NOTE: Extended to 9/30/2017 per PI and NSSC information (Ed., 7/22/16)

We have shown that space radiation inhibits angiogenesis and disrupts endothelial barrier function using human endothelial cells in 2- and 3-dimensional human tissue models. The doses and time course for radiation-induced events are now known which makes it possible to assay for joint effects with other environmental influences. Angiogenesis and barrier function are also affected by microgravity so there is a potential for further dysfunction of the human vasculature when applied in combination with radiation. Here, we propose a ground-based study using simulated microgravity to determine the combined effects of space radiation and microgravity on human blood vessel models and its impact on degeneration by testing for angiogenesis and endothelial barrier function using our established assays.

The development of 3-Dimensional human tissue models from normal human cells and stem cells has great potential in many fields of medical research. Tissue models can more accurately depict human tissue since the cells can be arranged spatially as they would be in vivo and can interact with each other as they would in the human body. A neurovascular unit can be used for basic research on many aspects of the human brain. These include regeneration, synaptic function, and degeneration. Because the tissue model is derived from individual cells, each cell type can be altered genetically before it is incorporated into the model. The effects of radiation combined with simulated microgravity can be of benefit to the health of astronauts.

A second set of experiments investigated the effects of simulated microgravity (SMG) on the entire process of capillary formation, from seeding of cells in the matrices to the final vessels structure after 7 days of culture. In this case there was a significant effect on capillary formation. We tested several types of rotation including vertical spins at 1, 5, and 10 rpm, a horizontal spin at 10 rpm, and 3D clinostat rotation at 10 rpm for each axis. There was a clear inhibition of vessel formation after all vertical rotations and the 3D rotation whereas vessel formation after horizontal rotation was unaffected and similar to the stationary control. There are 2 main conclusions from these data: 1) Since vertical rotation is as effective as 3D rotation, the data indicates that removal of the vector of gravity is sufficient to cause an inhibition of vessel formation. It is not necessary to distribute the vector of gravity globally to cause an effect, removal by vertical spin is sufficient. This suggests that a mechanism is held in place by the force of gravity on Earth and can be disrupted by rotation and cause the kinds of effects seen in the space environment. 2) Since a 1 rpm spin was as effective as faster rotations even though there is little turbulence at such a speed it is unlikely that turbulence is causing the inhibition. Further experiments showed that vessel development in disruptive spins was normal for up to 3 days but affected after this point in development when the vessels became more mature with widened. Examining the history of microvessel growth by looking at the collagen deposited by microvessels shows that microvessels complete maturity before collapsing.

Effects of microgravity on mature vessels: Human capillary models were first grown to maturity, then subjected to the same variety of rotations used in the angiogenesis studies. These models represent the capillaries already present in vivo. The effect of rotation on these cultures is similar to that on developing cultures, that is, all the vertical and the 3D rotations are effective at changing the final capillary structure. After 48 hours rotation there was a reduction in total vessel length although further studies are required to determine how this response translates in vivo. Morphological studies show that mature vessels collapsed. These studies and the angiogenesis studies do, however, prove that in isolation, human capillaries are responsive to alterations in gravity. Therefore, it highly likely that there is some kind of response in vivo.

Low dose inhibition of human angiogenesis by charged particles: LET (linear energy transfer) ranges and synergistic effect.

Since both space radiation and simulated microgravity are disruptive to human capillary models we expected that their combined effects would occur at lower doses of charged particle radiation. Previous data from several years ago indicated that heavy and light ions inhibit angiogenesis with a 50% effect at around 40 cGy for both types of radiation albeit via distinct mechanisms. In order to investigate the combined effect we carried out experiments with much lower doses than previously used with an improved assay. Surprisingly, we found that even without simulated microgravity the charged particles inhibited angiogenesis at much lower concentrations than previously detected. For angiogenesis: Light and heavy ions show distinct responses at different stages of angiogenesis and LET range studies showed that light ion effects occur with an LET of < 3 KeV/AMU and heavy ion effects occur with an LET of > 8 KeV/AMU or greater. Light ions caused significant inhibition at 1.25 cGy and Heavy ions coincidentally also caused significant inhibition at 1.25 cGy. These doses were considered to be much more relevant to those in space and because of the nature of mixed ion species in the space environment; experiments were carried out using mixed heavy and light ion beams at NASA Space Radiation Laboratory (NSRL). For angiogenesis, a surprising result was seen. A 1:1 ratio of light and heavy ions shows a synergistic effect that is significant at a dose of 0.3 cGy (0.15 cGy each of Fe ions 1GeV and protons 1GeV). This is the lowest known dose effect of space radiation at the cell and tissue level and represents a novel synergistic effect of heavy and light ions.

Experiments using mixed ions experiment have been repeated with different ions--Helium (1GeV) and Si ions (600 MeV) and a similar result confirms the synergistic response. This is considered an important unexpected finding and a manuscript is being prepared together with data on LET ranges from a previous grant award. Acknowledgements will be made to both grant awards. Combined effect of microgravity and space radiation on developing vessels: Since the LET range studies delineated the response to radiation into 2 groups we used both heavy ions and light ions in combination with SMG for these studies. Vessel models were exposed to radiation on day 1 after seeding the cells in 3D matrices then either grown to maturity while stationary or revolving vertically at 5 rpm. For all particle radiation (including 1 GeV He ions, 1 GeV protons, and 1 GeV Fe ions) combined with SMG there is an additive effect--the shape of the dose curve is the same but shifted lower in the presence of SMG. Thus, there is the potential for the combined effects of the space environment to cause more damage to micro-capillaries.

Combined effect of microgravity and space radiation on mature vessels: The data proves that simulated microgravity together with heavy ion radiation is at least additive in breaking down the structure of mature vessels. There was a distinct morphology when both agents are applied together. There are a number of vessels ending in dead ends and in fact very little tube structure left. Experiments with protons and Helium ions, which do not affect vessels up to 4 Gy, showed that microgravity acts alone in this case.

In conclusion we have shown that simulated microgravity alone has little effect on the early motile tip stage of angiogenesis but does have an effect on the entire process of capillary formation and is also effective on mature vessels. Both light and heavy ions are more effective than previously thought and can inhibit angiogenesis detectible at 1.25 cGy and that there is a synergistic response of mixed ions that brings the effective doses even lower (lowest significant effect with 0.15 cGy each ion). In mature vessel models, simulated microgravity has a profound effect of reducing full width vessel models at 1.25 cGy. Furthermore, the combined effect of microgravity is additive to that of charged particles making the space environment. Taken together the radiation and microgravity in the space environment has the potential to damage the microvasculature, which could cause pathologies such as CVD (cardiovascular disease) and neurodegeneration in astronauts.

Several manuscripts are in preparation.

NOTE: Extended to 9/30/2017 per PI and NSSC information (Ed., 7/22/16)

We have shown that space radiation inhibits angiogenesis and disrupts endothelial barrier function using human endothelial cells in 2- and 3-dimensional human tissue models. The doses and time course for radiation-induced events are now known which makes it possible to assay for joint effects with other environmental influences. Angiogenesis and barrier function are also affected by microgravity so there is a potential for further dysfunction of the human vasculature when applied in combination with radiation. Here, we propose a ground-based study using simulated microgravity to determine the combined effects of space radiation and microgravity on human blood vessel models and its impact on degeneration by testing for angiogenesis and endothelial barrier function using our established assays.

The development of 3-Dimensional human tissue models from normal human cells and stem cells has great potential in many fields of medical research. Tissue models can more accurately depict human tissue since the cells can be arranged spatially as they would be in vivo and can interact with each other as they would in the human body. A neurovascular unit can be used for basic research on many aspects of the human brain. These include regeneration, synaptic function, and degeneration. Because the tissue model is derived from individual cells, each cell type can be altered genetically before it is incorporated into the model. The effects of radiation combined with simulated microgravity can be of benefit to the health of astronauts.

Task progress

Experiments addressing the effect of simulated microgravity alone on the development of human capillaries (angiogenesis) and the effects on mature capillary models are now completed. This material is being prepared as a manuscript for submission to the new Journal – npgMicrogravity. Both angiogenesis (capillary formation) and the effect on mature vessels were examined.

Effects of microgravity on developing microvessels

We first tested the effect of a 3D clinostat spin on the activity of motile tips – the initial structures that facilitate the extension of pioneer tunnels, which later become capillary tubes. The results showed little effect--only a slight increase in the number of motile tips in vertical and 3D rotations.

A second set of experiments investigated the effects of simulated microgravity (SMG) on the entire process of capillary formation, from seeding of cells in the matrices to the final vessels structure after 7 days of culture. In this case there was a significant effect on capillary formation. We tested several types of rotation including vertical spins at 1, 5, and 10 rpm, a horizontal spin at 10 rpm and 3D clinostat rotation at 10 rpm for each axis. There was a clear inhibition of vessel formation after all vertical rotations and the 3D rotation whereas vessel formation after horizontal rotation was unaffected and similar to the stationary control. There are 2 main conclusions from these data. 1) Since vertical rotation is as effective as 3D rotation, the data indicates that removal of the vector of gravity is sufficient to cause an inhibition of vessel formation. It is not necessary to distribute the vector of gravity globally to cause an effect; removal by vertical spin is sufficient. This suggests that a mechanism is held in place by the force of gravity on Earth can be disrupted by rotation and cause the kinds of effects seen in the space environment. 2) Since a 1 rpm spin was as effective as faster rotations even though there is little turbulence at such a speed it is unlikely that turbulence is causing the inhibition. Further experiments showed that vessel development in disruptive spins was normal for up to 3 days but affected after this point in development when the vessels became more mature with widened. Examining the history of microvessel growth by looking at the collagen deposited by microvessels shows that microvessels complete maturity before collapsing.

Effects of microgravity on mature vessels

Human capillary models were first grown to maturity and then subjected to the same variety of rotations used in the angiogenesis studies. These models represent the capillaries already present in vivo. The effect of rotation on these cultures is similar to that on developing cultures, that is, all the vertical and the 3D rotations are effective at changing the final capillary structure. After 48 hours rotation there was a reduction in total vessel length although further studies are required to determine how this response translates in vivo. Morphological studies show that mature vessels collapsed. These studies and the angiogenesis studies do, however, prove that in isolation, human capillaries are responsive to alterations in gravity. Therefore, it highly likely that there is some kind of response in vivo.

Low dose inhibition of human angiogenesis by charged particles: LET (linear energy transfer) ranges and synergistic effect

Since both space radiation and simulated microgravity are disruptive to human capillary models we expected that their combined effects would occur at lower doses of charged particle radiation. Previous data from several years ago indicated that heavy and light ions inhibit angiogenesis with a 50% effect at around 40 cGy for both types of radiation albeit via distinct mechanisms. In order to investigate the combined effect we carried out experiments with much lower doses than previously used with an improved assay. Surprisingly, we found that even without simulated microgravity the charged particles inhibited angiogenesis at much lower concentrations than previously detected.

For angiogenesis: Light and heavy ions show distinct responses at different stages of angiogenesis and LET range studies showed that light ion effects occur with an LET of < 3 KeV/AMU and heavy ion effects occur with an LET of > 8 KeV/AMU or greater. Light ions caused significant inhibition at 1.25 cGy and Heavy ions coincidentally also caused significant inhibition at 1.25 cGy. These doses were considered to be much more relevant to those in space and because of the nature of mixed ion species in the space environment, experiments were carried out using mixed heavy and light ion beams at NASA Space Radiation Laboratory (NSRL). For angiogenesis, a surprising result was seen. A 1:1 ratio of light and heavy ions shows a synergistic effect that is significant at a dose of 0.3 cGy (0.15 cGy each of Fe ions 1GeV and protons 1GeV). This is the lowest known dose effect of space radiation at the cell and tissue level and represents a novel synergistic effect of heavy and light ions.

Experiments using mixed ions experiment have been repeated with different ions- Helium (1GeV) and Si ions (600 MeV) and a similar result confirms the synergistic response.

This is considered this an important unexpected finding and a manuscript is being prepared together with data on LET ranges from a previous grant award. Acknowledgements will be made to both grant awards. Combined effect of microgravity and space radiation on developing vessels

Since the LET range studies delineated the response to radiation into 2 groups we used both heavy ions and light ions in combination with SMG for these studies. Vessel models were exposed to radiation on day 1 after seeding the cells in 3D matrices then either grown to maturity while stationary or revolving vertically at 5 rpm. For all particle radiation (including 1 GeV He ions, 1 GeV protons, and 1 GeV Fe ions) combined with SMG there is an additive effect--the shape of the dose curve is the same but shifted lower in the presence of SMG. Thus, there is the potential for the combined effects of the space environment to cause more damage to micro-capillaries.

Combined effect of microgravity and space radiation on mature vessels

The data prove that simulated microgravity together with heavy ion radiation is at least additive in breaking down the structure of mature vessels. There was a distinct morphology when both agents are applied together. There are a number of vessels ending in dead ends and in fact very little tube structure left. Experiments with protons and Helium ions, which do not affect vessels up to 4 Gy showed that the microgravity acts alone in this case.

In conclusion we have shown that simulated microgravity alone has little effect on the early motile tip stage of angiogenesis but does have an effect on the entire process of capillary formation and is also effective on mature vessels. Both light and heavy ions are more effective than previously thought and can inhibit angiogenesis detectible at 1.25 cGy and that there is a synergistic response of mixed ions that brings the effective doses even lower (lowest significant effect with 0.15 cGy each ion). In mature vessel models, simulated microgravity has a profound effect of reducing full width vessel models at 1.25 cGy. Furthermore, the combined effect of microgravity is additive to that of charged particles making the space environment. Taken together the radiation and microgravity in the space environment have the potential to damage the microvasculature, which could cause pathologies such as cardiovascular disease (CVD) and neurodegeneration in astronauts.

We have shown that space radiation inhibits angiogenesis and disrupts endothelial barrier function using human endothelial cells in 2- and 3-dimensional human tissue models. The doses and time course for radiation-induced events are now known which makes it possible to assay for joint effects with other environmental influences. Angiogenesis and barrier function are also affected by microgravity so there is a potential for further dysfunction of the human vasculature when applied in combination with radiation. Here, we propose a ground-based study using simulated microgravity to determine the combined effects of space radiation and microgravity on human blood vessel models and its impact on degeneration by testing for angiogenesis and endothelial barrier function using our established assays.

The development of 3-Dimensional human tissue models from normal human cells and stem cells has great potential in many fields of medical research. Tissue models can more accurately depict human tissue since the cells can be arranged spatially as they would be in vivo and can interact with each other as they would in the human body. A neurovascular unit can be used for basic research on many aspects of the human brain. These include regeneration, synaptic function, and degeneration. Because the tissue model is derived from individual cells, each cell type can be altered genetically before it is incorporated into the model. The effects of radiation combined with simulated microgravity can be of benefit to the health of astronauts.

Experiments addressing the effect of simulated microgravity alone on the development of human capillaries (angiogenesis) and the effects on mature capillary models are now completed. This material is being prepared as a manuscript for submission to the new Journal – Microgravity. Both angiogenesis (capillary formation) and the effect on mature vessels were examined.

We first tested the effect of a 3D clinostat spin on the activity of motile tips – the initial structures that facilitate the extension of pioneer tunnels, which later become capillary tubes. The results showed little effect only a slight increase in the number of motile tips in vertical and 3D rotations. A second set of experiments investigated the effects of simulated microgravity on the entire process of capillary formation, from seeding of cells in the matrices to the final vessels structure after 7 days of culture. In this case there was a significant effect on capillary formation. We tested several types of rotation including vertical spins at 1, 5, and 10 rpm, a horizontal spin at 10 rpm and 3D clinostat rotation at 10 rpm for each axis. There was a clear inhibition of vessel formation after all vertical rotations and the 3D rotation whereas vessel formation after horizontal rotation was unaffected and similar to the stationary control. There are 2 main conclusions from these data:

1) Since vertical rotation is as effective as 3D rotation, the data indicates that removal of the vector of gravity is sufficient to cause an inhibition of vessel formation. It is not necessary to distribute the vector of gravity globally to cause an effect; removal by vertical spin is sufficient. This suggests that a mechanism is held in place by the force of gravity on Earth can be disrupted by rotation and cause the kinds of effects seen in the space environment.

2) Since a 1 rpm spin was as effective as faster rotations even though there is little turbulence at such a speed it is unlikely that turbulence is causing the inhibition. Further experiments showed that vessel development in disruptive spins was normal for up to 3 days but affected after this point in development when the vessels became more mature with widened.

Effects of microgravity on mature vessels

Human capillary models were first grown to maturity, then subjected to the same variety of rotations used in the angiogenesis studies. These models represent the capillaries already present in vivo. The effect of rotation on these cultures is similar to that on developing cultures, that is, all the vertical and the 3D rotations are effective at changing the final capillary structure. After 48 hours rotation there was a reduction in total vessel length although further studies are required to determine how this response translates in vivo. These studies and the angiogenesis studies do, however, prove that in isolation, human capillaries are responsive to alterations in gravity. Therefore, it is ighly likely that there is some kind of response in vivo.

Combined effect of microgravity and space radiation angiogenesis

Since both space radiation and simulated microgravity are disruptive to human capillary models we expected that their combined effects would occur at lower doses of charged particle radiation. Previous data from several years ago indicated that heavy and light ions inhibit angiogenesis with a 50% effect at around 40 cGy for both types of radiation albeit via distinct mechanisms. In order to investigate the combined effect we carried out experiments with much lower doses than previously used with an improved assay. Surprisingly, we found that even without simulated microgravity the charged particles inhibited angiogenesis at much lower concentrations than previously detected. Both Fe ions (1 GeV) and protons (1 GeV) were effective at doses well below 10 cGy and were first significant at a dose of 1.25 cGy for both ions. Such sensitivity is not often seen in charged particle radiobiology studies. In fact, these doses are among the lowest recorded to date. The improvement of the assay together with a need to assay using lower doses revealed this effect. We considered this an important unexpected finding and a manuscript is being prepared together with data on LET (linear energy transfer) ranges from a previous grant award. Acknowledgements will be made to both grant awards.

For the combined effects the results are clear and for both protons and Fe ions that simulated microgravity is additive to the inhibition of angiogenesis by charged particles. For heavy (Fe) ions the rotated samples showed a reduction of 500-1000 µm2 vessel/1000 µm2 across the dose range. A similar result with comparable doses was seen with protons.

Combined effect of microgravity and space radiation on mature vessels

These studies are not yet completed. Nevertheless, we have carried out preliminary studies with Fe ions, protons, and Helium ions for this aim. There is data so far that proves that simulated microgravity together with heavy ion radiation is at least additive in breaking down the structure of mature vessels. There was a distinct morphology when both agents are applied together. There are a number of vessels ending in dead ends and in fact very little tube structure left. Experiments with protons and Helium ions, which do not affect vessels up to 4 Gy showed that the microgravity acts alone in this case.

In conclusion we have shown that simulated microgravity alone has little effect on the early motile tip stage of angiogenesis but does have an effect on the entire process of capillary formation. The finding that both light and heavy ions are more effective than previously thought and can inhibit angiogenesis detectible at 1.25 cGy, (one of the lowest dose responses seen in space radiobiology) is considered to be highly important to studies on the effect of the space environment on humans. Furthermore, the combined effect of microgravity is additive to that of charged particles making the hazards of space travel greater than previously thought. In mature vessel models, simulated microgravity has a profound effect of reducing full width vessel models. Furthermore, together with heavy ions there is at least an additive effect of each agent. This study is continuing on a no-cost extension. At the end of the research we expect to have a comprehensive knowledge of the combined effects of radiation and microgravity on these model human microvessels.

We have shown that space radiation inhibits angiogenesis and disrupts endothelial barrier function using human endothelial cells in 2 and 3-dimensional human tissue models. The doses and time course for radiation-induced events are now known which makes it possible to assay for joint effects with other environmental influences. Angiogenesis and barrier function are also affected by microgravity so there is a potential for further dysfunction of the human vasculature when applied in combination with radiation. Here, we propose a ground-based study using simulated microgravity to determine the combined effects of space radiation and microgravity on human blood vessel models and its impact on degeneration by testing for angiogenesis and endothelial barrier function using our established assays.

The development of 3-Dimensional human tissue models from normal human cells and stem cells has great potential in many fields of medical research. Tissue models can more accurately depict human tissue since the cells can be arranged spatially as they would be in vivo and can interact with each other as they would in the human body. A neurovascular unit can be used for basic research on many aspects of the human brain. These include regeneration, synaptic function, and degeneration. Because the tissue model is derived from individual cells, each cell type can be altered genetically before it is incorporated into the model. The effects of radiation combined with simulated microgravity can be of benefit to the health of astronauts.

The first opportunity to irradiate the vessel models was on the spring run at NASA Space Radiation Laboratory (NSRL) so initial experiments in the fall of 2014 were carried out on the effects of microgravity alone. It soon became clear that simulated microgravity has an effect on our vessel models. However, there were problems with the 3D clinostat running for long (more than 24 hours) periods. Further development of the clinostat out at our machine shop here at Columbia was necessary. During this time we carried out some experiments with single axis rotation (vertical and horizontal) as controls. It was found that for the effects on mature vessels, a vertical spin was sufficient to break down the structure of the vessel models. In contrast, studies on angiogenesis showed that even a horizontal spin was sufficient to alter development suggesting that turbulence may be an influencing factor. Further experiments in which we reduced the speed of rotation from 15 rpm to 5 rpm (resulting in much less turbulence in the culture flasks) showed that mature vessel structure was broken down even at lower speeds.

These experiments indicate that removal of the vector of gravity is sufficient to cause an effect on mature vessels at least. These studies were not completed since the irradiations at NSRL Brookhaven started and it was necessary to carry out combined microgravity and radiation experiments. We expect to finish the studies on microgravity alone (comparing different spins axis directions with 3D rotation in developing and mature vessels) and fill in the gaps during the summer and fall after irradiations at NSRL (June 25th was our last irradiation).

We did however, establish a protocol for the combined studies. Data showed that 5 rpm in a vertical spin was effective at breaking down the structure of mature vessels. We therefore used this protocol for experiments investigating both simulated microgravity and radiation. This protocol was used through the radiations in the spring and summer runs at NSRL. With an average of one run per week or so we have generated a significant amount of samples and data. Each experiment has fixed and stained samples that are imaged and then analyzed and the time of processing is significant. Therefore, there are a large amount of experiments still being processed (the last run was on June the 25th and at the time of writing this experiment is still running). We expect to process these samples and data over the next 2-3 months.

Nevertheless, there is data so far that proves that simulated microgravity together with heavy ion radiation is at least additive in breaking down the structure of mature vessels. Thus, the adverse effects of radiation and simulated microgravity on angiogenesis and mature vessel integrity are distinct and are at least additive when applied together.

Using a protocol in which the mature vessel cultures are rotated at 5 rpm in vertical spin together with a known effect of 75 cGy of 1 GeV Fe ions we investigated the effects of each, simulated microgravity and heavy ions. Vessels were grown at 1g until mature then irradiated with the chosen dose of Fe ions (75 cGy is sufficient to breakdown vessel morphology by approximately 50%). As expected 75 cGy caused the loss of full width vessels (12.5 microns diameter and larger) and an increase in vessels narrower than 12.5 microns in diameter suggesting that the heavy ions are causing collapse of vessels.. A 5 rpm vertical spin alone caused the loss of full width vessels and an increase in narrow vessels. The number of narrow vessels in fact increased in simulated microgravity indicating that the effect is distinct from that of heavy ions and further that in addition to degrading existing vessels it may be stimulating the extension narrow pioneering structures. Both together had a profound effect both full width vessels and narrow vessels. There was a distinct morphology when both agents are applied together. There are a number of vessels ending in dead ends and in fact very little tube structure left.

In conclusion we have shown that simulated microgravity alone has little effect on the early motile tip stage of angiogenesis. In mature vessel models, simulated microgravity (rotation in the vertical plane only at 5 rpm) has a profound effect of reducing full width vessel models. Furthermore, together with heavy ions there is at least an additive effect of each agent.

We have shown that space radiation inhibits angiogenesis and disrupts endothelial barrier function using human endothelial cells in 2 and 3-dimensional human tissue models. The doses and time course for radiation-induced events are now known which makes it possible to assay for joint effects with other environmental influences. Angiogenesis and barrier function are also affected by microgravity so there is a potential for further dysfunction of the human vasculature when applied in combination with radiation. Here, we propose a ground-based study using simulated microgravity to determine the combined effects of space radiation and microgravity on human blood vessel models and its impact on degeneration by testing for angiogenesis and endothelial barrier function using our established assays.