Space radiation damage rescued by inhibition of key spaceflight associated miRNAs.

Our previous research revealed a key microRNA signature that is associated with spaceflight that can be used as a biomarker and to develop countermeasure treatments to mitigate the damage caused by space radiation. Here, we expand on this work to determine the biological factors rescued by the countermeasure treatment. We performed RNA-sequencing and transcriptomic analysis on 3D microvessel cell cultures exposed to simulated deep space radiation (0.5 Gy of Galactic Cosmic Radiation) with and without the antagonists to three microRNAs: miR-16-5p, miR-125b-5p, and let-7a-5p (i.e., antagomirs). Significant reduction of inflammation and DNA double strand breaks (DSBs) activity and rescue of mitochondria functions are observed after antagomir treatment. Using data from astronaut participants in the NASA Twin Study, Inspiration4, and JAXA missions, we reveal the genes and pathways implicated in the action of these antagomirs are altered in humans. Our findings indicate a countermeasure strategy that can potentially be utilized by astronauts in spaceflight missions to mitigate space radiation damage.

Combined ion beam irradiation platform and 3D fluorescence microscope for cellular cancer research.

To improve particle radiotherapy, we need a better understanding of the biology of radiation effects, particularly in heavy ion radiation therapy, where global responses are observed despite energy deposition in only a subset of cells. Here, we integrated a high-speed swept confocally-aligned planar excitation (SCAPE) microscope into a focused ion beam irradiation platform to allow real-time 3D structural and functional imaging of living biological samples during and after irradiation. We demonstrate dynamic imaging of the acute effects of irradiation on 3D cultures of U87 human glioblastoma cells, revealing characteristic changes in cellular movement and intracellular calcium signaling following ionizing irradiation.

Influence of Simulated Microgravity on Mammary Epithelial Cells Grown as 2D and 3D Cultures.

During space travel, astronauts will experience a unique environment that includes continuous exposure to microgravity and stressful living conditions. Physiological adaptation to this is a challenge and the effect of microgravity on organ development, architecture, and function is not well understood. How microgravity may impact the growth and development of an organ is an important issue, especially as space flight becomes more commonplace. In this work, we sought to address fundamental questions regarding microgravity using mouse mammary epithelial cells in 2D and 3D tissue cultures exposed to simulated microgravity. Mouse mammary HC11 cells contain a higher proportion of stem cells and were also used to investigate how simulated microgravity may impact mammary stem cell populations. In these studies, we exposed mouse mammary epithelial cells to simulated microgravity in 2D and then assayed for changes in cellular characteristics and damage levels. The microgravity treated cells were also cultured in 3D to form acini structures to define if simulated microgravity affects the cells' ability to organize correctly, a quality that is of key importance for mammary organ development. These studies identify changes occurring during exposure to microgravity that impact cellular characteristics such as cell size, cell cycle profiles, and levels of DNA damage. In addition, changes in the percentage of cells revealing various stem cell profiles were observed following simulated microgravity exposure. In summary, this work suggests microgravity may cause aberrant changes in mammary epithelial cells that lead to an increase in cancer risk.

Looking on the horizon; potential and unique approaches to developing radiation countermeasures for deep space travel.

Future lunar missions and beyond will require new and innovative approaches to radiation countermeasures. The Translational Research Institute for Space Health (TRISH) is focused on identifying and supporting unique approaches to reduce risks to human health and performance on future missions beyond low Earth orbit. This paper will describe three funded and complementary avenues for reducing the risk to humans from radiation exposure experienced in deep space. The first focus is on identifying new therapeutic targets to reduce the damaging effects of radiation by focusing on high throughput genetic screens in accessible, sometimes called lower, organism models. The second focus is to design innovative approaches for countermeasure development with special attention to nucleotide-based methodologies that may constitute a more agile way to design therapeutics. The final focus is to develop new and innovative ways to test radiation countermeasures in a human model system. While animal studies continue to be beneficial in the study of space radiation, they can have imperfect translation to humans. The use of three-dimensional (3D) complex in vitro models is a promising approach to aid the development of new countermeasures and personalized assessments of radiation risks. These three distinct and unique approaches complement traditional space radiation efforts and should provide future space explorers with more options to safeguard their short and long-term health.

Inhibition of autophagic flux differently modulates cannabidiol-induced death in 2D and 3D glioblastoma cell cultures.

Radiotherapy combined with chemotherapy is the major treatment modality for human glioblastoma multiforme (GBM). GBMs eventually relapse after treatment and the average survival of GBM patients is less than two years. There is some evidence that cannabidiol (CBD) can induce cell death and increases the radiosensitivity of GBM by enhancing apoptosis. Beside initiation of death, CBD has been demonstrated as an inducer of autophagy. In the present study, we address the question whether CBD simultaneously induces a protective effect in GBM by upregulating autophagy. Addition of chloroquine that suppressed autophagic flux to 2D GBM cultures increased CBD-induced cell death, presenting proof for the protective autophagy. Blockage of autophagy upregulated radiation-induced cytotoxicity but only modestly affected the levels of cell death in CBD- or CBD/γ-irradiated 3D GBM cultures. Furthermore, CBD enhanced the pro-apoptotic activities of JNK1/2 and MAPK p38 signaling cascades while partially downregulated the pro-survival PI3K-AKT cascade, thereby changing a balance between cell death and survival. Suppression of JNK activation partially reduced CBD-induced cell death in 3D GBM cultures. In contrast, co-treatment of CBD-targeted cells with inhibitors of PI3K-AKT-NF-κB, IKK-NF-κB or JAK2-STAT3 pathways killed surviving GBM cells in both 2D and 3D cultures, potentially improving the therapeutic ratio of GBM.

High-Dose Radiation Increases Notch1 in Tumor Vasculature.

PURPOSE: The aim of this study is to characterize the effects of high-dose radiation therapy (HDRT) on Notch signaling components of the tumor vasculature. METHODS AND MATERIALS: Human umbilical vein endothelial cells monolayers were exposed to different single fraction doses of irradiation; ribonucleic acid RNA was isolated and polymerase chain reaction was performed for Notch signaling components. The vascular response to radiation therapy was examined in a xenograft model of neuroblastoma. Tumors were treated with 0 Gy, 2 Gy, and 12 Gy single fraction doses and analyzed by double immunofluorescence staining for Notch1, Notch ligands Jagged1 and Dll4, and the endothelial cell (EC) marker endomucin. To assess the role of Notch in vivo, NGP xenograft tumors expressing Fc or Notch1-1-24-decoy (a novel Notch inhibitor) were treated with 0 Gy and 12 Gy. Immunofluorescence staining for endomucin and endomucin/αSMA was performed to analyze the effect of combination treatment on tumor EC and endothelial-to-mesenchymal-transition (EndMT), respectively. RESULTS: In human umbilical vein endothelial cells monolayers doses ≥8 Gy increased expression of NOTCH1, JAG1, and Notch target genes HEY1 and HEY2 as early as 6 hours after irradiation. In vivo, 12 Gy significantly increased Notch1 and Jagged1 in tumor ECs compared with 0 Gy or 2 Gy after 72 hours. Combining HDRT with Notch inhibition using the Notch1-1-24-decoy resulted in a greater loss of EC coverage of tumor vessels than HDRT alone at 6 hours and 72 hours post treatment. Notch inhibition reduced EndMT induced by HDRT, as indicated by diminished αSMA staining in ECs. CONCLUSIONS: HDRT induced Notch1 expression and increased Notch1 signaling in the endothelial component of tumor vasculature, which was not observed with lower doses. This increase in Notch1 activation might protect tumor vessels from HDRT induced damage and regulate EndMT process.

GeneLab Database Analyses Suggest Long-Term Impact of Space Radiation on the Cardiovascular System by the Activation of FYN Through Reactive Oxygen Species.

Space radiation has recently been considered a risk factor for astronauts' cardiac health. As an example, for the case of how to query and identify datasets within NASA's GeneLab database and demonstrate the database utility, we used an unbiased systems biology method for identifying key genes/drivers for the contribution of space radiation on the cardiovascular system. This knowledge can contribute to designing appropriate experiments targeting these specific pathways. Microarray data from cardiomyocytes of male C57BL/6 mice followed-up for 28 days after exposure to 900 mGy of 1 GeV proton or 150 mGy of 1 GeV/n 56Fe were compared to human endothelial cells (HUVECs) cultured for 7 days on the International Space Station (ISS). We observed common molecular pathways between simulated space radiation and HUVECs flown on the ISS. The analysis suggests FYN is the central driver/hub for the cardiovascular response to space radiation: the known oxidative stress induced immediately following radiation would only be transient and would upregulate FYN, which in turn would reduce reactive oxygen species (ROS) levels, protecting the cardiovascular system. The transcriptomic signature of exposure to protons was also much closer to the spaceflight signature than 56Fe's signature. To our knowledge, this is the first time GeneLab datasets were utilized to provide potential biological indications that the majority of ions on the ISS are protons, clearly illustrating the power of omics analysis. More generally, this work also demonstrates how to combine animal radiation studies done on the ground and spaceflight studies to evaluate human risk in space.

Short-Term Effects of Low-LET Radiation on the Endothelial Barrier: Uncoupling of PECAM-1 and the Production of Endothelial Microparticles.

A significant target for radiation-induced effects is the microvascular system, which is critical to healthy tissue function and its pathology is linked to disrupted endothelial barrier function. Low-linear energy transfer (LET) ionizing radiation is a source of noncancer pathologies in humans and little is known about the early events that could initiate subsequent diseases. However, it is well known that gamma radiation causes a very early disruption of the endothelial barrier at doses below those required for cytotoxic effects. After irradiation of human umbilical vein endothelial cells (HUVECs) to doses as low as 2 Gy, transendothelial electrical resistance (TEER) is transiently reduced at 3 h, and the platelet-derived endothothelial cell adhesion molecule (PECAM-1 or CD31) is uncoupled from the cells along with the release of endothelial microparticles (EMPs). In this study, we measured TEER reduction as an indicator of barrier function loss, and specifically examined the shedding of EMPs from human endothelial barrier models after a variety of low-LET irradiations, including photons and charged particles. Our findings showed two TEER responses, dependent on radiation type and environmental conditions. The first response was diminishing oscillations of TEER, which occurred during the first 10 h postirradiation. This response occurred after a 5 Gy proton or helium-ion (1 GeV/n) dose in addition to a 5 Gy gamma or X radiation dose. This occurred only in the presence of multiple growth factors and did not show a dose response, nor was it associated with EMP release. The second response was a single acute drop in TEER at 3 h after photon irradiation. Dose response was observed and was associated with the shedding of EMPs in 2D barrier cultures and in 3D vessel models. In this case, helium-ion and proton irradiations did not induce a drop in TEER or shedding of EMPs. The photon radiation effects was observed both in serum-free media and in the presence of multiple growth factors, indicating that it occurs under a range of environmental conditions. These results show an acute response of the human endothelial barrier that is relevant to photon irradiation. Significantly, it involves the release of EMPs, which have recently attracted attention due to their emerging clinical importance.

Reprogramming mediated radio-resistance of 3D-grown cancer cells.

In vitro 3D growth of tumors is a new cell culture model that more closely mimics the features of the in vivo environment and is being used increasingly in the field of biological and medical research. It has been demonstrated that cancer cells cultured in 3D matrices are more radio-resistant compared with cells in monolayers. However, the mechanisms causing this difference remain unclear. Here we show that cancer cells cultured in a 3D microenvironment demonstrated an increase in cells with stem cell properties. This was confirmed by the finding that cells in 3D cultures upregulated the gene and protein expression of the stem cell reprogramming factors such as OCT4, SOX2, NANOG, LIN28 and miR-302a, compared with cells in monolayers. Moreover, the expression of ß-catenin, a regulating molecule of reprogramming factors, also increased in 3D-grown cancer cells. These findings suggest that cancer cells were reprogrammed to become stem cell-like cancer cells in a 3D growth culture microenvironment. Since cancer stem cell-like cells demonstrate an increased radio-resistance and chemo-resistance, our results offer a new perspective as to why. Our findings shed new light on understanding the features of the 3D growth cell model and its application in basic research into clinical radiotherapy and medicine.

The effects of radiation on angiogenesis.

The average human body contains tens of thousands of miles of vessels that permeate every tissue down to the microscopic level. This makes the human vasculature a prime target for an agent like radiation that originates from a source and passes through the body. Exposure to radiation released during nuclear accidents and explosions, or during cancer radiotherapy, is well known to cause vascular pathologies because of the ionizing effects of electromagnetic radiations (photons) such as gamma rays. There is however, another type of less well-known radiation - charged ion particles, and these atoms stripped of electrons, have different physical properties to the photons of electromagnetic radiation. They are either found in space or created on earth by particle collider facilities, and are of significant recent interest due to their enhanced effectiveness and increasing use in cancer radiotherapy, as well as a health risk to the growing number of people spending time in the space environment. Although there is to date, relatively few studies on the effects of charged particles on the vascular system, a very different picture of the biological effects of these particles compared to photons is beginning to emerge. These under researched biological effects of ion particles have a large impact on the health consequences of exposure. In this short review, we will discuss the effects of charged particles on an important biological process of the vascular system, angiogenesis, which creates and maintains the vasculature and is highly important in tumor vasculogenesis.

Two distinct types of the inhibition of vasculogenesis by different species of charged particles.

BACKGROUND: Charged particle radiation is known to be more biologically effective than photon radiation. One example of this is the inhibition of the formation of human blood vessels. This effect is an important factor influencing human health and is relevant to space travel as well as to cancer radiotherapy. We have previously shown that ion particles with a high energy deposition, or linear energy transfer (LET) are more than four times more effective at disrupting mature vessel tissue models than particles with a lower LET. For vasculogenesis however, the relative biological effectiveness between particles is the same. This unexpected result prompted us to investigate whether the inhibition of vasculogenesis was occurring by distinct mechanisms. METHODS: Using 3-Dimensional human vessel models, we developed assays that determine at what stage angiogenesis is inhibited. Vessel morphology, the presence of motile tip structures, and changes in the matrix architecture were assessed. To confirm that the mechanisms are distinct, stimulation of Protein Kinase C (PKC) with phorbol ester (PMA) was employed to selectively restore vessel formation in cultures where early motile tip activity was inhibited. RESULTS: Endothelial cells in 3-D culture exposed to low LET protons failed to make connections with other cells but eventually developed a central lumen. Conversely, cells exposed to high LET Fe charged particles extended cellular processes and made connections to other cells but did not develop a central lumen. The microtubule and actin cytoskeletons indicated that motility at the extending tips of endothelial cells is inhibited by low LET but not high LET particles. Actin-rich protrusive structures that contain bundled microtubules showed a 65% decrease when exposed to low LET particles but not high LET particles, with commensurate changes in the matrix architecture. Stimulation of PKC with PMA restored tip motility and capillary formation in low but not high LET particle treated cultures. CONCLUSION: Low LET charged particles inhibit the early stages of vasculogenesis when tip cells have motile protrusive structures and are creating pioneer guidance tunnels through the matrix. High LET charged particles do not affect the early stages of vasculogenesis but they do affect the later stages when the endothelial cells migrate to form tubes.

Short term effects of gamma radiation on endothelial barrier function: uncoupling of PECAM-1.

A limiting factor in the treatment of cancer with radiotherapy is the damage to surrounding normal tissue, particularly the vasculature. Vessel pathologies are a major feature of the side effects of radiotherapy and little is known about early events that could initiate subsequent diseases. We tested the hypothesis that gamma radiation has early damaging effects on the human endothelial barrier. Two models were used; Human Brain Microcapillary Endothelial Cells (HBMEC), and Human Umbilical Vein Endothelial Cells (HUVEC). Endpoints included Trans-Endothelial Electrical Resistance (TEER), barrier permeability to 10 kDa and 70 kDa tracer molecules, and the localization of F-actin, and junction proteins and the Platelet Endothelial Cell Adhesion Molecule (PECAM-1). Radiation induced a rapid and transient decrease in TEER at 3 h, with effects also seen at the radiotherapy doses. This dip in resistance correlated to the transient loss of PECAM-1 in discrete areas where cells often detached from the monolayer leaving gaps. Redistribution of PECAM-1 was also seen in 3-D human tissue models. By 6 h, the remaining cells had migrated to reseal the barrier, coincident with TEER returning to control levels. Resealed monolayers contained fewer cells per unit area and their barrier function was weakened as evidenced by an increased permeability over 24 h. This is the first demonstration of a transient and rapid effect of gamma radiation on human endothelial barriers that involves cell detachment and the loss of PECAM-1. Considering the association of cell adhesion molecules with vasculopathies, such an effect has the potential to be clinically relevant to the longer-term effects of radiotherapy.

DNA damage foci formation and decline in two-dimensional monolayers and in three-dimensional human vessel models: differential effects according to radiation quality.

PURPOSE: To analyze the effect of different radiation qualities on the kinetics of p53 Binding Protein 1 (53BP1) formation and decline in human three-dimensional (3-D) vessel models. MATERIAL AND METHODS: Two-dimensional (2-D) and 3-D cultures of human umbilical vein cells were exposed to 80 cGy of Gamma radiation and high-energy protons and Fe ions. 53BP1 antibodies were used for foci visualization via immunocytochemistry. Computer analysis was used to determine the number and the size of foci up to 48 hours after irradiation. RESULTS: DNA foci kinetics in 2-D and 3-D human vessel cultures show that foci formation and removal were the same in each type of culture. After 48 h, the number of foci induced by high-energy protons and gamma rays reduced to almost control levels while high linear energy transfer (LET) Fe particles produced more persistent damage. CONCLUSION: The kinetics of radiation-induced 53BP1 foci in 3-D vessel models is essentially the same as in 2-D monolayers. Since the basal level of spontaneous foci is low in these differentiated non-proliferating cultures, the persistence of radiation-induced 53BP1 foci is detected longer than previously noted. Furthermore, analysis of foci sizes revealed that abnormal radiation-induced foci can persist even when foci frequencies are close to basal levels. The detection of these latent abnormalities could be useful for a more sensitive dosimetry.

Effects of ionizing radiation on three-dimensional human vessel models: differential effects according to radiation quality and cellular development.

Little is known about the effects of space radiation on the human body. There are a number of potential chronic and acute effects, and one major target for noncarcinogenic effects is the human vasculature. Cellular stress, inflammatory response, and other radiation effects on endothelial cells may affect vascular function. This study was aimed at understanding the effects of space ionizing radiation on the formation and maintenance of capillary-like blood vessels. We used a 3D human vessel model created with human endothelial cells in a gel matrix to assess the effects of low-LET protons and high-LET iron ions. Iron ions were more damaging and caused significant reduction in the length of intact vessels in both developing and mature vessels at a dose of 80 cGy. Protons had no effect on mature vessels up to a dose of 3.2 Gy but did inhibit vessel formation at 80 cGy. Comparison with γ radiation showed that photons had even less effect, although, as with protons, developing vessels were more sensitive. Apoptosis assays showed that inhibition of vessel development or deterioration of mature vessels was not due to cell death by apoptosis even in the case of iron ions. These are the first data to show the effects of radiation with varying linear energy transfer on a human vessel model.

Cytoplasmic dynein and LIS1 are required for microtubule advance during growth cone remodeling and fast axonal outgrowth.

Recent evidence has implicated dynein and its regulatory factors dynactin and LIS1 in neuronal and non-neuronal cell migration. In the current study we sought to test whether effects on neuronal cell motility might reflect, in part, a role for these proteins in the growth cone. In chick sensory neurons subjected to acute laminin treatment dynein, dynactin, and LIS1 were mobilized strikingly and rapidly to the leading edge of the growth cone, where they were seen to be associated with microtubules converging into the laminin-induced axonal outgrowths. To interfere acutely with LIS1 and dynein function and to minimize secondary phenotypic effects, we injected antibodies to these proteins just before axon initiation. Antibody to both proteins produced an almost complete block of laminin-induced growth cone remodeling and the underlying reorganization of microtubules. Penetration of microtubules into the peripheral zone of differentiating axonal growth cones was decreased dramatically by antibody injection, as judged by live analysis of enhanced green fluorescent protein-tubulin and the microtubule tip-associated EB3 (end-binding protein 3). Dynein and LIS1 inhibition had no detectable effect on microtubule assembly but reduced the ability of microtubules to resist retrograde actin flow. In hippocampal neurons dynein, dynactin, and LIS1 were enriched in axonal growth cones at stage 3, and both growth cone organization and axon elongation were altered by LIS1 RNA interference. Together, our data indicate that dynein and LIS1 play a surprisingly prominent role in microtubule advance during growth cone remodeling associated with axonogenesis. These data may explain, in part, the role of these proteins in brain developmental disease and support an important role in diverse aspects of neuronal differentiation and nervous system development.