Our NSBRI-supported research project can be considered quite productive--seven peer-reviewed primary papers have been published to date, one published review article, and numerous talks and posters were presented at national and international scientific conferences. Two of the published papers garnered considerable public interest and exposure in the popular press, while one was the subject of both a commentary and a recent article in JAMA. A new manuscript is now in review at a journal (Radiation Research). Three additional primary research papers acknowledging this grant's support are still in preparation (all experimental work complete), and a review article is also being prepared that integrates both our new findings over the life of this project and the contributions of others to this field of research. In the course of this project, two NASA postdoctoral fellowships (funded by the Space Biology program, 3-yr in duration) were awarded to perform work related to this grant. Further, results obtained in the course of executing this grant's research contributed key preliminary results to two new grants that were awarded recently by NASA. Thus, considerable scientific advances and leverage were realized as a consequence of this NSBRI award.

Using mice to simulate weightlessness and space-relevant radiation, results from the series of studies supported by this grant demonstrate that both of these environmental conditions interact to induce early impairment of endothelium-dependent vasodilation and cancellous bone loss. However, the only sustained vascular endothelial cell dysfunction is that mediated by exposure to High-Z-High Energy ions (HZE) and not by simulated weightlessness. If such results translate to the human condition, then long-term dysfunction of the vascular endothelium induced by HZE particles could be a major contributor to the development of atherosclerotic cardiovascular disease in astronauts, as well as contribute to the long-term bone loss.

We find that simulated weightlessness causes decrements in both slow-turnover cortical bone tissue and high turnover cancellous tissue, whereas ionization radiation (0.5-2Gy) causes decrements only in cancellous tissue. Whereas the radiation-induced deficits in skeletal microarchitecture diminish over a period of 6-7 months due to age-related bone loss in control animals, dysfunction in cell populations persists. HZE but not protons or gamma (<2Gy) cause defects in osteoblastogenesis from bone marrow derived stem cells and progenitors. This defect can be attributed to persistent deficits in progenitor cell proliferation and colony growth, whereas the capacity to differentiate into osteoblast-like cells and mineralize an extracellular matrix (the hallmark of osteoblasts) is retained. In addition, bones from HZE-irradiated animals can respond later to anabolic loading stimulus with improved bone formation, although there is some evidence from analyses by dynamic histomorphometry and gene expression that there may be persistent defects in osteoprogenitor cell populations localized to regions adjacent to the periosteal surfaces of bone tissue. Together, these findings on marrow-derived progenitors and periosteal cell behavior lead us to predict that fracture healing and perhaps other wound healing processes that depend mesenchymal stem cells derived from the marrow and/or periosteal bone surfaces are deficient after exposure to HZE at space relevant doses. This prediction is both consistent with a few reports in the scientific literature and may have relevance to regenerative medicine in space, thus represents a potentially important area for future study. With respect to prevention, either mechanical stimulation (resembling vigorous exercise) or feeding a diet containing dried plum, can improve bone structure despite prior exposure to HZE. In contrast, treatment with antioxidants that have displayed at least some radioprotective properties (lipoic acid injections, anti-oxidant cocktail, or treatment with an anti-inflammatory (Ibuprofen)) failed to prevent radiation-induced bone loss. These findings imply treatment with antioxidants alone are unlikely to prove fully protective to the skeleton exposed to ionizing radiation.

In sum, findings from our studies show that in the short term, ionizing radiation and simulated weightlessness cause greater deficits in blood vessels when combined compared to either challenge alone. In the long term, HZE but not unloading, can lead to persistent, adverse consequences for bone cell and vessel function, possibly due to oxidative stress-related pathways. Novel countermeasures to radiation-induced damage to the skeleton identified in the course of this project include both mechanical stimulation and a dietary supplement.

On the basis of the mechanistic insight gained, potential anti-oxidant countermeasures will be tested in the following specific aims. Aim 1: Determine how prolonged weightlessness and space radiation (simulated spaceflight) cause functional and structural changes in skeletal vasculature and bone. Aim 2. Determine the extent to which specific countermeasures protect against weightlessness and space radiation-induced bone loss and vascular dysfunction. Aim 3. Determine how low dose space radiation influences later skeletal recovery from prolonged weightlessness. Aim 4. Determine if transient treatment with countermeasures protects from bone loss caused by weightlessness and radiation during subsequent aging. These studies will provide important new insight into the bone loss that is caused by musculoskeletal disuse and radiation at the molecular, cell, and tissue level, with biomedical applications to Earth (e.g., radiotherapy, accidental exposures), as well as space.

This last year, four published papers describe results from our experiments with mice testing various aspects of our hypothesis. We examined the effects of radiation and/or simulated weightlessness by hindlimb unloading on bone and blood vessel function either after a short period or at a later time after transient exposures. In short term studies the combination of weightlessness and heavy ion radiation together cause worse deficits in blood vessel function than either factor alone, and these deficits appear to be mediated via free radical-related pathways. In contrast, long-term studies show that bones and vessels can recover from exposure to transient simulated weightlessness, but cannot recover fully from heavy ion radiation. With respect to prevention, either mechanical stimulation (resembling vigorous exercise) or feeding a diet containing dried plum, can improve bone structure despite prior exposure to heavy ion radiation. In sum, recent findings from our studies show that in the short term, ionizing radiation and simulated weightlessness cause greater deficits in blood vessels when combined compared to either challenge alone. In the long term, heavy ion radiation, but not unloading, can lead to persistent, adverse consequences for bone and vessel function, possibly due to oxidative stress-related pathways.

(2) Key findings were:

• Both weightlessness and ionizing radiation) caused rapid but transient increase in skeletal expression levels of the global antioxidant transcription factor, nrf2 expression, indicating that weightlessness and radiation pose an acute oxidative challenge to skeletal tissue.

• Total-body irradiation (gamma or heavy-ion) caused temporal, concerted regulation of gene expression within both marrow and mineralized tissue for select, osteoclastogenic cytokines and markers of bone resorption; this is likely to account for the rapid and progressive deterioration of cancellous microarchitecture observed following exposure to ionizing radiation.

• A dietary countermeasure with high antioxidant activity showed an impressive ability to maintain the integrity of their bone structure after high LET irradiation, making it a strong candidate to mitigate radiation-induced tissue damage.

• High LET radiation did not completely impair ability of the bone to respond to a potent anabolic mechanical stimulus, rest-inserted axial loading.

• Simulated weightlessness and iron radiation each impaired peak endothelium-dependent vasodilation and the combination of HU and IR further impaired endothelium-dependent vasodilation.

(3) Impact of key findings on hypotheses, technology requirements, objectives, and specific aims of the original proposal.

• Further experiments and analysis of tissues from the dietary intervention and mechanical stimulation experiments needed.

• Results from the iron radiation experiment support the theory of a bone-vascular coupling for bone remodeling in response to simulated spaceflight.

(4) Proposed research plan for the coming year. In the coming year, we plan to complete analysis of tissues and bones from recent extensive experimentation conducted at NASA Space Radiation Laboratory/Brookhaven National Lab (Aim 1 and 2). We also will conduct experiments at both NSRL/BNL and ARC for this coming year that will: 1) evaluate the effects of the new dietary intervention on vascular reactivity and bone structure following 2 wks of simulated weightlessness alone and combined with simulated space radiation (protons + iron) (Aim 2); 2) define the ability of bone and vascular responses to recover over the long term (2-6 mos) from the adverse effects of protons + iron on a standard vs. supplemented diet (Aim 2, 3) determine the time- and radiation- dependence of anabolic mechanical loading during recovery from simulated space radiation (Aim 1, 3).

(2) Key Findings. Substantial progress was made in completing Aims 1 and 2 of the original proposal. A standard protocol for simulating the spaceflight factors of space radiation (dual-ion; proton+iron) and weightlessness (hindlimb unloading) was established, and the influence of possible nutritional countermeasures (antioxidant cocktail and flaxseed) tested. The following provides a summary of key findings.

• Dietary supplementation of male, 16wk old C57Bl/6J mice with an antioxidant cocktail or flaxseed did not prevent decrements in cancellous bone volume and microarchitecture caused by exposure to ionizing radiation.

• Gamma irradiation and hindlimb unloading each reduced endothelium-dependent vasodilation of the gastrocnemius feed artery, which correlated well with cancellous tissue decrements. Signaling pathways in addition to those related to nitric oxide (NO) release may contribute to observed changes in vascular reactivity during simulated spaceflight.

(3) Impact of key findings on hypotheses, technology requirements, objectives, and specific aims of the original proposal.

• Further analysis of tissues and results from the dietary intervention strategies tested to date are needed before definitive conclusions can be drawn and impact on hypotheses and aims determined. Results obtained to date lead us to focus on evaluating tissues at the mRNA and biochemical levels for evidence of both oxidative damage and the impact of the dietary countermeasures.

• Results from the gamma radiation experiment support the theory of a bone-vascular coupling for bone remodeling in response to simulated spaceflight.

(4) Proposed research plan for the coming year. In the coming year, we plan to complete analysis of tissues and bones from the dietary countermeasure experiments using ionizing radiation (Aim 1 and 2). We also will conduct experiments at both NSRL/BNL and ARC for this coming year that will: continue analysis of tissues and data from vascular reactivity experiments using simulated space radiation at NSRL/BNL with Dr. Delp's team and perform additional in-depth experiments to evaluate vascular responses (Aim 1); analyze skeletal gene expression after exposure to simulated space radiation (Aim 1 and 2); determine the influence of dietary antioxidants on skeletal responses to simulated weightlessness (Aim 2); assess the ability of axial loading to stimulate bone formation after exposure of the mice to simulated space radiation in recovery experiments (Aim 3); perform additional follow-on experiments to determine the ability of dietary countermeasures to prevent bone loss caused by simulated spaceflight (Aim 2, 3).

(2) Key Findings. Progress has been made on multiple fronts during the first year of the grant. We have confirmed structural and cellular phenotypes with previous results. Work remains to study the cellular and molecular mechanisms in more detail and to investigate how antioxidants can effectively modulate skeletal radioresponses. The following provides a summary of key findings.

Bone Structure and Vascular Reactivity Acute Effects

• Radiation-induced bone loss results above a cumulative dose of 100 cGy from low- or high-LET ion constituents

• Vasodilation responses to acetylcholine were diminished in gastrocnemius muscle feed arteries in hindlimb unloaded and irradiated mice relative to that in control animals. The combined effects of hindlimb unloading and irradiation did not further depress endothelium-dependent vasodilation.

Simulated Spaceflight Effects on Osteoblast Cell Cultures

• These results suggest that osteoprogenitor growth from marrow progenitors may be impaired above a threshold dose of heavy-ions between 20 and 50 cGy

(3) These experiments inform the radiation doses and duration of hindlimb unloading to be utilized in future work, as part of Milestone 1, 2, and 3 of our grant proposal, and to establish treatment schedules to modulate bone structure and vascular reactivity

(4) In the coming year, we plan to test antioxidant countermeasures to radiation-induced bone loss and to protect osteoprogenitors. Pilot studies will be conducted at ARC and NSRL/BNL to establish the efficacy of different antioxidant compounds and additional experiments will be conducted to investigate if simulated space-irradiation affects vascular reactivity, responses to unloading, and oxidative mechanisms.

ESTIMATED % COMPLETION PER AIM: AIM 1: ~75% complete AIM 2: ~10% complete. AIM 3: ~2% complete As outlined in our proposal, we have been actively involved with several Education & Outreach activities, including the STEM Teacher And Researcher (STAR), the Education Associates' Program (EAP) programs, and the Space Settlement Design Contest.

Our plan has three aims.

AIM 1: Determine the functional and structural consequences of prolonged weightlessness and space radiation (simulated spaceflight) for bone and skeletal vasculature in the context of bone cell function and oxidative stress.

AIM 2: Determine the extent to which an anti-oxidant protects against weightlessness and space radiation-induced bone loss and vascular dysfunction.

AIM 3: Determine how space radiation influences later skeletal and vascular recovery from prolonged weightlessness and the potential of anti-oxidants to improve or prevent this recovery.

This proposal addresses the current research announcement and NASA's HRP-Integrated Research Plan risks for the bone system by defining mechanisms and countermeasures designed to prevent the effects of combined unloading and radiation on bone, with the goal of enabling human space exploration.