and radiation exposure. This will determine whether the effect of radiation is modified by microgravity and, conversely, whether the response to microgravity is altered by radiation.
The transcriptomic response will be investigated using bulk RNA sequencing across the complete 2 × 2 experimental design. As in the preceding axes, RNA will be collected from the lymphatic vessel-on-chip samples and pooled across the three independent biological experiments to obtain sufficient material for sequencing while preserving biological reproducibility.
Transcriptomic analysis will combine differential expression with formal interaction analyses. This will allow genes and pathways to be classified according to whether they are primarily associated with microgravity, primarily associated with radiation, or specifically dependent on the interaction between the two stressors. Particular emphasis will be placed on interaction-dependent transcriptional responses, as these may identify molecular mechanisms that would not be detected by studying microgravity or radiation independently.
Integration of the transcriptomic data with the functional datasets from all three axes will then be used to prioritize candidate molecular mechanisms. Candidate selection will be primarily driven by the strongest and most reproducible RNA-seq responses, while incorporating pathway relevance, functional phenotype and evidence of interaction between the two stressors. Molecular pathways capable of integrating mechanical, oxidative and inflammatory signals will be of particular interest as potential convergence points between altered gravity and radiation-induced stress.
The highest-priority candidates will subsequently be tested using complementary loss- and gain-of-function approaches. siRNA and/or shRNA-mediated silencing will determine whether candidate pathways are required for the combined phenotype, while lentiviral overexpression will assess whether their modulation is sufficient to modify or rescue the response. Importantly, candidates will be evaluated using functional endpoints rather than transcriptomic changes alone.
Finally, the most compelling candidates will be validated in the three-dimensional lymphatic vessel-on-chip model. Candidate manipulation will be combined with permeability measurements and high-resolution imaging of endothelial junctional organization to determine whether modulation of the identified pathway can prevent or rescue the functional consequences of combined microgravity and GCR exposure.
A successful rescue of endothelial barrier integrity specifically under combined exposure would provide strong causal evidence that the identified pathway represents a mechanistic determinant of lymphatic endothelial adaptation to the space environment. Conversely, identification of interaction-dependent pathways without a simple additive phenotype would provide evidence that simultaneous exposure to microgravity and radiation generates a distinct biological state rather than merely increasing the magnitude of two independent stress responses