NOTE: Received no-cost extension to 9/30/2011 per C. Guidry/JSC (10/2010)

NOTE: Received no-cost extension to 9/30/2010 per J. Dardano/JSC (8/09)

We have successfully developed 3-D human micro-vasculature models from normal human umbilical vein endothelial cells. These structures have been characterized by multi- photon microscopy and have been irradiated with high energy iron ions and protons. Irradiation of mature vessels led to a breakdown of the vessels after low doses of iron ions [<1 Gy] but no effect of protons out to 3.2 Gray. Monitoring vessel structures over time led to the observation that the 3-D mature vessel network is essentially restored by 12 days, even after 3.2 Gy of iron ions. By contrast the irradiation of developing vessels showed that both protons and iron ions at 1GeV similarly disrupt network vessel development, with ~50% loss of intact vessel length [relative to control] at 3 days after 0.8 Gy. The full pattern of vessel recovery remains to be determined.

DNA damage foci [53BP-1] formation was examined in endothelial cell nuclei in 2-D monolayers and in the cell nuclei of the 3-D micro-vasculature structures. Both protons at 0.22 keV/micrometer and Fe ions at 150 keV/micrometer showed similar kinetics of foci formation with peak yields at 1 hr and a 10 fold decline at 48hrs. However there were dramatic differences in the efficiency of focus formation. At the peak there was ~ one 53 BP-1 focus for each Fe ion traversal and ~ 1 focus for every 1,000 proton traversals. Human endothelial cells can form 3-D micro-vasculature like structures and show quantitative morphological and DNA damage responses after space-like radiations at moderate to high doses. At the fluences likely to be experienced in cells by man in space the question might reasonably be asked about the micro-vasculature; does this matter? Probably not.

The most significant achievement from the body of work carried out under support from this grant was the development and introduction to space radiation biology of a human model tissue system. Normal human endothelial cells were encouraged to develop into the capillary-like structures of the human circulatory system. At various stages of development these structures were exposed to proscribed doses of energetic iron particles or protons as found in space, and responses assessed. Along with continued participation in national and international meetings where information was presented and encouragingly discussed, three peer reviewed publications resulted. The last of these was published in June 2012, after submission in December 2011, and acceptance in March 2012. Two highlights resulting from the publications are worth of note. The paper published in the International Journal of Radiation Biology in June 2012 was accorded the honor of having a representative figure of treated capillary structures from the manuscript on the front cover. The paper published in the journal, Radiation Research, in January 2011, was chosen by members of the Radiation Research Society as the most scientifically compelling of the issue. This led to an interview by Marjan Boerman, a student representative, with the PI and colleague Dr. Peter Grabham. The interview is available as a Podcast from the Radiation Research Society.

NOTE: Received no-cost extension to 9/30/2010 per J. Dardano/JSC (8/09)

We have successfully developed 3-D human micro-vasculature models from normal human umbilical vein endothelial cells. These structures have been characterized by multi- photon microscopy and have been irradiated with high energy iron ions and protons. Irradiation of mature vessels led to a breakdown of the vessels after low doses of iron ions [<1 Gy] but no effect of protons out to 3.2 Gray. Monitoring vessel structures over time led to the observation that the 3-D mature vessel network is essentially restored by 12 days, even after 3.2 Gy of iron ions. By contrast the irradiation of developing vessels showed that both protons and iron ions at 1GeV similarly disrupt network vessel development, with ~50% loss of intact vessel length [relative to control] at 3 days after 0.8 Gy. The full pattern of vessel recovery remains to be determined. DNA damage foci [53BP-1] formation was examined in endothelial cell nuclei in 2-D monolayers and in the cell nuclei of the 3-D micro-vasculature structures. Both protons at 0.22 keV/micrometer and Fe ions at 150 keV/micrometer showed similar kinetics of foci formation with peak yields at 1 hr and a 10 fold decline at 48hrs. However there were dramatic differences in the efficiency of focus formation. At the peak there was ~ one 53 BP-1 focus for each Fe ion traversal and ~ 1 focus for every 1,000 proton traversals. Human endothelial cells can form 3-D micro-vasculature like structures and show quantitative morphological and DNA damage responses after space-like radiations at moderate to high doses. At the fluences likely to be experienced in cells by man in space the question might reasonably be asked about the micro-vasculature; does this matter? Probably not.

Fe ions affect mature and developing vessel structures with the same dose response. The 3 dimensional micro-vascular structure is however reconstituted with time.

Mature vessels are unaffected by protons up to 3.2 Gy.

Developing vessel structure formation is sensitive to protons.

Compared to protons Fe ions are highly efficient at causing DNA damage. At the peak there is ~ one 53 BP-1 focus for each Fe ion traversal and ~ 1 focus for every 1,000 proton traversals.

Both particles produce DNA damage tracks and foci. Tracks are longer and broader after Fe ion exposure and persist longer.

Human endothelial cells can form 3 D micro-vasculature like structures and show quantitative morphological and DNA damage responses after space-like radiations at moderate to high doses.

At this time, it is uncertain whether these responses reflect damage that may be of consequence in the space environment.

We have successfully developed 3-D human micro-vasculature models from normal human umbilical vein endothelial cells. These structures have been characterized by multi- photon microscopy and have been irradiated with high energy iron ions and protons. Irradiation of mature vessels led to a breakdown of the vessels after low doses of iron ions [<1 Gy] but no effect of protons out to 3.2 Gray. Monitoring vessel structures over time led to the observation that the 3-D mature vessel network is essentially restored by 12 days, even after 3.2 Gy of iron ions. By contrast the irradiation of developing vessels showed that both protons and iron ions at 1GeV similarly disrupt network vessel development, with ~50% loss of intact vessel length [relative to control] at 3 days after 0.8 Gy. The full pattern of vessel recovery remains to be determined. DNA damage foci [53BP-1] formation was examined in endothelial cell nuclei in 2-D monolayers and in the cell nuclei of the 3-D micro-vasculature structures. Both protons at 0.22 keV/micrometer and Fe ions at 150 keV/micrometer showed similar kinetics of foci formation with peak yields at 1 hr and a 10 fold decline at 48hrs. However there were dramatic differences in the efficiency of focus formation. At the peak there was ~ one 53 BP-1 focus for each Fe ion traversal and ~ 1 focus for every 1,000 proton traversals. Human endothelial cells can form 3-D micro-vasculature like structures and show quantitative morphological and DNA damage responses after space-like radiations at moderate to high doses. At the fluences likely to be experienced in cells by man in space the question might reasonably be asked about the micro-vasculature; does this matter? Probably not.

DNA damage foci [53BP-1] formation was examined in endothelial cell nuclei in 2-D monolayers and in the cell nuclei of the 3-D micro-vasculature structures. Both protons at 0.22 keV/micrometer and Fe ions at 150 keV/micrometer showed similar kinetics of foci formation with peak yields at 1 hr and a 10 fold decline at 48hrs. However there were dramatic differences in the efficiency of focus formation. At the peak there was ~ one 53 BP-1 focus for each Fe ion traversal and ~ 1 focus for every 1,000 proton traversals. Human endothelial cells can form 3-D micro-vasculature like structures and show quantitative morphological and DNA damage responses after space-like radiations at moderate to high doses. At the fluences likely to be experienced in cells by man in space the question might reasonably be asked about the micro-vasculature; does this matter? Probably not.

Both high LET Fe ions and low LET protons, in addition to gamma radiation cause chromosome damage in HUVECS growing in 2D cultures. mFISH is being used to identify chromosome specific aberrations. For example, in response to 0.8 Gy Fe ions, cells showed a large number of aberrations [94% of cells] involving primarily chromosomes 1, 2, 4, 5, 7, 7, 9, and X. These include stable aberrations such as reciprocal translocations.

Endothelial cells [HUVEC`s] have been successfully cultured in 3D matrices, and in the presence of appropriate growth factors found to differentiate and assemble into capillary tubes. Cells were fluorescently labelled with a long-live cyto-tracker, suspended in collagen gels and stimulated to differentiate and form vessels. Live 3D imaging of cells showed distinct stages of development starting with the formation of vacuoles, followe by cell elongation and cellular coalecence leading to the formation of capillary tube structures. Having established this scenario the effects of space-related radiations on vessel formation from individual cells, and the integrity of mature vessels was assessed. Irradiation of cells while dividing showed that a dose of 0.2 Gy of Fe ions can cause a significant decrease in vessel formation.

Irradiation of mature vessels revealed that they were much more sensitive to Fe ions than they were to low LET protons. Doses of 0.8 to 1.6 Gy causing significant loss of vessel integrity, while after the highest dose of protons [3.2 Gy], vessels were indistiguishable from controls. Formed vessels are also highly resitant to gamma radiation, with limited effects at doses up to 16 Gy.

A technique was developed to monitor cellular apoptosis in the 3D cultures , along with the 2D system and will be applied to radiation-type comparisons in the future. The appproach outlined has succeeded in providing the first information on the effects of space related radiations on human endothelial cells in a 3D tissue configuration. There are clear differences between the effectiveness of different radiations in these non-dividing differentiated cells. The meaning, and the mechanistic bases of these findings will be explored in future cycles, and will lead to assessments of potential consequences to the cardio-vascular system of the space traveller.