Menu

 

The NASA Task Book
Advanced Search     

Project Title:  Ionizing Radiation and its Effects on Cardiovascular Function in the Context of Space Flight Reduce
Fiscal Year: FY 2012 
Division: Human Research 
Research Discipline/Element:
HRP SR:Space Radiation
Start Date: 07/01/2005  
End Date: 12/30/2011  
Task Last Updated: 02/13/2012 
Download report in PDF pdf
Principal Investigator/Affiliation:   Berkowitz, Dan E. M.D. / The Johns Hopkins University 
Address:  Department of Anesthesiology & Critical Care Medicine 
600 North Wolfe Street 
Baltimore , MD 21287-8711 
Email: dberkow1@jhmi.edu 
Phone: 410-614-1517  
Congressional District:
Web:  
Organization Type: UNIVERSITY 
Organization Name: The Johns Hopkins University 
Joint Agency:  
Comments:  
Co-Investigator(s)
Affiliation: 
Nyhan, Daniel  Johns Hopkins 
Shoukas, Artin  Johns Hopkins 
Vazquez, Marcello  Brookhaven National Laboratory  
Project Information: Grant/Contract No. NNJ05HF03G 
Responsible Center: NASA JSC 
Grant Monitor: Simonsen, Lisa  
Center Contact:  
lisa.c.simonsen@nasa.gov 
Solicitation / Funding Source: 2004 Radiation Biology NNH04ZUU005N 
Grant/Contract No.: NNJ05HF03G 
Project Type: GROUND 
Flight Program:  
TechPort: No 
No. of Post Docs:
No. of PhD Candidates:
No. of Master's Candidates:
No. of Bachelor's Candidates:
No. of PhD Degrees:
No. of Master's Degrees:
No. of Bachelor's Degrees:
Human Research Program Elements: (1) SR:Space Radiation
Human Research Program Risks: (1) Degen:Risk Of Cardiovascular Disease and Other Degenerative Tissue Effects From Radiation Exposure (IRP Rev F)
Human Research Program Gaps: (1) Degen01:How can tissue specific experimental models be developed for the major degenerative tissue risks, including cardiovascular, lens, digestive, endocrine, and other tissue systems in order to estimate space radiation risks for degenerative diseases? (IRP Rev F)
(2) Degen02:What are the adverse outcome pathways associated with degenerative tissues changes in the cardiovascular, cerebrovascular, lens, immune, digestive, endocrine, and other tissue systems? What are the key events or hallmarks, their time sequence, and their associated biomarkers? (IRP Rev J)
Flight Assignment/Project Notes: NOTE: End date changed per 11/11/2011 HRP Master Task List information (Ed., 2/2/2012)

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)

Task Description: An appropriate examination of the health risks associated with manned space flight necessitates an understanding of the molecular consequences of exposure to the radiations encountered in space. Human radio-epidemiologic data and animal studies indicate that irradiation of the heart can cause a spectrum of cardiovascular complications. The mechanisms suggested for these alterations are chronic inflammation induced by oxidative stress. It is well known that ionizing radiation (IR) produces biological damage by direct effect on DNA and indirectly by generation of reactive oxygen species (ROS) in the cellular milieu. The xanthine oxidoreductase (XOD) system is one of the major sources of free radicals in biologic systems. Since the XOD system is present primarily in the reduced XDH form in normal tissue, the production of free radicals is negligible. However, emerging data demonstrates that IR irreversibly converts the xanthine dehydrogenase (XDH) to xanthine oxidase (XO) leading to amplification and persistence of IR induced, ROS dependent cell damage. It is well known that ROS interferes with cellular signaling (nitrosylation and phosphorylation) and is pro-apoptotic (releases mitochondrial cytochrome-C and activates apoptotic pathways). One of the postulated mechanisms of radiation related tissue injury is endothelial cell damage. However little is known regarding other cellular and molecular targets in the pathophysiology of radiation-induced cardiovascular system dysfunction. Furthermore, little is known regarding the response of endothelial cells and cardiac myocytes to high LET (linear energy transfer) radiation. In this proposal we intend to use established in vivo and in vitro bioassays to characterize the radiation response to charged particle exposure. Furthermore, mechanistically we will focus on the interaction between ROS and nitric oxide (NO) pathways in the regulation of myocardial and vascular structure and function following oxidative stress (OS) induced by high LET radiation. Our group have demonstrated the important reciprocal interaction between NO and O2- (derived from XO) in the regulation of myocardial contractility and endothelial function. We will utilize our expertise to determine the effect of radiation on these important signaling pathways in the cardiovascular system. 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, 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) 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.

Research Impact/Earth Benefits: Our research primarily studies space-related radiation effects. However, the majority of our iron-radiation studies are paired with similar studies investigating gamma-radiation biological effects. Gamma-radiation is a very prevalent source of radiation on earth, particularly in medical radiotherapy. Our research focuses on cardiovascular diseases and complications caused by radiation exposure. Many medical radiotherapies target the body core, where the heart and major veins and arteries are located. This is true in cardiac imaging techniques and treatment for cancers, such as Hodgkin's Disease. Thus, radiotherapy has potential to be very damaging to the cardiovascular system.

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.

Task Progress & Bibliography Information FY2012 
Task Progress: Supplement to grant: Combined effect of Radiation and Hyperoxia on Cardiovascular Function

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.

Bibliography Type: Description: (Last Updated: 01/13/2014) 

Show Cumulative Bibliography Listing
 
Articles in Peer-reviewed Journals Soucy KG, Lim HK, Kim JH, Oh Y, Attarzadeh DO, Sevinc B, Kuo MM, Shoukas AA, Vazquez ME, Berkowitz DE. "HZE 56Fe-ion irradiation induces endothelial dysfunction in rat aorta: role of xanthine oxidase." Radiat Res. 2011 Oct;176(4):474-85. Epub 2011 Jul 25. PubMed PMID: 21787183 , Oct-2011
Project Title:  Ionizing Radiation and its Effects on Cardiovascular Function in the Context of Space Flight Reduce
Fiscal Year: FY 2010 
Division: Human Research 
Research Discipline/Element:
HRP SR:Space Radiation
Start Date: 07/01/2005  
End Date: 12/30/2011  
Task Last Updated: 07/26/2010 
Download report in PDF pdf
Principal Investigator/Affiliation:   Berkowitz, Dan E. M.D. / The Johns Hopkins University 
Address:  Department of Anesthesiology & Critical Care Medicine 
600 North Wolfe Street 
Baltimore , MD 21287-8711 
Email: dberkow1@jhmi.edu 
Phone: 410-614-1517  
Congressional District:
Web:  
Organization Type: UNIVERSITY 
Organization Name: The Johns Hopkins University 
Joint Agency:  
Comments:  
Co-Investigator(s)
Affiliation: 
Nyhan, Daniel  Johns Hopkins 
Shoukas, Artin  Johns Hopkins 
Vazquez, Marcello  Brookhaven National Laboratory  
Project Information: Grant/Contract No. NNJ05HF03G 
Responsible Center: NASA JSC 
Grant Monitor: Cucinott1a, Francis  
Center Contact: 281-483-0968 
noaccess@nasa.gov 
Solicitation / Funding Source: 2004 Radiation Biology NNH04ZUU005N 
Grant/Contract No.: NNJ05HF03G 
Project Type: GROUND 
Flight Program:  
TechPort: No 
No. of Post Docs:
No. of PhD Candidates:
No. of Master's Candidates:
No. of Bachelor's Candidates:
No. of PhD Degrees:  
No. of Master's Degrees:  
No. of Bachelor's Degrees:
Human Research Program Elements: (1) SR:Space Radiation
Human Research Program Risks: (1) Degen:Risk Of Cardiovascular Disease and Other Degenerative Tissue Effects From Radiation Exposure (IRP Rev F)
Human Research Program Gaps: (1) Degen01:How can tissue specific experimental models be developed for the major degenerative tissue risks, including cardiovascular, lens, digestive, endocrine, and other tissue systems in order to estimate space radiation risks for degenerative diseases? (IRP Rev F)
(2) Degen02:What are the adverse outcome pathways associated with degenerative tissues changes in the cardiovascular, cerebrovascular, lens, immune, digestive, endocrine, and other tissue systems? What are the key events or hallmarks, their time sequence, and their associated biomarkers? (IRP Rev J)
Flight Assignment/Project Notes: NOTE: End date changed per 11/11/2011 HRP Master Task List information (Ed., 2/2/2012)

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)

Task Description: An appropriate examination of the health risks associated with manned space flight necessitates an understanding of the molecular consequences of exposure to the radiations encountered in space. Human radio-epidemiologic data and animal studies indicate that irradiation of the heart can cause a spectrum of cardiovascular complications. The mechanisms suggested for these alterations are chronic inflammation induced by oxidative stress. It is well known that ionizing radiation (IR) produces biological damage by direct effect on DNA and indirectly by generation of reactive oxygen species (ROS) in the cellular milieu. The xanthine oxidoreductase (XOD) system is one of the major sources of free radicals in biologic systems. Since the XOD system is present primarily in the reduced XDH form in normal tissue, the production of free radicals is negligible. However, emerging data demonstrates that IR irreversibly converts the xanthine dehydrogenase (XDH) to xanthine oxidase (XO) leading to amplification and persistence of IR induced, ROS dependent cell damage. It is well known that ROS interferes with cellular signaling (nitrosylation and phosphorylation) and is pro-apoptotic (releases mitochondrial cytochrome-C and activates apoptotic pathways). One of the postulated mechanisms of radiation related tissue injury is endothelial cell damage. However little is known regarding other cellular and molecular targets in the pathophysiology of radiation-induced cardiovascular system dysfunction. Furthermore, little is known regarding the response of endothelial cells and cardiac myocytes to high LET (linear energy transfer) radiation. In this proposal we intend to use established in vivo and in vitro bioassays to characterize the radiation response to charged particle exposure. Furthermore, mechanistically we will focus on the interaction between ROS and nitric oxide (NO) pathways in the regulation of myocardial and vascular structure and function following oxidative stress (OS) induced by high LET radiation. Our group have demonstrated the important reciprocal interaction between NO and O2- (derived from XO) in the regulation of myocardial contractility and endothelial function. We will utilize our expertise to determine the effect of radiation on these important signaling pathways in the cardiovascular system. 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, 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) 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.

Research Impact/Earth Benefits: Our research primarily studies space-related radiation effects. However, the majority of our iron-radiation studies are paired with similar studies investigating gamma-radiation biological effects. Gamma-radiation is a very prevalent source of radiation on earth, particularly in medical radiotherapy. Our research focuses on cardiovascular diseases and complications caused by radiation exposure. Many medical radiotherapies target the body core, where the heart and major veins and arteries are located. This is true in cardiac imaging techniques and treatment for cancers, such as Hodgkin's Disease. Thus, radiotherapy has potential to be very damaging to the cardiovascular system.

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.

Task Progress & Bibliography Information FY2010 
Task Progress: Our most current findings demonstrate that the aortic endothelium develops reduced migratory or proliferative capacity shortly after whole-body radiation exposure. The anti-angiogenic effect is maintained at later time points after radiation. Furthermore, a similar angiogenic impairment is produced with whole-body HZE Fe irradiation and by direct ex vivo irradiation of aorta. This may contribute to radiation-induced endothelial dysfunction or it may leave the vasculature vulnerable to future vascular injury.

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.

Bibliography Type: Description: (Last Updated: 01/13/2014) 

Show Cumulative Bibliography Listing
 
Articles in Peer-reviewed Journals Soucy KG, Lim HK, Attarzadeh DO, Santhanam L, Kim JH, Bhunia AK, Sevinc B, Ryoo S, Vazquez ME, Nyhan D, Shoukas AA, Berkowitz DE. "Dietary inhibition of xanthine oxidase attenuates radiation-induced endothelial dysfunction in rat aorta." J Appl Physiol. 2010 May;108(5):1250-8. PMID: 20167676 , May-2010
Articles in Peer-reviewed Journals Soucy KG, Attarzadeh DO, Ramachandran R, Soucy PA, Romer LH, Shoukas AA, Berkowitz DE. "Single exposure to radiation produces early anti-angiogenic effects in mouse aorta." Radiat Environ Biophys. 2010 Aug;49(3):397-404. Epub 2010 Apr 18. PMID: 20401726 , Aug-2010
Project Title:  Ionizing Radiation and its Effects on Cardiovascular Function in the Context of Space Flight Reduce
Fiscal Year: FY 2009 
Division: Human Research 
Research Discipline/Element:
HRP SR:Space Radiation
Start Date: 07/01/2005  
End Date: 06/30/2011  
Task Last Updated: 06/15/2009 
Download report in PDF pdf
Principal Investigator/Affiliation:   Berkowitz, Dan E. M.D. / The Johns Hopkins University 
Address:  Department of Anesthesiology & Critical Care Medicine 
600 North Wolfe Street 
Baltimore , MD 21287-8711 
Email: dberkow1@jhmi.edu 
Phone: 410-614-1517  
Congressional District:
Web:  
Organization Type: UNIVERSITY 
Organization Name: The Johns Hopkins University 
Joint Agency:  
Comments:  
Co-Investigator(s)
Affiliation: 
Nyhan, Daniel  Johns Hopkins 
Shoukas, Artin  Johns Hopkins 
Vazquez, Marcello  Brookhaven National Laboratory  
Project Information: Grant/Contract No. NNJ05HF03G 
Responsible Center: NASA JSC 
Grant Monitor: Cucinott1a, Francis  
Center Contact: 281-483-0968 
noaccess@nasa.gov 
Solicitation / Funding Source: 2004 Radiation Biology NNH04ZUU005N 
Grant/Contract No.: NNJ05HF03G 
Project Type: GROUND 
Flight Program:  
TechPort: No 
No. of Post Docs:
No. of PhD Candidates:
No. of Master's Candidates:
No. of Bachelor's Candidates:
No. of PhD Degrees:  
No. of Master's Degrees:  
No. of Bachelor's Degrees:
Human Research Program Elements: (1) SR:Space Radiation
Human Research Program Risks: (1) Degen:Risk Of Cardiovascular Disease and Other Degenerative Tissue Effects From Radiation Exposure (IRP Rev F)
Human Research Program Gaps: (1) Degen01:How can tissue specific experimental models be developed for the major degenerative tissue risks, including cardiovascular, lens, digestive, endocrine, and other tissue systems in order to estimate space radiation risks for degenerative diseases? (IRP Rev F)
(2) Degen02:What are the adverse outcome pathways associated with degenerative tissues changes in the cardiovascular, cerebrovascular, lens, immune, digestive, endocrine, and other tissue systems? What are the key events or hallmarks, their time sequence, and their associated biomarkers? (IRP Rev J)
Flight Assignment/Project Notes: 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)

Task Description: An appropriate examination of the health risks associated with manned space flight necessitates an understanding of the molecular consequences of exposure to the radiations encountered in space. Human radio-epidemiologic data and animal studies indicate that irradiation of the heart can cause a spectrum of cardiovascular complications. The mechanisms suggested for these alterations are chronic inflammation induced by oxidative stress. It is well known that ionizing radiation (IR) produces biological damage by direct effect on DNA and indirectly by generation of reactive oxygen species (ROS) in the cellular milieu. The xanthine oxidoreductase (XOD) system is one of the major sources of free radicals in biologic systems. Since the XOD system is present primarily in the reduced XDH form in normal tissue, the production of free radicals is negligible. However, emerging data demonstrates that IR irreversibly converts the xanthine dehydrogenase (XDH) to xanthine oxidase (XO) leading to amplification and persistence of IR induced, ROS dependent cell damage. It is well known that ROS interferes with cellular signaling (nitrosylation and phosphorylation) and is pro-apoptotic (releases mitochondrial cytochrome-C and activates apoptotic pathways). One of the postulated mechanisms of radiation related tissue injury is endothelial cell damage. However little is known regarding other cellular and molecular targets in the pathophysiology of radiation-induced cardiovascular system dysfunction. Furthermore, little is known regarding the response of endothelial cells and cardiac myocytes to high LET (linear energy transfer) radiation. In this proposal we intend to use established in vivo and in vitro bioassays to characterize the radiation response to charged particle exposure. Furthermore, mechanistically we will focus on the interaction between ROS and nitric oxide (NO) pathways in the regulation of myocardial and vascular structure and function following oxidative stress (OS) induced by high LET radiation. Our group have demonstrated the important reciprocal interaction between NO and O2- (derived from XO) in the regulation of myocardial contractility and endothelial function. We will utilize our expertise to determine the effect of radiation on these important signaling pathways in the cardiovascular system. 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, 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) 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.

Research Impact/Earth Benefits: Our research primarily studies space-related radiation effects. However, the majority of our iron-radiation studies are paired with similar studies investigating gamma-radiation biological effects. Gamma-radiation is a very prevalent source of radiation on earth, particularly in medical radiotherapy. Our research focuses on cardiovascular diseases and complications caused by radiation exposure. Many medical radiotherapies target the body core, where the heart and major veins and arteries are located. This is true in cardiac imaging techniques and treatment for cancers, such as Hodgkin's Disease. Thus, radiotherapy has potential to be very damaging to the cardiovascular system.

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.

Task Progress & Bibliography Information FY2009 
Task Progress: Radiation exposure and the associated risks are important concerns in medical radiotherapy, occupational exposure, and manned space flight. Dose limits in particular fields have been established, but mostly in terms of excess cancer mortality. The risks of other fatal consequences and diseases still remain largely uncharacterized. Regardless, irradiation of the heart and vasculature has been implicated in the development of significant cardiovascular complications. While epidemiologic studies indicate a strong relationship between ionizing radiation and cardiovascular events, little is known about the pathobiology of this phenomenon. Consequently, potential therapies and countermeasures are sorely lacking. It is well known that radiation generates biological injury through DNA damage and production of reactive oxygen species (ROS). ROS is a critical signaling molecule at low levels, however, at high levels it can damage biomolecules, induce cellular death, and disrupt vital signaling pathways. In fact, oxidative stress is an accepted marker of poor vascular health observed in aging, hypertension, and other cardiovascular dysfunctions. More specifically, in the endothelial cell layer of the vasculature, ROS has been shown to scavenge the protective molecule, nitric oxide (NO). Through this common feature of radiation effect and cardiovascular disease, we have examined the molecular mechanisms of radiation-induced vascular injury and repair.

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.

Bibliography Type: Description: (Last Updated: 01/13/2014) 

Show Cumulative Bibliography Listing
 
Abstracts for Journals and Proceedings Soucy KG, Kim JH, Bugaj L, Ryoo S, Vandegaer KM, Nyhan D, Shoukas AA, Berkowitz DE. "Xanthine oxidase inhibition attenuates vascular endothelial dysfunction in irradiated rats." Presented at the NASA Human Research Program Investigators’ Workshop, Houston, TX, February 4-6, 2008.

NASA Human Research Program Investigators’ Workshop, February 4-6, 2008. , Feb-2008

Abstracts for Journals and Proceedings Soucy KG, Bhunia AK, Kim JH, Ryoo S, Vandegaer KM, Shoukas AA, Berkowitz DE. "Radiation impairs nitric oxide bioavailability and induces cellular damage through xanthine oxidase activation." Presented at the NASA Space Radiation Investigators’ Workshop, Philadelphia, PA, June 30 - July 2, 2008.

NASA Space Radiation Investigators’ Workshop, Philadelphia, PA, June 30 - July 2, 2008. , Jul-2008

Abstracts for Journals and Proceedings Soucy KG, Lim HK, Santhanam L, Bhunia A, Ryoo S, Kim JH, Lim HK, Nyhan D, Shoukas AA, Berkowitz DE. "Xanthine oxidase inhibition attenuates vascular endothelial dysfunction in irradiated rats." Presented at the Biomedical Engineering Society Fall Meeting, St. Louis, MO, October 1-4, 2008.

Biomedical Engineering Society Fall Meeting, St. Louis, MO, October 1-4, 2008. , Oct-2008

Abstracts for Journals and Proceedings Soucy KG, Bhunia AK, Chang F, Attarzadeh D, Romer LH, Shoukas AA, Berkowitz DE. "Radiation-induced endothelial dysfunction: Investigating the balance of injury and repair." Presented at NASA Human Research Program Investigators’ Workshop, Houston, TX, February 2-4, 2009.

NASA Human Research Program Investigators’ Workshop, February 2009. , Feb-2009

Abstracts for Journals and Proceedings Soucy KG, Bhunia AK, Attarzadeh D, Sevinc B, Chang F, Romer L, Shoukas AA, Berkowitz DE. "High-dose radiation produces anti-angiogenic effects potentially through impaired Nrf2-antioxidant defenses." Presented at Heavy Ions Symposium, Cologne, Germany, July 6-10, 2009.

Heavy Ions Symposium, Cologne, Germany, July 6-10, 2009. , Jul-2009

Awards Soucy KG, Bhunia AK, Attarzadeh D, Sevinc B, Chang F, Romer L, Shoukas AA, Berkowitz DE. "NASA Student Travel Award, July 2009." Jul-2009
Dissertations and Theses Soucy KG. "Radiation Induces Vascular Dysfunction Through Xanthine Oxidase Activation." Thesis Proposal, Johns Hopkins University, May 2009. , May-2009
Project Title:  Ionizing Radiation and its Effects on Cardiovascular Function in the Context of Space Flight Reduce
Fiscal Year: FY 2007 
Division: Human Research 
Research Discipline/Element:
HRP SR:Space Radiation
Start Date: 07/01/2005  
End Date: 06/30/2009  
Task Last Updated: 07/24/2007 
Download report in PDF pdf
Principal Investigator/Affiliation:   Berkowitz, Dan E. M.D. / The Johns Hopkins University 
Address:  Department of Anesthesiology & Critical Care Medicine 
600 North Wolfe Street 
Baltimore , MD 21287-8711 
Email: dberkow1@jhmi.edu 
Phone: 410-614-1517  
Congressional District:
Web:  
Organization Type: UNIVERSITY 
Organization Name: The Johns Hopkins University 
Joint Agency:  
Comments:  
Co-Investigator(s)
Affiliation: 
Hare, Joshua  Johns Hopkins 
Nyhan, Daniel  Johns Hopkins 
Shoukas, Artin  Johns Hopkins 
Vazquez, Marcello  Brookhaven National Laboratory  
Project Information: Grant/Contract No. NNJ05HF03G 
Responsible Center: NASA JSC 
Grant Monitor:  
Center Contact:   
Solicitation / Funding Source: 2004 Radiation Biology NNH04ZUU005N 
Grant/Contract No.: NNJ05HF03G 
Project Type: GROUND 
Flight Program:  
TechPort: No 
No. of Post Docs:
No. of PhD Candidates:
No. of Master's Candidates:
No. of Bachelor's Candidates:
No. of PhD Degrees:  
No. of Master's Degrees:  
No. of Bachelor's Degrees:  
Human Research Program Elements: (1) SR:Space Radiation
Human Research Program Risks: (1) Degen:Risk Of Cardiovascular Disease and Other Degenerative Tissue Effects From Radiation Exposure (IRP Rev F)
Human Research Program Gaps: (1) Degen01:How can tissue specific experimental models be developed for the major degenerative tissue risks, including cardiovascular, lens, digestive, endocrine, and other tissue systems in order to estimate space radiation risks for degenerative diseases? (IRP Rev F)
(2) Degen02:What are the adverse outcome pathways associated with degenerative tissues changes in the cardiovascular, cerebrovascular, lens, immune, digestive, endocrine, and other tissue systems? What are the key events or hallmarks, their time sequence, and their associated biomarkers? (IRP Rev J)
Task Description: An appropriate examination of the health risks associated with manned space flight necessitates an understanding of the molecular consequences of exposure to the radiations encountered in space. Human radio-epidemiologic data and animal studies indicate that irradiation of the heart can cause a spectrum of cardiovascular complications. The mechanisms suggested for these alterations are chronic inflammation induced by oxidative stress. It is well known that ionizing radiation (IR) produces biological damage by direct effect on DNA and indirectly by generation of reactive oxygen species (ROS) in the cellular milieu. The xanthine oxidoreductase (XOD) system is one of the major sources of free radicals in biologic systems. Since the XOD system is present primarily in the reduced XDH form in normal tissue, the production of free radicals is negligible. However, emerging data demonstrates that IR irreversibly converts the xanthine dehydrogenase (XDH) to xanthine oxidase (XO) leading to amplification and persistence of IR induced, ROS dependent cell damage. It is well known that ROS interferes with cellular signaling (nitrosylation and phosphorylation) and is pro-apoptotic (releases mitochondrial cytochrome-C and activates apoptotic pathways). One of the postulated mechanisms of radiation related tissue injury is endothelial cell damage. However little is known regarding other cellular and molecular targets in the pathophysiology of radiation-induced cardiovascular system dysfunction. Furthermore, little is known regarding the response of endothelial cells and cardiac myocytes to high LET (linear energy transfer) radiation. In this proposal we intend to use established in vivo and in vitro bioassays to characterize the radiation response to charged particle exposure. Furthermore, mechanistically we will focus on the interaction between ROS and nitric oxide (NO) pathways in the regulation of myocardial and vascular structure and function following oxidative stress (OS) induced by high LET radiation. Our group have demonstrated the important reciprocal interaction between NO and O2- (derived from XO) in the regulation of myocardial contractility and endothelial function. We will utilize our expertise to determine the effect of radiation on these important signaling pathways in the cardiovascular system. 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, 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) 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.

Research Impact/Earth Benefits: 0

Task Progress & Bibliography Information FY2007 
Task Progress: Our data strongly supports the hypothesis that a single, high-dose gamma-irradiation can induce endothelial dysfunction. We have confirmed that this phenomenon is observed. The effects observed two-weeks post-radiation include: increased in vivo aortic stiffness and impaired endothelial-dependent vasorelaxation. In addition, we demonstrate a substantial increase of XO activity and a restoration of vasorelaxation after XO-specific inhibition. Furthermore, we have demonstrated that ROS production is increased in irradiated rats and that the source of the ROS is most likely XO. Finally, and most importantly, administration of Oral Oxp, the XO inhibitor results in a significant attenuation of endothelial dysfunction and prevents the development of increased vascular stiffness in irradiated rats. Thus, radiation insult can produce endothelial dysfunction and vascular stiffening, with the XO upregulation system being a probably contributory mechanism. More importantly, xanthine oxido-reductase appears to be a valuable target for radiation-induced vascular pathology.

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.

Bibliography Type: Description: (Last Updated: 01/13/2014) 

Show Cumulative Bibliography Listing
 
Articles in Peer-reviewed Journals Soucy KG, Lim HK, Benjo A, Santhanam L, Ryoo S, Shoukas AA, Vazquez ME, Berkowitz DE. "Single exposure gamma-irradiation amplifies xanthine oxidase activity and induces endothelial dysfunction in rat aorta." Radiat Environ Biophys. 2007 Jun;46(2):179-86. Epub 2007 Jan 26. PMID: 17256177 , Jun-2007
Articles in Peer-reviewed Journals Berkowitz DE. "Myocyte nitroso-redox imbalance in sepsis: NO simple answer." Circ Res. 2007 Jan 5;100(1):1-4. Review. PMID: 17204656 , Jan-2007
Project Title:  Ionizing Radiation and its Effects on Cardiovascular Function in the Context of Space Flight Reduce
Fiscal Year: FY 2006 
Division: Human Research 
Research Discipline/Element:
HRP SR:Space Radiation
Start Date: 07/01/2005  
End Date: 06/30/2009  
Task Last Updated: 08/15/2006 
Download report in PDF pdf
Principal Investigator/Affiliation:   Berkowitz, Dan E. M.D. / The Johns Hopkins University 
Address:  Department of Anesthesiology & Critical Care Medicine 
600 North Wolfe Street 
Baltimore , MD 21287-8711 
Email: dberkow1@jhmi.edu 
Phone: 410-614-1517  
Congressional District:
Web:  
Organization Type: UNIVERSITY 
Organization Name: The Johns Hopkins University 
Joint Agency:  
Comments:  
Co-Investigator(s)
Affiliation: 
Hare, Joshua  Johns Hopkins 
Nyhan, Daniel  Johns Hopkins 
Shoukas, Artin  Johns Hopkins 
Vazquez, Marcello  Brookhaven National Laboratory  
Project Information: Grant/Contract No. NNJ05HF03G 
Responsible Center: NASA JSC 
Grant Monitor:  
Center Contact:   
Solicitation / Funding Source: 2004 Radiation Biology NNH04ZUU005N 
Grant/Contract No.: NNJ05HF03G 
Project Type: GROUND 
Flight Program:  
TechPort: No 
No. of Post Docs:
No. of PhD Candidates:
No. of Master's Candidates:
No. of Bachelor's Candidates:
No. of PhD Degrees:  
No. of Master's Degrees:  
No. of Bachelor's Degrees:  
Human Research Program Elements: (1) SR:Space Radiation
Human Research Program Risks: (1) Degen:Risk Of Cardiovascular Disease and Other Degenerative Tissue Effects From Radiation Exposure (IRP Rev F)
Human Research Program Gaps: (1) Degen01:How can tissue specific experimental models be developed for the major degenerative tissue risks, including cardiovascular, lens, digestive, endocrine, and other tissue systems in order to estimate space radiation risks for degenerative diseases? (IRP Rev F)
(2) Degen02:What are the adverse outcome pathways associated with degenerative tissues changes in the cardiovascular, cerebrovascular, lens, immune, digestive, endocrine, and other tissue systems? What are the key events or hallmarks, their time sequence, and their associated biomarkers? (IRP Rev J)
Task Description: An appropriate examination of the health risks associated with manned space flight necessitates an understanding of the molecular consequences of exposure to the radiations encountered in space. Human radio-epidemiologic data and animal studies indicate that irradiation of the heart can cause a spectrum of cardiovascular complications. The mechanisms suggested for these alterations are chronic inflammation induced by oxidative stress. It is well known that ionizing radiation (IR) produces biological damage by direct effect on DNA and indirectly by generation of reactive oxygen species (ROS) in the cellular milieu. The xanthine oxidoreductase (XOD) system is one of the major sources of free radicals in biologic systems. Since the XOD system is present primarily in the reduced XDH form in normal tissue, the production of free radicals is negligible. However, emerging data demonstrates that IR irreversibly converts the xanthine dehydrogenase (XDH) to xanthine oxidase (XO) leading to amplification and persistence of IR induced, ROS dependent cell damage. It is well known that ROS interferes with cellular signaling (nitrosylation and phosphorylation) and is pro-apoptotic (releases mitochondrial cytochrome-C and activates apoptotic pathways). One of the postulated mechanisms of radiation related tissue injury is endothelial cell damage. However little is known regarding other cellular and molecular targets in the pathophysiology of radiation induced cardiovascular system dysfunction. Furthermore little is known regarding the response of endothelial cells and cardiac myocytes to high LET radiation. In this proposal we intend to use established in vivo and in vitro bioassays to characterize the radiation response to charged particle exposure. Furthermore, mechanistically will focus on the interaction between ROS and nitric oxide (NO) pathways in the regulation of both myocardial and vascular structure and function following OS induced by high LET radiation. Our group have demonstrated the important reciprocal interaction between NO and O2- (derived from XO) in the regulation of myocardial contractility and endothelial function. We will utilize our expertise to determine the effect of radiation on these important signaling pathways in the cardiovascular system.

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.

Research Impact/Earth Benefits: 0

Task Progress & Bibliography Information FY2006 
Task Progress: This report represents the report of the first year of the proposed studies. As such, our first animal experiments will take place this summer (06/23/06) for which we applied and were awarded beamline time at the Brookhaven National Laboratory. In the interim, we have conducted pilot experiments at the Johns Hopkins University Medical campus using conventional Gamma radiation facilities. Our findings are exciting and are consistent with our primary hypothesis. We hope to corroborate our findings in the pilot experiments. Below we describe the methods used in the conventional radiation studies. The endpoint measures are the same as will be used in the heavy ion radiation protocols.

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.

Bibliography Type: Description: (Last Updated: 01/13/2014) 

Show Cumulative Bibliography Listing
 
 None in FY 2006
Project Title:  Ionizing Radiation and its Effects on Cardiovascular Function in the Context of Space Flight Reduce
Fiscal Year: FY 2005 
Division: Human Research 
Research Discipline/Element:
HRP SR:Space Radiation
Start Date: 07/01/2005  
End Date: 06/30/2009  
Task Last Updated: 02/08/2006 
Download report in PDF pdf
Principal Investigator/Affiliation:   Berkowitz, Dan E. M.D. / The Johns Hopkins University 
Address:  Department of Anesthesiology & Critical Care Medicine 
600 North Wolfe Street 
Baltimore , MD 21287-8711 
Email: dberkow1@jhmi.edu 
Phone: 410-614-1517  
Congressional District:
Web:  
Organization Type: UNIVERSITY 
Organization Name: The Johns Hopkins University 
Joint Agency:  
Comments:  
Project Information: Grant/Contract No. NNJ05HF03G 
Responsible Center: NASA JSC 
Grant Monitor:  
Center Contact:   
Solicitation / Funding Source: 2004 Radiation Biology NNH04ZUU005N 
Grant/Contract No.: NNJ05HF03G 
Project Type: GROUND 
Flight Program:  
TechPort: No 
No. of Post Docs:
No. of PhD Candidates:
No. of Master's Candidates:
No. of Bachelor's Candidates:
No. of PhD Degrees:  
No. of Master's Degrees:  
No. of Bachelor's Degrees:  
Human Research Program Elements: (1) SR:Space Radiation
Human Research Program Risks: (1) Degen:Risk Of Cardiovascular Disease and Other Degenerative Tissue Effects From Radiation Exposure (IRP Rev F)
Human Research Program Gaps: (1) Degen01:How can tissue specific experimental models be developed for the major degenerative tissue risks, including cardiovascular, lens, digestive, endocrine, and other tissue systems in order to estimate space radiation risks for degenerative diseases? (IRP Rev F)
(2) Degen02:What are the adverse outcome pathways associated with degenerative tissues changes in the cardiovascular, cerebrovascular, lens, immune, digestive, endocrine, and other tissue systems? What are the key events or hallmarks, their time sequence, and their associated biomarkers? (IRP Rev J)
Task Description: An appropriate examination of the health risks associated with manned space flight necessitates an understanding of the molecular consequences of exposure to the radiations encountered in space. Human radio-epidemiologic data and animal studies indicate that irradiation of the heart can cause a spectrum of cardiovascular complications. The mechanisms suggested for these alterations are chronic inflammation induced by oxidative stress. Its well known that ionizing radiation (IR) produces biological damage by direct effect on DNA and indirectly by generation of reactive oxygen species (ROS) in the cellular milieu. The xanthine oxidoreductase (XOD) system is one of the major sources of free radicals in biologic systems. Since the XOD system is present primarily in the reduced XDH form in normal tissue, the production of free radicals is negligible. However, emerging data demonstrates that IR irreversibly converts the xanthine dehydrogenase (XDH) to xanthine oxidase (XO) leading to amplification and persistence of IR induced, ROS dependent cell damage. It is well known that ROS interferes with cellular signaling (nitrosylation and phosphorylation) and is pro-apoptotic (releases mitochondrial cytochrome-C and activates apoptotic pathways). One of the postulated mechanisms of radiation related tissue injury is endothelial cell damage. However little is known regarding other cellular and molecular targets in the pathophysiology of radiation induced cardiovascular system dysfunction. Furthermore little is known regarding the response of endothelial cells and cardiac myocytes to high LET radiation. In this proposal we intend to use established in vivo and in vitro bioassays to characterize the radiation response to charged particle exposure. Furthermore, mechanistically will focus on the interaction between ROS and nitric oxide (NO) pathways in the regulation of both myocardial and vascular structure and function following OS induced by high LET radiation. Our group have demonstrated the important reciprocal interaction between NO and O2- (derived from XO) in the regulation of myocardial contractility and endothelial function. We will utilize our expertise to determine the effect of radiation on these important signaling pathways in the cardiovascular system. 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, arginase pathways play a critical role in the response to radiation induced OS.

Research Impact/Earth Benefits: 0

Task Progress & Bibliography Information FY2005 
Task Progress: Please note that this is a new grant for the FY 2005 year. The investigator will provide a task progress at the time of the one year anniversary of the grant. If you need more information, please contact the Task Book Help Desk at taskbook@nasaprs.com.

Bibliography Type: Description: (Last Updated: 01/13/2014) 

Show Cumulative Bibliography Listing
 
 None in FY 2005