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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.

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.

From our previous work we have shown a key miRNA signature that is associated with spaceflight can be used as a biomarker and countermeasure to mitigate the damage caused by space radiation. Here, we have further expanded on this work to determine key biological factors that are being rescued by the countermeasure treatment. We performed RNA-sequencing and transcriptomic analysis on our established 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 (i.e. antagomirs). Significant reduction of inflammation and DNA DSBs activity is observed, along with key mitochondria functions being rescued after antagomir treatment. Lastly, we compared the key genes and pathways involved with the antagomirs with astronaut data from Inspiration4 and JAXA missions to demonstrate that the key genes and pathways associated with these experiments occur in humans and this countermeasure strategy can potentially be utilized in astronauts to mitigate the space radiation response.

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., Summary High-dose radiation (HDRT) not only has a direct cytotoxic effect on tumor cells but can also effect the tumor vasculature. Our study examined HDRT and Notch signaling in a xenograft model of neuroblastoma. We observed an increased Notch signaling in endothelial cells after HDRT. Notch1 inhibition augments the effect of HDRT on endothelial cell loss and reduces radiation-induced endothelial-to-mesenchymal transition.

MicroRNAs (miRNAs) are small non-coding RNAs involved in post-transcriptional gene regulation that have a major impact on many diseases and provide an exciting avenue toward antiviral therapeutics. From patient transcriptomic data, we determined that a circulating miRNA, miR-2392, is directly involved with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) machinery during host infection. Specifically, we show that miR-2392 is key in driving downstream suppression of mitochondrial gene expression, increasing inflammation, glycolysis, and hypoxia, as well as promoting many symptoms associated with coronavirus disease 2019 (COVID-19) infection. We demonstrate that miR-2392 is present in the blood and urine of patients positive for COVID-19 but is not present in patients negative for COVID-19. These findings indicate the potential for developing a minimally invasive COVID-19 detection method. Lastly, using in vitro human and in vivo hamster models, we design a miRNA-based antiviral therapeutic that targets miR-2392, significantly reduces SARS-CoV-2 viability in hamsters, and may potentially inhibit a COVID-19 disease state in humans., Graphical abstract, McDonald et al. uncover a role of a circulating microRNA, miR-2392, as a potential biomarker for COVID-19 and begin development of a potential COVID-19 therapeutic and antiviral to inhibit miR-2392.

Summary Space radiation inhibits angiogenesis by two mechanisms depending on the linear energy transfer (LET). Using human 3D micro-vessel models, blockage of the early motile stage of angiogenesis was determined to occur after exposure to low LET ions (8 KeV/AMU). Strikingly, the combined effect is synergistic, detectible as low as 0.06 Gy making mixed ion space radiation more potent. Candidates for bystander transmission are microRNAs (miRNAs), and analysis on miRNA-seq data from irradiated mice shows that angiogenesis would in theory be downregulated. Further analysis of three previously identified miRNAs showed downregulation of their targets associated with angiogenesis and confirmed their involvement in angiogenesis pathways and increased health risks associated with cardiovascular disease. Finally, synthetic molecules (antagomirs) designed to inhibit the predicted miRNAs were successfully used to reverse the inhibition of angiogenesis., Graphical Abstract, Highlights • Space radiation inhibits angiogenesis synergistically at low doses by 2 mechanisms • Candidates for bystander transmission are microRNAs • Three previously identified miRNAs showed downregulation of their angiogenesis targets • Synthetic miRNA inhibitors were used to reverse the inhibition of angiogenesis, Radiation Biology; Molecular Biology; Space Sciences

Research on astronaut health and model organisms have revealed six features of spaceflight biology that guide our current understanding of fundamental molecular changes that occur during space travel. The features include oxidative stress, DNA damage, mitochondrial dysregulation, epigenetic changes (including gene regulation), telomere length alterations, and microbiome shifts. Here we review the known hazards of human spaceflight, how spaceflight affects living systems through these six fundamental features, and the associated health risks of space exploration. We also discuss the essential issues related to the health and safety of astronauts involved in future missions, especially planned long-duration and Martian missions.

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.

We have identified and validated a spaceflight-associated microRNA (miRNA) signature that is shared by rodents and humans in response to simulated and real spaceflight and regulates vascular damage by simulated deep space radiation. In previous studies, we identified miRNAs that are predicted to regulate rodent responses to spaceflight in low-Earth orbit. Here we have validated the expression of key spaceflight-associated miRNAs in rodents reacting to simulated spaceflight conditions (exposure to ionizing radiation combined with simulated microgravity) and in astronaut samples from the NASA Twin Study. To demonstrate their physiological relevance, we have utilized antagomirs to target a subset of miRNAs from the spaceflight associated signature to successfully rescue simulated deep space radiation-mediated damage in human 3D vascular constructs.

Space Radiation inhibits angiogenesis by two mechanisms depending on the linear energy transfer (LET). Using human 3D micro-vessel models, blockage of the early motile stage of angiogenesis was determined to occur after exposure to low LET ions ( 8 KeV/AMU). Strikingly, the combined effect is synergistic, detectible as low as 0.6 cGy making mixed ion Space Radiation more potent. Candidates for bystander transmission are microRNAs (miRNAs) and analysis on miRNA-seq data from irradiated mice shows that angiogenesis would in theory, be downregulated. Further analysis of three previously identified miRNAs showed downregulation of their targets associated with angiogenesis and confirmed their involvement in angiogenesis pathways and increased health risks associated with cardiovascular disease. Finally, synthetic molecules (Antagomirs) designed to inhibit the predicted miRNAs were successfully used to reverse the inhibition of angiogenesis.

Spaceflight missions can cause immune system dysfunction in astronauts with little understanding of immune outcomes in deep space. This study assessed immune responses following ground-based, simulated deep spaceflight conditions, compared to data from astronauts on ISS missions. For ground studies, we simulated microgravity using the hindlimb unloaded mouse model alone or in combination with acute simulated galactic cosmic rays or solar particle events irradiation. Immune profiling results revealed unique immune diversity following each experimental condition, suggesting each stressor results in distinct circulating immune responses, with clear consequences for deep spaceflight. Circulating plasma microRNA sequence analysis revealed involvment in immune system dysregulation. Furthermore, a large astronaut cohort showed elevated inflammation during LEO missions, thereby supporting our simulated ground experiments. Herein, circulating immune biomarkers are defined by distinct deep space irradiation types coupled to simulated microgravity and could be targets for future space health initiatives.

Space radiation has recently been considered a risk factor for astronauts&rsquo, cardiac health. As an example, for the case of how to query and identify datasets within NASA&rsquo, 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&rsquo, 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.

Spaceflight can cause immune system dysfunction in astronauts with little understanding of immune outcomes in deep space. This study assessed immune responses in mice following simulated deep spaceflight, along with retrospective data from astronauts on the International Space Station (ISS). We simulated microgravity using the hindlimb unloaded mouse model alone or in combination with the newly developed simulated galactic cosmic rays or solar particle events irradiation at NASA’s Space Radiation Laboratory. Immune differentials revealed unique immune diversity following each experimental platform, suggesting consequences for deep spaceflight. Furthermore, circulating plasma microRNA sequencing analysis revealed immune system dysregulation. An ISS astronaut cohort showed elevated inflammation during low-Earth orbit missions, which supports our simulated ground experiments. Herein, distinct circulating immune biomarkers are defined by irradiation type, coupled to simulated microgravity, which can be promising targets for countermeasure developments and programmatic queries.

SUMMARY We have identified and validated a spaceflight-associated microRNA (miRNA) signature that is shared by rodents and humans in response to simulated, short-duration and long-duration spaceflight. Previous studies have identified miRNAs that regulate rodent responses to spaceflight in low-Earth orbit, and we have confirmed the expression of these proposed spaceflight-associated miRNAs in rodents reacting to simulated spaceflight conditions. Moreover, astronaut samples from the NASA Twins Study confirmed these expression signatures in miRNA sequencing, single-cell RNA sequencing (scRNA-seq), and single-cell assay for transposase accessible chromatin (scATAC-seq) data. Additionally, a subset of these miRNAs (miR-125, miR-16, and let-7a) was found to regulate vascular damage caused by simulated deep space radiation. To demonstrate the physiological relevance of key spaceflight-associated miRNAs, we utilized antagomirs to inhibit their expression and successfully rescue simulated deep-space-radiation-mediated damage in human 3D vascular constructs., Graphical Abstract, In Brief Malkani et al. uncover the role of circulating microRNAs as both a potential biomarker for health risks associated with spaceflight and a countermeasure to mitigate the damage caused to the body by the space environment.

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 β-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.

Space travel involves exposure to biologically effective heavy ion radiation and there is consequently a concern for possible degenerative disorders in humans. A significant target for radiation effects is the microvascular system, which is crucial to healthy functioning of the tissues. Its pathology is linked to disrupted endothelial barrier function and is not only a primary event in a range of degenerative diseases but also an important influencing factor in many others. Thus, an assessment of the effects of heavy ion radiation on endothelial barrier function would be useful for estimating the risks of space travel. This study was aimed at understanding the effects of high LET Fe particles (1 GeV/n) and is the first investigation of the effects of charged particles on the function of the human endothelial barrier. We used a set of established and novel endpoints to assess barrier function after exposure. These include, trans-endothelial electrical resistance (TEER), morphological effects, localization of adhesion and cell junction proteins (in 2D monolayers and in 3D tissue models), and permeability of molecules through the endothelial barrier. A dose of 0.50 Gy was sufficient to cause a progressive reduction in TEER measurements that were significant 48 hours after exposure. Concurrently, there were morphological changes and a 14% loss of cells from monolayers. Gaps also appeared in the normally continuous cell-border localization of the tight junction protein – ZO-1 but not the Platelet endothelial cell adhesion molecule (PECAM-1) in both monolayers and in 3D vessel models. Disruption of barrier function was confirmed by increased permeability to 3 kDa and 10 kDa dextran molecules. A dose of 0.25 Gy caused no detectible change in cell number, morphology, or TEER, but did cause barrier disruption since there were gaps in the cell border localization of ZO-1 and an increased permeability to 3 kDa dextran. These results indicate that Fe particles potently have impact on human endothelial barrier function and represent a risk for degenerative diseases in the space environment.

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.

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.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.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.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.

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.

In Aplysia, the growth of axonal arbor and the formation of new presynaptic varicosities are thought to contribute to long-term facilitation (LTF) produced by serotonin (5-HT). While it is known that there is a requirement for both transcription and translation in LTF and in the accompanying morphological changes, the mechanisms mediating the initiation and maintenance of these changes are poorly understood. We used long-term labeling of the presynaptic sensory neuron to carry out repeated imaging of axonal morphology, coupled with electrophysiology, to further elucidate the macromolecular requirements of this process. Robust synaptic facilitation, axonal growth, and the formation of axonal varicosities were elicited by 5-HT even when transcription was blocked with actinomycin. Increases in synaptic efficacy and varicosity number were detected 12 h after exposure to 5-HT but did not persist to 24 h. Even when sensory neuron cell bodies were removed, eliminating the contributions of both somal transcription and translation, 5-HT elicited these transient morphological and electrophysiological responses. New sensory varicosities contacting the postsynaptic neuron were filled with the neuropeptide sensorin. Under all conditions, global inhibition of protein synthesis completely blocked the formation of new axonal branches and varicosities. These results demonstrate that neither transcription nor somal translation is required to initiate the axonal growth that often accompanies long-term synaptic plasticity-protein synthesis in the axon is sufficient. Macromolecular synthesis in the cell body is, however, required to maintain the enlarged arbor.

The growth of axons and dendrites during development and regeneration is regulated by cues in the environment. Many of these cues regulate the actin cytoskeleton of the protrusive structures (like filopodia) of the growth cone that are essential for detecting and responding to cues. Nerve growth factor, which promotes the formation of protrusive structures, stimulated actin polymerization in rat sympathetic growth cones, resulting within 1–2 min in accumulations of F-actin at the distal edge and in splotches of F-actin farther back. We examined the potential involvement of a protein machinery important in at least certain types of actin polymerization in non-neuronal cells. Members of the Arp2/3 complex, p34-Arc and p21-Arc, heavily concentrated in the early accumulations of F-actin, as did one member of the Ena/VASP family (Mena) but not another (VASP). Retention of Arc proteins at preferred sites of actin polymerization did not require polymerization itself. Growth cones of differentiated PC12 cells were similar to sympathetic growth cones in their response to NGF. Introduction into these cells of a peptide that should block the binding of Ena/VASP family proteins to the protein complex at sites of actin polymerization reduced the formation of splotches and filopodia in response to NGF. These results point to the early involvement of the Arp2/3 complex and the Ena/VASP family in growth factor-stimulated actin polymerization that gives rise to protrusive structures at the growth cone. J. Neurosci. Res. 4:458–467, 2000 © 2000 Wiley-Liss, Inc.

Addition of nerve growth factor (NGF) to sympathetic neurons that have been starved of it causes a rapid induction of growth cone motility and the resumption of neurite growth. Using immunofluorescence staining, we show that within 10 min, NGF stimulated the accumulation of dense aggregates of β1 integrin [a receptor for extracellular matrix (ECM) proteins] at most of the tips of either newly extended or preexisting filopodia. This effect occurred in the absence of ECM proteins and in the presence of 1 mg/ml Arg-Gly-Asp-Ser peptide, which blocks ECM binding to integrin, indicating that occupation of the integrin receptor is not necessary for tip localization. In fact, addition of either laminin or fibronectin caused a rapid withdrawal of β1 integrin aggregates from filopodial tips at a rate comparable to that of the rearward flow of actin filaments in the periphery of the growth cone. Surface labeling of the extracellular domain of β1 integrin while aggregated at the tips of filopodia or withdrawing in response to ECM proteins showed that the receptor is positioned within the membrane. The drug butanedione monoxime, an inhibitor of myosins, blocked the accumulation of β1 integrin at the tips of filopodia without inhibiting the formation of filo-podia, suggesting the involvement of a myosin motor in β1 integrin transport. These results provide the first evidence of NGF-mediated accumulation of ECM receptors to sensory elements of the growth cone and suggest one mechanism whereby soluble and substrate-bound cues coordinate to produce directed neurite growth.

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.

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.

Exposure to ionizing radiation may induce a heritable genomic instability phenotype that results in a persisting and enhanced genetic and functional change among the progeny of irradiated cells. Since radiation-induced bystander effects have been demonstrated with a variety of biological end points under both in vitro and in vivo conditions, this raises the question whether cytoplasmic irradiation or the radiation-induced bystander effect can also lead to delayed genomic instability. In the present study, we used the Radiological Research Accelerator Facility charged-particle microbeam for precise nuclear or cytoplasmic irradiation. The progeny of irradiated and the bystander human hamster hybrid (A(L)) cells were analyzed using multicolor banding (mBAND) to examine persistent chromosomal changes. Our results showed that the numbers of metaphase cells involving changes of human chromosome 11 (including rearrangement, deletion and duplication) were significantly higher than that of the control in the progeny of both nuclear and cytoplasmic targeted cells. These chromosomal changes could also be detected among the progeny of bystander cells. mBAND analyses of clonal isolates from nuclear and cytoplasm irradiations as well as the bystander cell group showed that chromosomal unstable clones were generated. Analyses of clonal stability after long-term culture indicated no significant change in the number of unstable clones for the duration of culture in each irradiated group. These results suggest that genomic instability that is manifested after ionizing radiation exposure is not dependent on direct damage to the cell nucleus.

The human vasculature is critical to healthy functioning of the tissues of the body and a major factor in maintaining homeostasis is the endothelial barrier. In the brain, the blood–brain barrier (BBB) is highly specialized in order to sustain the neural tissue. Here, we have examined the effects of radiation on BBB models using a unique variety of endpoints to assess barrier function. These include trans-endothelial electrical resistance (TEER), morphological effects, localization of adhesion and cell junction proteins (in two-dimensional monolayers and in three-dimensional tissue models) and permeability of molecules through the endothelial barrier. Two culture conditions were used to represent conditions on the inside or the lumen of vessels and conditions on the outside or ablumenal side of vessels. For the lumen, cells were cultured in serum and growth factor containing media, and for the ablumenal side of vessels, cells were cultured in serum-free defined media. Initial experiments with gamma rays in serum-free conditions revealed a previously unknown acute effect involving cell detachment and the loss of the clinically relevant cell adhesion molecule—cell platelet endothelial adhesion molecule (PECAM)-1 [ 1]. Gamma radiation (5 Gy) induced a rapid and transient decrease in TEER at 3 h, with effects also seen at the lower radiotherapy dose of 2 Gy. This dip in resistance correlated with the transient loss PECAM-1 in discrete areas where cells often detached from the monolayer leaving gaps. Loss in PECAM-1 occurred at least in part as detached microparticles. Redistribution of PECAM-1 microparticles was also seen in three-dimensional 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 corroborated by an increased permeability over 24 h. Because PECAM-1 is involved in barrier function and platelet aggregation, this effect is likely highly relevant to cancer radiotherapy using gamma rays. These studies were extended to include low linear energy transfer (LET) photons of X-rays and ion particles present in the space environment—low LET ion particles including high energy (1 GeV) protons and helium ions. X-rays under serum-free conditions also showed an acute response involving a dip in TEER at 3 h and the loss of PECAM-1 between cells as microparticles. Ion particles, however, did not show these effects of photons under serum-free conditions. Both protons and helium ions at doses up to 5 Gy did not produce this transient change in TEER or PECAM-1, although some longer term effects in TEER were noted. In the presence of serum and growth factors, however, all radiations tested showed short-term effects in TEER, that of a series of symmetrical peaks which diminished in size over several hours. For 1 GeV protons and helium ions, this effect could be fitted to an equation for under-damped oscillation, a pattern typical of a mechanism for timing of events in periodic processes. For gamma and X-rays, the underdamped oscillation was present but superimposed on a drop in resistance at 3 h similar to that seen in serum-free conditions. In conclusion, we have shown two acute effects of low LET radiation on the human endothelial barrier. First, a short-term effect of photons but not ion particles involving a single dip in TEER and the loss of PECAM-1 at 3 h after irradiation, and second an underdamped oscillation of TEER induced by both photons and ion particles that to date does not appear to be associated with the loss of PECAM-1 or any other junction molecules.

Caveolin-3 the muscle-specific caveolin isoform, acts like the more ubiquitously expressed caveolin-1 to sculpt caveolae, specialized membrane microdomains that serve as platforms to organize signal transduction pathways. Caveolin-2 is a structurally related isoform that alone does not drive caveolae biogenesis; rather, caveolin-2 cooperates with caveolin-1 to form caveolae in nonmuscle cells. Although caveolin-2 might be expected to interact in an fashion analogous to that of caveolin-3, it generally has not been detected in cardiomyocytes. This study shows that caveolin-2 and caveolin-3 are detected at low levels in ventricular myocardium and increase dramatically with age or when neonatal cardiomyocytes are placed in culture. In contrast, flotillins (caveolin functional homologs) are expressed at relatively constant levels in these preparations. In neonatal cardiac cultures, caveolin-2 and -3 expression is not influenced by thyroid hormone (a postnatal regulator of other cardiac gene products). The further evidence that caveolin-2 coimmunoprecipitates with caveolin-3 and floats with caveolin-3 by isopycnic centrifugation in cardiomyocyte cultures suggests that caveolin-2 may play a role in caveolae biogenesis and influence cardiac muscle physiology.

The cycling of membrane receptors for substrate-bound proteins via their interaction with the actin cytoskeleton at the leading edge of growth cones and other motile cells is important for neurite outgrowth and cell migration. Receptor delivered to the leading edge binds to its ligand, which induces coupling of the receptor to a rearward flowing network of actin filaments. This coupling is thought to facilitate advance. We show here that a soluble growth factor stimulates this cycling. We have used single particle tracking to monitor the effects of nerve growth factor (NGF) on the movements of beta1 integrin in the plane of the plasma membrane of the filopodia of growth cones. Beta1 integrin was visualized by its binding of 0.2 microm beads coated with a monoclonal Ab directed against an extracellular epitope distant from the binding site for extracellular matrix ligands. The beads were observed by video microscopy. Beads coated with a low concentration of antibody, and therefore bound to unliganded receptor with little cross-linking, showed an increase in both diffusion and directed forward transport in response to NGF. Transport had a net velocity of 37 microm/minute and was characterized by brief periods of sustained forward excursions with a velocity of 75–150 microm/minute. There was a 2-fold increase in the number of beads accumulated at the tips of filopodia after 10 minutes, indicating that NGF enhanced the delivery of beta1 integrin to the periphery. Forward transport was dependent on an intact actin cytoskeleton and myosin ATPase, since treatment with cytochalasin D or the myosin ATPase inhibitor butanedione monoxime inhibited the transport but not the diffusion of receptors. NGF also greatly increased the steady rearward migration of beads coated with a high density of (β)1 integrin antibody, indicating that coupling of cross-linked receptor to the retrograde flow of actin is also enhanced. The rate of the retrograde flow of actin was unaffected by NGF. These studies show that a soluble factor can stimulate the coupling of a receptor for substrate-bound factor to two actomyosin-based transport mechanisms and thus facilitate the response of the growth cone to the substrate-bound factor by increasing cycling of the receptor at the periphery.

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.