NOTE: New end date will be 6/30/2011, per C. Guidry/JSC (6/2010)

NOTE: Received NCE to 12/31/2009 per J. Dardano/JSC (7/2009)

Therefore, our Specific Aims are:

Hypothesis 1: Charged particles (iron ions) will produce an acute oxidative stress event characterized by cellular and tissue injury expressed by endothelial and myocardial dysfunction.

Specific Aim 1: Time- and dose-responses for multiple indices of endothelial and myocardial function will be established in adult Wistar rats exposed to 600 MeV/n Fe (iron) beams at the NASA Space Radiation Laboratory, Brookhaven National Laboratory (BNL). Animals will studied non-invasively and tissues will be collected for histological, functional and molecular analyses using methods established in our laboratory at different time points. Indices of normal tissue function and homeostasis to be investigated include:

a) Endothelium: 1) vascular stiffness by Doppler effect using pulse wave velocity; 2) endothelial function in isolated vascular ring tissue and microvessels; 3) markers of apoptosis in vascular tissue.

b) Heart: 1) myocardial contractile function and contractile reserve in vivo; 2) contractility and contractile reserve in vitro in isolated cardiac myocytes; 3) markers of apoptosis in cardiac tissue (as above).

Hypothesis 2: Iron irradiation-induced endothelial and myocardial contractile dysfunction results from the specific imbalance in NO signaling induced by increased ROS production.

Specific Aim 2: To determine the whether low-fluences of iron ions alter the balance in NO signaling as a function of increased ROS production thereby impairing endothelial and myocardial function. Radiation doses will be selected based on results of Aim 1 and animals will be sacrificed for detailed analyses at various time points as in Aim 1. Vascular and heart tissues from adult Wistar rats exposed to 600 MeV/n Fe ions will be collected and we will measure:

1) NO bioavailability in vascular rings and NOx in plasma , 2) NOS activity using fluorescent dye in heart and blood vessels, 3) ROS levels using chemiluminescence and fluorescence bioassays, 4) Nitroso-tyrosine expression in vascular and cardiac tissue using Western blot analysis.

Hypothesis 3: XO, NOS, and arginase pathways play a critical role in the cardiovascular response to HZE particle radiation.

Specific Aim 3: Rats will be exposed to 600 MeV/n iron ions to determine the specific roles of XO, NOS and arginase in modulating cellular and tissue response to charge particle-induced oxidative stress. Radiation doses will be selected based on results of Aims 1-2 and animals will be sacrificed for detailed analyses at various time points as in Aim 1 for the following endpoints:

1) expression and activity of NOS, Arginase and XO at an RNA and protein level using quatitative PCR, Western blot and immunohistochemistry in heart and blood vessels; 2) Enzyme activity using specific inhibitors of each of the enzymes both alone and in combination with our in vitro vascular ring bioassay and isolated cardiac myocytes; 3) The effect of specific inhibitors on bioassays of ROS and NO (as in Aim 2). Hypothesis 4: Enzyme inhibitors and ROS scavengers will modulate early and late cardiovascular toxicity of low-fluences of iron ions.

Specific Aim 4: To determine if enzyme inhibitors and ROS scavengers can modulate the cardio-vascular effects of iron ions, Wistar rats and/or tissue preparations will be treated with enzyme inhibitors or ROS scavengers prior to and following 600 MeV/n Fe beam irradiation. We will use in vivo and in vitro bioassays of endothelial and myocardial function to test whether the XO inhibitor allopurinol, and the arginase inhibitors S-(2-boronoethyl)-L-cysteine (BEC), or difluoromethylornithine (DFMO) will attenuate radiation-induced cardiovascular effects.

While IR may have parallel effects on peripheral vasculature endothelium and cardiac contractile tissue, the interaction between the blood vessels and heart (ventricular-vascular coupling) has further profound effects on each of these systems. It is for this reason that an approach which incorporates both in vivo (integrated cardiovascular measures such as PWV and P-V loops), as well as isolated cellular and tissue measures of function is so important. Our methodologies will allow us to assess the contribution of each component (heart and vasculature) to the integrated system response to charged particle exposure.

Although our research has found high doses of gamma radiation to cause some vascular injuries, we are also interested in vascular protection. We are studying how large of a radiation dose a biological system can absorb before its defenses are overwhelmed. This knowledge would be very helpful in radiotherapy and occupational radiation exposure control. Also, we have identified a drug that can potentially protect against radiation injury. This can be very valuable in the cases of accidental radiation exposures, such as nuclear accidents. In conclusion, our research is very applicable to life on Earth.

INTRODUCTION: Spaceflight missions entail up to 24 hours of extravehicular activity (EVA) per week. Astronauts are exposed to 100% oxygen during EVA activities in the space environment. The tissue response to hyperoxia in the space environment is influenced by exposure to radiation, mostly in the form of Galactic Cosmic Radiation. There are no obvious sequelae evident shortly after the EVA, however it is unknown if prolonged and repeated EVAs cause later tissue damage. Previously we have demonstrated that radiation induces an oxidative stress in vessels which leads to impaired endothelial function and vascular stiffness. We tested the hypothesis that hyperoxia represents an added or synergistic risk with radiation for vascular endothelial oxidative stress and dysfunction.

METHODS AND RESULTS: We developed an animal model incorporating 4 groups of mice: one group exposed to a hyperoxic environment, one exposed to radiation alone and one group exposed to hyperoxia in addition to radiation. A group without exposure to hyperoxia or radiation served as controls. Hyperoxia treatment was performed by exposing mice to >95% oxygen for 8 hours 3 times during one week in special chambers. Chambers have been designed and used by NASA to study hyperoxia / hypoxia in animal model. Radiation treatment was performed by exposing mice to 1 Gy gamma radiation at rate of 100 rad/minute. Mice were exposed for a period of 2 weeks. At the end of the 2 week period, terminal endpoints were determined 1) In vivo integrated cardiovascular function was measured using non-invasive Doppler to measure pulse wave velocity (PWV) 2) Ex-vivo endothelial function by measuring endothelial dependent vasodilation in aortic rings. Compared to unexposed animals PWV was increased both following exposure to hyperoxia and to radiation alone. Combined exposure to both hyperoxia and radiation resulted in additive effect and resulted in worse cardiovascular function as evidenced by maximum increase in PWV. In ex-vivo experiments of endothelial function we did not find any difference between control and exposed to radiation and hyperoxia groups.

CONCLUSION: Our data confirm that both hyperoxia and radiation adversely affect cardiovascular function and provides evidence that their negative effects are additive in nature.

FUTURE STUDIES: The current results in animal model provide sufficient evidence to justify future studies in astronauts.

NOTE: New end date will be 6/30/2011, per C. Guidry/JSC (6/2010)

NOTE: Received NCE to 12/31/2009 per J. Dardano/JSC (7/2009)

Therefore, our Specific Aims are:

Hypothesis 1: Charged particles (iron ions) will produce an acute oxidative stress event characterized by cellular and tissue injury expressed by endothelial and myocardial dysfunction.

Specific Aim 1: Time- and dose-responses for multiple indices of endothelial and myocardial function will be established in adult Wistar rats exposed to 600 MeV/n Fe (iron) beams at the NASA Space Radiation Laboratory, Brookhaven National Laboratory (BNL). Animals will studied non-invasively and tissues will be collected for histological, functional and molecular analyses using methods established in our laboratory at different time points. Indices of normal tissue function and homeostasis to be investigated include:

a) Endothelium: 1) vascular stiffness by Doppler effect using pulse wave velocity; 2) endothelial function in isolated vascular ring tissue and microvessels; 3) markers of apoptosis in vascular tissue.

b) Heart: 1) myocardial contractile function and contractile reserve in vivo; 2) contractility and contractile reserve in vitro in isolated cardiac myocytes; 3) markers of apoptosis in cardiac tissue (as above).

Hypothesis 2: Iron irradiation-induced endothelial and myocardial contractile dysfunction results from the specific imbalance in NO signaling induced by increased ROS production.

Specific Aim 2: To determine the whether low-fluences of iron ions alter the balance in NO signaling as a function of increased ROS production thereby impairing endothelial and myocardial function. Radiation doses will be selected based on results of Aim 1 and animals will be sacrificed for detailed analyses at various time points as in Aim 1. Vascular and heart tissues from adult Wistar rats exposed to 600 MeV/n Fe ions will be collected and we will measure:

1) NO bioavailability in vascular rings and NOx in plasma , 2) NOS activity using fluorescent dye in heart and blood vessels, 3) ROS levels using chemiluminescence and fluorescence bioassays, 4) Nitroso-tyrosine expression in vascular and cardiac tissue using Western blot analysis.

Hypothesis 3: XO, NOS, and arginase pathways play a critical role in the cardiovascular response to HZE particle radiation.

Specific Aim 3: Rats will be exposed to 600 MeV/n iron ions to determine the specific roles of XO, NOS and arginase in modulating cellular and tissue response to charge particle-induced oxidative stress. Radiation doses will be selected based on results of Aims 1-2 and animals will be sacrificed for detailed analyses at various time points as in Aim 1 for the following endpoints:

1) expression and activity of NOS, Arginase and XO at an RNA and protein level using quatitative PCR, Western blot and immunohistochemistry in heart and blood vessels; 2) Enzyme activity using specific inhibitors of each of the enzymes both alone and in combination with our in vitro vascular ring bioassay and isolated cardiac myocytes; 3) The effect of specific inhibitors on bioassays of ROS and NO (as in Aim 2). Hypothesis 4: Enzyme inhibitors and ROS scavengers will modulate early and late cardiovascular toxicity of low-fluences of iron ions.

Specific Aim 4: To determine if enzyme inhibitors and ROS scavengers can modulate the cardio-vascular effects of iron ions, Wistar rats and/or tissue preparations will be treated with enzyme inhibitors or ROS scavengers prior to and following 600 MeV/n Fe beam irradiation. We will use in vivo and in vitro bioassays of endothelial and myocardial function to test whether the XO inhibitor allopurinol, and the arginase inhibitors S-(2-boronoethyl)-L-cysteine (BEC), or difluoromethylornithine (DFMO) will attenuate radiation-induced cardiovascular effects.

While IR may have parallel effects on peripheral vasculature endothelium and cardiac contractile tissue, the interaction between the blood vessels and heart (ventricular-vascular coupling) has further profound effects on each of these systems. It is for this reason that an approach which incorporates both in vivo (integrated cardiovascular measures such as PWV and P-V loops), as well as isolated cellular and tissue measures of function is so important. Our methodologies will allow us to assess the contribution of each component (heart and vasculature) to the integrated system response to charged particle exposure.

Although our research has found high doses of gamma radiation to cause some vascular injuries, we are also interested in vascular protection. We are studying how large of a radiation dose a biological system can absorb before its defenses are overwhelmed. This knowledge would be very helpful in radiotherapy and occupational radiation exposure control. Also, we have identified a drug that can potentially protect against radiation injury. This can be very valuable in the cases of accidental radiation exposures, such as nuclear accidents. In conclusion, our research is very applicable to life on Earth.

Our finding of significantly decreased cell outgrowth from aorta following radiation exposure can be explained by two mechanisms: 1) radiation-impaired endothelial proliferation or migration, or 2) radiation-induced endothelial cell death. Previous studies support both of these as potential mechanisms. In the coronary vasculature of Sprague-Dawley and Wistar rats, capillary density was diminished following 10, 15, 20, and 25Gy irradiation. This finding mimics pathological signs of heart disease and demonstrates a loss of angiogenic ability. On et al. observed reduced endothelial cell quantity in aortas of female Sprague-Dawley rats exposed to 10Gy gamma radiation after 1-week, through histological evaluation and immunoblotting for the endothelial-specific protein, von Willebrand factor. In contrast, a study of 45Gy irradiation of rabbit ear artery only reported changes in endothelial cell morphology 6 weeks after exposure, and no changes were detected following 10 and 20Gy doses. This same study showed a drastic decrease in endothelial nitric oxide synthase (eNOS) protein abundance 1-week post-irradiation. eNOS-produced nitric oxide is a critical signaling molecule promoting endothelial cell proliferation and health.

In our current results, anti-angiogenic effects were observed at radiation doses much lower than those mentioned above. Furthermore, the data of Chapter 3 demonstrated that acute chemical treatments were able to fully restore aortic endothelial dependent relaxation after radiation, indicating that the endothelium is still present but in altered nitroso-redox state (decreased NO production and increased reactive oxygen species). This was confirmed through measurement of aortic NO and superoxide. Finally, our group (unpublished observation) and Oh et al. observed that radiation induces senescence in cultured endothelial cells, which is characterized primarily by arrested cell cycle and reduced proliferation. Considering these results, we hypothesize that the observed radiation-impaired cell outgrowth is due to reduced endothelial proliferation or migration as a consequence of impaired NO signaling.

References:

On YK, Kim HS, Kim SY, Chae IH, Oh BH, Lee MM, Park YB, Choi YS, Chung MH. Vitamin C prevents radiation-induced endothelium-dependent vasomotor dysfunction and de-endothelialization by inhibiting oxidative damage in the rat. Clin Exp Pharmacol Physiol. 2001;28:816-821.

Oh CW, Bump EA, Kim JS, Janigro D, Mayberg MR. Induction of a senescence-like phenotype in bovine aortic endothelial cells by ionizing radiation. Radiat Res. 2001;156:232-240.

NOTE: Received NCE to 12/31/2009 per J. Dardano/JSC (7/2009)

Therefore, our Specific Aims are:

Hypothesis 1: Charged particles (iron ions) will produce an acute oxidative stress event characterized by cellular and tissue injury expressed by endothelial and myocardial dysfunction.

Specific Aim 1: Time- and dose-responses for multiple indices of endothelial and myocardial function will be established in adult Wistar rats exposed to 600 MeV/n Fe (iron) beams at the NASA Space Radiation Laboratory, Brookhaven National Laboratory (BNL). Animals will studied non-invasively and tissues will be collected for histological, functional and molecular analyses using methods established in our laboratory at different time points. Indices of normal tissue function and homeostasis to be investigated include:

a) Endothelium: 1) vascular stiffness by Doppler effect using pulse wave velocity; 2) endothelial function in isolated vascular ring tissue and microvessels; 3) markers of apoptosis in vascular tissue.

b) Heart: 1) myocardial contractile function and contractile reserve in vivo; 2) contractility and contractile reserve in vitro in isolated cardiac myocytes; 3) markers of apoptosis in cardiac tissue (as above).

Hypothesis 2: Iron irradiation-induced endothelial and myocardial contractile dysfunction results from the specific imbalance in NO signaling induced by increased ROS production.

Specific Aim 2: To determine the whether low-fluences of iron ions alter the balance in NO signaling as a function of increased ROS production thereby impairing endothelial and myocardial function. Radiation doses will be selected based on results of Aim 1 and animals will be sacrificed for detailed analyses at various time points as in Aim 1. Vascular and heart tissues from adult Wistar rats exposed to 600 MeV/n Fe ions will be collected and we will measure:

1) NO bioavailability in vascular rings and NOx in plasma , 2) NOS activity using fluorescent dye in heart and blood vessels, 3) ROS levels using chemiluminescence and fluorescence bioassays, 4) Nitroso-tyrosine expression in vascular and cardiac tissue using Western blot analysis.

Hypothesis 3: XO, NOS, and arginase pathways play a critical role in the cardiovascular response to HZE particle radiation.

Specific Aim 3: Rats will be exposed to 600 MeV/n iron ions to determine the specific roles of XO, NOS and arginase in modulating cellular and tissue response to charge particle-induced oxidative stress. Radiation doses will be selected based on results of Aims 1-2 and animals will be sacrificed for detailed analyses at various time points as in Aim 1 for the following endpoints:

1) expression and activity of NOS, Arginase and XO at an RNA and protein level using quatitative PCR, Western blot and immunohistochemistry in heart and blood vessels; 2) Enzyme activity using specific inhibitors of each of the enzymes both alone and in combination with our in vitro vascular ring bioassay and isolated cardiac myocytes; 3) The effect of specific inhibitors on bioassays of ROS and NO (as in Aim 2). Hypothesis 4: Enzyme inhibitors and ROS scavengers will modulate early and late cardiovascular toxicity of low-fluences of iron ions.

Specific Aim 4: To determine if enzyme inhibitors and ROS scavengers can modulate the cardio-vascular effects of iron ions, Wistar rats and/or tissue preparations will be treated with enzyme inhibitors or ROS scavengers prior to and following 600 MeV/n Fe beam irradiation. We will use in vivo and in vitro bioassays of endothelial and myocardial function to test whether the XO inhibitor allopurinol, and the arginase inhibitors S-(2-boronoethyl)-L-cysteine (BEC), or difluoromethylornithine (DFMO) will attenuate radiation-induced cardiovascular effects.

While IR may have parallel effects on peripheral vasculature endothelium and cardiac contractile tissue, the interaction between the blood vessels and heart (ventricular-vascular coupling) has further profound effects on each of these systems. It is for this reason that an approach which incorporates both in vivo (integrated cardiovascular measures such as PWV and P-V loops), as well as isolated cellular and tissue measures of function is so important. Our methodologies will allow us to assess the contribution of each component (heart and vasculature) to the integrated system response to charged particle exposure.

Although our research has found high doses of gamma radiation to cause some vascular injuries, we are also interested in vascular protection. We are studying how large of a radiation dose a biological system can absorb before its defenses are overwhelmed. This knowledge would be very helpful in radiotherapy and occupational radiation exposure control. Also, we have identified a drug that can potentially protect against radiation injury. This can be very valuable in the cases of accidental radiation exposures, such as nuclear accidents. In conclusion, our research is very applicable to life on Earth.

We previously found radiation to damage vascular function in rats. We also determined the ROS producing enzyme, xanthine oxidase (XO), to contribute significantly to this damage. We continued to investigate if XO inhibition through a special diet could protect against radiation injury. In animals exposed to gamma radiation, this diet began 1 week before irradiation. In animals exposed to iron radiation, the diet began immediately after irradiation. In both cases, the XO inhibition diet provided significant protection for vascular function. After irradiation, we found that rat aorta could not relax tension as well and we determined that the aorta was stiffer than un-irradiated blood vessels. These are both symptoms of cardiovascular diseases, such as hypertension and aging. In the iron irradiated aorta, we did not observe any geometric changes in the vessels, indicating that mechanical properties of the aorta are responsible for the increased stiffness. Also, with gamma-radiation, we tested passive mechanical properties of aorta. Even after removing active muscle control, we found that the irradiated aorta was less compliant. All of these parameters were greatly improved with the dietary XO inhibition.

We continued to investigate the reasons for these vascular problems. We determined that after radiation, rat aorta produced significantly less NO compared to un-irradiated aortas. Once again, the dietary inhibition of XO completely restored the NO production levels. In addition, after iron radiation these aorta also produced significantly more ROS. Through short-term XO inhibition, we could decrease this ROS production to amounts equal to un-irradiated aorta. We next look specifically at XO activity. After gamma radiation exposure, the XO activity was significantly elevated in rat aorta. There is also less damaging form of the XO enzyme, called xanthine dehydrogenase (XDH). The XDH activity was also increased, but to a lesser extent than XO activity. As a result, the XO-to-XDH ratio was greater in irradiated aortas, compared to un-irradiated aorta. As expected, dietary XO inhibition caused a decrease of XO and XDH activities, and the XO-to-XDH ratio. We have collected preliminary data showing an increase of both XO and XDH protein expression in response to radiation. Through continuing studies of XO activity and protein amount, we hope to determine the radiation-induced XO conversion mechanism. In conclusion, the inhibition of xanthine oxidase provides significant protection against radiation exposure.

We also examined how radiation affected blood vessels’ ability to produce new blood vessels, known as angiogenesis. To accomplish this we implemented an aortic angiogenesis assay in which aorta is embedded in a three-dimension biological substrate. After embedding the aortic sections, of both un-irradiated and irradiated rats, we would store the aorta in conditions to promote cell growth. After 4 days of incubation we would measure the cellular outgrowth from the aortic section. Before quantifying growth, we were able to determine that the cell outgrowth is endothelial dependent and that the cells sprouting from the aorta are mostly endothelial cells. We found that 1 day after single high dose of gamma radiation, the cell outgrowth was significantly reduces. This implies that blood vessels lose the ability to repair themselves or make new blood vessels after radiation injury. As a result, the vasculature is vulnerable to future complications. We will continue these studies and measure angiogenesis at lower radiation doses.

Understanding radiation dose limits and thresholds is important for biological safety. These thresholds are most likely dependent on both damage pathways and endogenous protective mechanisms. Above, we described some potential damaging pathways in the cardiovascular system in response to radiation. However, we also investigated a potent antioxidant defense mechanism. When exposed to oxidative stress, a master transcription factor protein, Nrf2, moves from the cellular cytosol to the nucleus and binds with the antioxidant response element (ARE) on numerous antioxidant genes. As a result, numerous antioxidant genes are produced to provide defense against the oxidative stress that triggered the response. Using a cellular model of human aortic endothelial cells (HAEC) we found that gamma irradiation can cause the translocation of Nrf2 into the cellular nucleus. However, at our selected radiation dose range, we did not observe increased protein amounts for two of the downstream antioxidant proteins. We supported this finding with Taqman gene expression analysis. We did not see an increase of protein amount or gene expression of heme oxygenase or NAD(P)H:quinine oxidoreductase. We are interested if these genes can be induced at lower radiation doses.

In summary, we’ve made significant progress in understanding the role of XO in radiation-induced damage. We also found that long term dietary inhibition delivers significant radiation protection. We’ve successfully implemented the established angiogenesis assay in our laboratory to assess endothelial damage and repair capability. In addition, we’ve acquired preliminary data indicating that the Nrf2 antioxidant defense system is either impaired by radiation or is not a viable defense system for radiation exposure, at least over our tested dose range. We plan to advance all these studies.

Therefore, our Specific Aims are:

Hypothesis 1: Charged particles (iron ions) will produce an acute oxidative stress event characterized by cellular and tissue injury expressed by endothelial and myocardial dysfunction.

Specific Aim 1: Time- and dose-responses for multiple indices of endothelial and myocardial function will be established in adult Wistar rats exposed to 600 MeV/n Fe (iron) beams at the NASA Space Radiation Laboratory, Brookhaven National Laboratory (BNL). Animals will studied non-invasively and tissues will be collected for histological, functional and molecular analyses using methods established in our laboratory at different time points. Indices of normal tissue function and homeostasis to be investigated include:

a) Endothelium: 1) vascular stiffness by Doppler effect using pulse wave velocity; 2) endothelial function in isolated vascular ring tissue and microvessels; 3) markers of apoptosis in vascular tissue.

b) Heart: 1) myocardial contractile function and contractile reserve in vivo; 2) contractility and contractile reserve in vitro in isolated cardiac myocytes; 3) markers of apoptosis in cardiac tissue (as above).

Hypothesis 2: Iron irradiation-induced endothelial and myocardial contractile dysfunction results from the specific imbalance in NO signaling induced by increased ROS production.

Specific Aim 2: To determine the whether low-fluences of iron ions alter the balance in NO signaling as a function of increased ROS production thereby impairing endothelial and myocardial function. Radiation doses will be selected based on results of Aim 1 and animals will be sacrificed for detailed analyses at various time points as in Aim 1. Vascular and heart tissues from adult Wistar rats exposed to 600 MeV/n Fe ions will be collected and we will measure:

1) NO bioavailability in vascular rings and NOx in plasma , 2) NOS activity using fluorescent dye in heart and blood vessels, 3) ROS levels using chemiluminescence and fluorescence bioassays, 4) Nitroso-tyrosine expression in vascular and cardiac tissue using Western blot analysis.

Hypothesis 3: XO, NOS, and arginase pathways play a critical role in the cardiovascular response to HZE particle radiation.

Specific Aim 3: Rats will be exposed to 600 MeV/n iron ions to determine the specific roles of XO, NOS and arginase in modulating cellular and tissue response to charge particle-induced oxidative stress. Radiation doses will be selected based on results of Aims 1-2 and animals will be sacrificed for detailed analyses at various time points as in Aim 1 for the following endpoints:

1) expression and activity of NOS, Arginase and XO at an RNA and protein level using quatitative PCR, Western blot and immunohistochemistry in heart and blood vessels; 2) Enzyme activity using specific inhibitors of each of the enzymes both alone and in combination with our in vitro vascular ring bioassay and isolated cardiac myocytes; 3) The effect of specific inhibitors on bioassays of ROS and NO (as in Aim 2). Hypothesis 4: Enzyme inhibitors and ROS scavengers will modulate early and late cardiovascular toxicity of low-fluences of iron ions.

Specific Aim 4: To determine if enzyme inhibitors and ROS scavengers can modulate the cardio-vascular effects of iron ions, Wistar rats and/or tissue preparations will be treated with enzyme inhibitors or ROS scavengers prior to and following 600 MeV/n Fe beam irradiation. We will use in vivo and in vitro bioassays of endothelial and myocardial function to test whether the XO inhibitor allopurinol, and the arginase inhibitors S-(2-boronoethyl)-L-cysteine (BEC), or difluoromethylornithine (DFMO) will attenuate radiation-induced cardiovascular effects.

While IR may have parallel effects on peripheral vasculature endothelium and cardiac contractile tissue, the interaction between the blood vessels and heart (ventricular-vascular coupling) has further profound effects on each of these systems. It is for this reason that an approach which incorporates both in vivo (integrated cardiovascular measures such as PWV and P-V loops), as well as isolated cellular and tissue measures of function is so important. Our methodologies will allow us to assess the contribution of each component (heart and vasculature) to the integrated system response to charged particle exposure.

In summary, we have demonstrated that:

1) Both conventional gamma and Fe56 ion radiation results in significant endothelial dysfunction.

2) This endothelial dysfunction results in a significant increase in vascular stiffness. 3) Endothelial dysfunction results in part from an increase in ROS production.

4) The source of the ROS is predominantly but most likely not exclusively xanthine oxidase.

5) Inhibition of XO prior to following radiation with Oxp results in a significant improvement in endothelial function and a decrease in vascular stiffness in irradiated rats.

Thus, XO appears to be a critical target in countermeasure design for radiation-induced cardiovascular dysfunction.

We hypothesize that charged particles will produce an acute oxidative stress event with cellular injury and possible death with early and late consequences that are dose, LET (linear energy transfer), and time-dependent. Endothelial and myocardial dysfunction represent integrated cumulative indicators of this cellular injury. We further hypothesize that radiation induced endothelial and myocardial contractile dysfunction results from the specific imbalance in NO signaling induced by increased ROS production. In addition, we hypothesize that the XO, NOS (Nitric Oxide Synthase), arginase pathways play a critical role in the response to radiation induced OS.

Therefore, our Specific Aims are:

Hypothesis 1: Charged particles (iron ions) will produce an acute oxidative stress event characterized by cellular and tissue injury expressed by endothelial and myocardial dysfunction.

Specific Aim 1: Time- and dose-responses for multiple indices of endothelial and myocardial function will be established in adult Wistar rats exposed to 600 MeV/n Fe (iron) beams at the NASA Space Radiation Laboratory Brookhaven National Laboratory (BNL). Animals will studied non-invasively and tissues will be collected for histological, functional and molecular analyses using methods established in our laboratory at different time points. Indices of normal tissue function and homeostasis to be investigated include:

a) Endothelium: 1) vascular stiffness by Doppler effect using pulse wave velocity; 2) endothelial function in isolated vascular ring tissue and microvessels; 3) markers of apoptosis in vascular tissue.

b) Heart: 1) myocardial contractile function and contractile reserve in vivo ; 2) contractility and contractile reserve in vitro in isolated cardiac myocytes; 3) markers of apoptosis in cardiac tissue (as above) .

Hypothesis 2: Iron irradiation-induced endothelial and myocardial contractile dysfunction results from the specific imbalance in NO signaling induced by increased ROS production.

Specific Aim 2: To determine the whether low-fluences of iron ions alter the balance in NO signaling as a function of increased ROS production thereby impairing endothelial and myocardial function. Radiation doses will be selected based on results of Aim 1 and animals will be sacrificed for detailed analyses at various time points as in Aim 1. Vascular and heart tissues from adult Wistar rats exposed to 600 MeV/n Fe ions will be collected and we will measure:

1 )NO bioavailability in vascular rings and NOx in plasma , 2) NOS activity using Fluorescent dye in heart and blood vessels, 3) ROS levels using chemiluminescence and fluorescence bioassays, 4) Nitrosotyrosine expression in vascular and cardiac tissue using Western blot analysis.

Hypothesis 3: XO, NOS, and arginase pathways play a critical role in the cardiovascular response to HZE particle radiation.

Specific Aim 3: Rats will be exposed to 600 MeV/n iron ions to determine the specific roles of XO, NOS and arginase in modulating cellular and tissue response to charge particle induced oxidative stress. Radiation doses will be selected based on results of Aim 1-2 and animals will be sacrificed for detailed analyses at various time points as in Aim 1 for the following endpoints:

1) expression and activity of NOS, Arginase and XO at an RNA and protein level using quatitative PCR, Western blot and immunohistochemistry in heart and blood vessels ; 2) Enzyme activity using specific inhibitors of each of the enzymes both alone and in combination with our in vitro vascular ring bioassay and isolated cardiac myocytes ; 3) The effect of specific inhibitors on bioassays of ROS and NO (as in SA2) .

Hypothesis 4: Enzyme inhibitors and ROS scavengers will modulate the early and late cardiovascular toxicity of low fluences of iron ions.

Specific Aim 4: To determine if enzyme inhibitors and ROS scavengers can modulate the cardio-vascular effects of iron ions, Wistar rats and/or tissue preparations will be treated with enzyme inhibitors or ROS scavengers prior to and following 600 MeV/n Fe beam irradiation. We will use in vivo and in vitro bioassays of endothelial and myocardial function to test whether the XO inhibitor allopurinol, and the arginase inhibitors S-(2-boronoethyl)-L-cysteine (BEC), or difluoromethylornithine (DFMO) will attenuate radiation induced cardiovascular effects.

While IR may have parallel effects on peripheral vasculature endothelium) and cardiac contractile tissue, the interaction between the blood vessels and heart (ventricular-vascular coupling) has further profound effects on each of these systems. It is for this reason that an approach which incorporates both in vivo (integrated cardiovascular measures such as PWV and P-V loops) as well as isolated cellular and tissue measures of function is so important. Our methodologies will allow us to assess the contribution of each component (heart and vasculature) to the integrated system response to charge particle exposure.

Male Sprague-Dawley rats were exposed to different doses of gamma-ray irradiation. Measurements of vascular stiffness, vasocontractility, vasorelaxation, and Xanthine Oxidase activity and expression were studied.

Epidemiologic data and limited experimental data support the notion that radiation has significant effects on the cardiovascular system. These effects include vascular pathologies including accelerated atherosclerosis and hypertension, as well as primary myocardial dysfunction as a result of impaired myocytes contractility. Our current preliminary pilot study is one of the first studies in vivo and ex vivo to demonstrate that radiation induces changes in vascular stiffness (a well known independent predictor of cardiovascular events) as well as vascular endothelial dysfunction. Moreover, the impaired endothelial function could be reversed in vitro in the presence of the XO inhibitor oxypurinol. As highlighted in the background it is week established that XO is one of the primary sources of ROS in the cardiovascular system. In the blood vessels, an upregulation to XO contributes to endothelial dysfunction and vascular pathobiology in diabetes and aging. In the heart, and upregulation of XO contributes to the pathobiology of heart failure and the XO inhibitor markedly attenuates developed heart failure pathophysiology. Our pilot study supports the hypothesis that and activation/upregulation of XO may be an important/the important pathophysiologic consequence of radiation.

The preliminary data demonstrated here is consistent with our original hypothesis. However, this data is observed with Low LET radiation. We await the results of the studies to be preformed at NSRL in the summer to confirm this hypothesis. If indeed upregulation of XO contributes to oxidative stress and endothelial dysfunction, XO may be a target for prevention and possible treatment of radiation-induced cardiovascular function.

Presentations: 4th International Workshop on Space Radiation Research and 17th Annual NASA Space Radiation Health Investigator’s Workshop, Moscow and St. Petersburg, June 5-9, 2006. Abstract title: Single exposure gamma-irradiation amplifies xanthine oxidase activity and induces endothelial dysfunction in rat aorta.

2006 NASA Space Radiation Summer School Lecture: Cardiovascular Tissue Responses.