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Project Title:  Targeting NO/IKK Signaling to Counteract Hemodynamic Flow-Dependent Endothelial Dysfunction and Vascular Damage after Space Radiation Reduce
Fiscal Year: FY 2016 
Division: Human Research 
Research Discipline/Element:
HRP HHC:Human Health Countermeasures
Start Date: 11/01/2012  
End Date: 10/31/2015  
Task Last Updated: 03/10/2016 
Download report in PDF pdf
Principal Investigator/Affiliation:   Natarajan, Mohan  Ph.D. / The University of Texas Health Science Center at San Antonio 
Address:  Pathology 
7703 Floyd Curl Dr 
San Antonio , TX 78229-3901 
Email: natarajan@uthscsa.edu 
Phone: 210-567-5663  
Congressional District: 21 
Web:  
Organization Type: UNIVERSITY 
Organization Name: The University of Texas Health Science Center at San Antonio 
Joint Agency:  
Comments:  
Co-Investigator(s)
Affiliation: 
Prihoda, Tom  The University of Texas Health Science Center at San Antonio 
Mohan, Sumathy  The University of Texas Health Science Center at San Antonio 
Blakely, Eleanor  Lawrence Berkeley National Laboratory 
Project Information: Grant/Contract No. NCC 9-58-CA02802 
Responsible Center: NSBRI 
Grant Monitor:  
Center Contact:   
Solicitation / Funding Source: 2011 Crew Health NNJ11ZSA002NA 
Grant/Contract No.: NCC 9-58-CA02802 
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) HHC:Human Health Countermeasures
Human Research Program Risks: (1) Arrhythmia:Risk of Cardiac Rhythm Problems
Human Research Program Gaps: (1) CV08:Can manifestations of sub-clinical or environmentally induced cardiovascular diseases during spaceflight be predicted?
Flight Assignment/Project Notes: NOTE: Period of performance change to 11/1/2012-10/31/2015 per NSBRI; previous POP was 9/1/2012-8/31/2015 (Ed., 11/13/12)

Task Description: The development of subclinical vascular abnormalities, which have been known to occur during space missions, is largely due to functional alterations of endothelial cells (inner lining of the vessels). Endothelial dysfunction is regarded as a primary sub-clinical condition that could progress into cardiovascular diseases over the life of the astronauts. Our hypothesis is that space radiation at low doses may impair the interplay between three key proteins (eNOS, Hsp-90, and IKK-beta) and cause functional alterations of endothelial cells. This dysfunctional endothelium fails to regulate vascular healing processes and negates cell migration/motility. When unchecked, this may predispose the vascular bed to become a sustained pro-inflammatory milieu for the initiation of cardiovascular abnormalities. Radiation exposure can simultaneously also have an impact on endothelial progenitor cells (EPCs) and thereby attenuate EPC-dependent repair and re-endothelialization. The specific aims proposed in this study address the Human Research Program (HRP)-identified Integrated Research Plan (IRP) risks, gaps, and deliverables.

In Aim 1, we proposed to study the significance of relevant doses of three qualitatively different high linear energy transfer (LET) radiation exposures on endothelial dysfunction that leads to vessel denudation, loss of migration and proliferation potential, inefficient damage-induced repair progression, and inept vascular wound healing.

In Aim 2, we proposed to establish the intrinsic mechanism involved in radiation mediated endothelial dysfunction. The interplay between HSP-90, eNOS, and IKK-b will be examined. This will be validated with genetic and pharmacological blockers.

As indicated in the HRP-Integrated Research Program road map, in Aim 3, we proposed to study the influence of countermeasure agents in limiting the radiation-related damage to endothelium.

This study emphasizes a multi-stage approach (in vitro, ex vivo, and in vivo) to understand the underlying mechanism of functional alteration of flow-adapted endothelial cells in response to space radiation. The findings, whilst allowing us to gain knowledge on the mechanism of cardiovascular alterations by high LET radiation exposure, would lead us to develop and quantitatively assess biological countermeasures for cardiovascular risks. In this final year study on eNOS/NO signaling: We established space radiation (56Fe) at low doses (0.2 - 0.8 Gy) at 600 MeV energy could alter the delayed vascular function at molecular level (preferential binding of IKK-beta-Hsp-90 versus eNOS-HSP-90) cellular level (endothelial dysfunction), and tissue level (vascular relaxation/contractile function). Experiments revealed that eNOS activity is necessary but not sufficient to maintain the vasorelaxation function. Overexpression of vascular-specific expression of eNOS further worsens the relaxation function of the vessels in irradiated animals. We validated in vivo the potential countermeasure by duel approach of simultaneous upregulation of eNOS activity and at the same time negate the activity of IKK-ß with physiological eNOS inducer(s) and IKK-ß inhibitor(s), respectively. Currently there is no suitable animal model available to understand early biomarkers of radiation-induced cardiovascular complication. Since modulating the fluid dynamics of the artery has been shown to be associated with the accelerated atherosclerosis, we developed and optimized a partial ligation mouse model in wild type C57Bl/6 mice. Low or disturbed flow achieved in carotid artery by partial ligation will be used for the first time for radiation studies.

Since the results were encouraging from the initial experiments with carotid artery ligation carried out last year, a second set of carotid artery ligation was carried out to determine the reproducibility. The results clearly showed infiltration of macrophages and intimal thickness that are responsible for atherosclerotic plaque formation. This novel approach, repurposed for radiation delayed effect, will help to understand whether or not the galactic cosmic radiation (GCR) mediates an accelerated atherosclerosis at low doses in normal individuals. Finally, a set of animals were exposed to 56Fe (600 MeV) at a total dose of 0.2 Gy for cardiotoxicity. In this experiment only the heart is targeted and rest of the body was shielded. Cardiac functional studies were performed in these mice after 3-6 months of post exposure period.

Risk reduction & Gap closure: We have clearly identified the molecular mediators involved in space radiation-mediated vascular dysfunction. We have also proved that these molecular mediators are involved in vascular function in the arterial segments. These studies set a stage to move forward and validate the agents that target these selective mediators of the pathway as a potential countermeasure approach to alleviate the space radiation–induced endothelial-mediated vascular dysfunction. Recently we participated in a study as a co-author and submitted the findings to American Journal of Pathology - Heart and Circulatory Physiology. The manuscript was accepted for publication [Coleman MA, P Sasi SP, Onufrak J, Natarajan M, et al (2015) Am J Physiol Heart Circ Physiol 309: H1947–H1963, 2015] and was selected for podcast report. The corresponding author of this paper was invited to participate in AJP-Heart and Cir editorial Podcast. Part of this work was supported by the grant NSBRI - CA02802.

Research Impact/Earth Benefits: Increased longevity in cancer patients due to advancement in chemo and radiotherapy brought the awareness of possible therapy-induced vascular complications as one of the main latent side effects. Treatment-induced latent cardiovascular disease in surviving cancer patients is significantly limiting effective clinical management and quality of life. Being one of the primary cancer treatment modalities, the development of strategies that improve the efficacy of radiation therapy could benefit a significant number of cancer patients. Radio-therapeutic strategies can be significantly improved when the mechanism of therapy-induced cardiovascular effects are clearly understood and strategies developed to simultaneously intervene the involvement of adverse pathway mediators. The overall outcome from this study proposed to the National Space Biomedical Research Institute (NSBRI) will add a significant advancement towards treatment of cancer patients.

Task Progress & Bibliography Information FY2016 
Task Progress: Milestones

1. Established space radiation (56Fe) at low doses (0.2 - 0.8 Gy) at 600 MeV energy could alter the delayed vascular function at molecular level (preferential binding of IKK-beta-Hsp-90 versus eNOS-HSP-90) cellular level (endothelial dysfunction), and tissue level (vascular relaxation/contractile function).

2. Experiments revealed that eNOS activity is necessary but not sufficient to maintain the vasorelaxation function. Overexpression of vascular-specific expression of eNOS further worsens the relaxation function of the vessels in irradiated animals. We validated in vivo the potential countermeasure by duel approach of simultaneous upregulation of eNOS activity and at the same time negate the activity of IKK-ß with physiological eNOS inducer(s) and IKK-ß inhibitor(s), respectively.

3. Currently there is no suitable animal model available to understand early biomarkers of radiation-induced cardiovascular complication. Since modulating the fluid dynamics of the artery has been shown to be associated with the accelerated atherosclerosis, we developed and optimized a partial ligation mouse model in wild type C57Bl/6 mice. Low or disturbed flow achieved in carotid artery by partial ligation will be used for the first time for radiation studies. This novel approach repurposed for radiation delayed effect will help to understand whether or not the GCR mediates an accelerated atherosclerosis at low doses in normal individuals.

Risk reduction & Gap closure

We have clearly identified the molecular mediators involved in space radiation-mediated vascular dysfunction. We have also proved that these molecular mediators are involved in vascular function in the arterial segments. These studies set a stage to move forward and validate the agents that target these selective mediators of the pathway as a potential countermeasure approach to alleviate the space radiation–induced endothelial-mediated vascular dysfunction.

Significant Media Coverage

Recently we participated in a study as a co-author and submitted the findings to American Journal of Pathology - Heart and Circulatory Physiology. The manuscript was accepted for publication [Coleman MA, P Sasi SP, Onufrak J, Natarajan M, et al (2015) Am J Physiol Heart Circ Physiol 309: H1947–H1963, 2015] and was selected for podcast report. The corresponding author of this paper was invited to participate in AJP-Heart and Cir editorial Podcast. Part of this work was supported by the grant NSBRI - CA02802.

Bibliography Type: Description: (Last Updated: 04/11/2021) 

Show Cumulative Bibliography Listing
 
Articles in Peer-reviewed Journals Coleman MA, Sasi SP, Onufrak J, Natarajan M, Manickam K, Schwab J, Muralidharan S, Peterson LE, Alekseyev YO, Yan X, Goukassian DA. "Low-dose radiation affects cardiac physiology: gene networks and molecular signaling in cardiomyocytes." Am J Physiol Heart Circ Physiol. 2015 Dec 1;309(11):H1947-63. Epub 2015 Sep 25. http://dx.doi.org/10.1152/ajpheart.00050.2015 ; PubMed PMID: 26408534; PubMed Central PMCID: PMC4698384 , Dec-2015
Articles in Peer-reviewed Journals Krishnan M, Janardhanan P, Roman L, Reddick R, Natarajan M, van Haperen R, Habib S, de Crom R, Mohan S. "Enhancing eNOS activity with simultaneous inhibition of IKKß restores vascular function in Ins2(Akita+/-) type-1 diabetic mice." Lab Invest. 2015 Oct;95(10):1092-104. Epub 2015 Jul 27. http://dx.doi.org/10.1038/labinvest.2015.96 ; PubMed PMID: 26214584 , Oct-2015
Articles in Peer-reviewed Journals Natarajan M, Konopinski R, Krishnan M, Roman L, Bera A, Hongying Z, Habib SL, Mohan S. "Inhibitor-kB kinase attenuates Hsp90-dependent endothelial nitric oxide synthase function in vascular endothelial cells." Am J Physiol Cell Physiol. 2015 Apr 15;308(8):C673-83. Epub 2015 Feb 4. http://dx.doi.org/10.1152/ajpcell.00367.2014 ; PubMed PMID: 25652452; PubMed Central PMCID: PMC4398846 , Apr-2015
Articles in Peer-reviewed Journals Natarajan M, Aravindan N, Sprague EA, Mohan S. "Hemodynamic flow-induced mechanotransduction signaling influences the radiation response of the vascular endothelium." Radiat Res. 2016 Aug;186(2):175-88. https://doi.org/10.1667/RR14410.1 ; PMID: 27387860 , Aug-2016
Project Title:  Targeting NO/IKK Signaling to Counteract Hemodynamic Flow-Dependent Endothelial Dysfunction and Vascular Damage after Space Radiation Reduce
Fiscal Year: FY 2015 
Division: Human Research 
Research Discipline/Element:
HRP HHC:Human Health Countermeasures
Start Date: 11/01/2012  
End Date: 10/31/2015  
Task Last Updated: 11/17/2014 
Download report in PDF pdf
Principal Investigator/Affiliation:   Natarajan, Mohan  Ph.D. / The University of Texas Health Science Center at San Antonio 
Address:  Pathology 
7703 Floyd Curl Dr 
San Antonio , TX 78229-3901 
Email: natarajan@uthscsa.edu 
Phone: 210-567-5663  
Congressional District: 21 
Web:  
Organization Type: UNIVERSITY 
Organization Name: The University of Texas Health Science Center at San Antonio 
Joint Agency:  
Comments:  
Co-Investigator(s)
Affiliation: 
Prihoda, Tom  The University of Texas Health Science Center at San Antonio 
Mohan, Sumathy  The University of Texas Health Science Center at San Antonio 
Blakely, Eleanor  Lawrence Berkeley National Laboratory 
Project Information: Grant/Contract No. NCC 9-58-CA02802 
Responsible Center: NSBRI 
Grant Monitor:  
Center Contact:   
Solicitation / Funding Source: 2011 Crew Health NNJ11ZSA002NA 
Grant/Contract No.: NCC 9-58-CA02802 
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) HHC:Human Health Countermeasures
Human Research Program Risks: (1) Arrhythmia:Risk of Cardiac Rhythm Problems
Human Research Program Gaps: (1) CV08:Can manifestations of sub-clinical or environmentally induced cardiovascular diseases during spaceflight be predicted?
Flight Assignment/Project Notes: NOTE: Period of performance change to 11/1/2012-10/31/2015 per NSBRI; previous POP was 9/1/2012-8/31/2015 (Ed., 11/13/12)

Task Description: It is important to determine the effect of long-duration space flight on the heart and blood vessels and research ways to counteract those risks in order to subdue the onset/manifestation of any vascular abnormalities during the mission. In this study, we propose to test the hypothesis that space radiation at low doses may impair the interplay between three key proteins (eNOS, Hsp-90, and IKK-ß) in the vascular endothelial cells, the cell type that covers the inner lining of the blood vessels. This dysfunctional endothelium undergoes functional alterations. When unchecked, this may predispose the vascular bed to become a sustained pro-inflammatory milieu for the initiation of cardiovascular abnormalities. We proposed to address these above concerns by investigating how these molecular mediators are functionally interrelated and how they coordinately provide a niche for the development of cardiovascular abnormalities upon high LET radiation exposure. The objectives are: (i) to investigate the significance of high LET radiation on causing endothelial dysfunction and associated damages on vascular bed, impairment of cell migration/motility and inhibition of vascular healing processes. Three different HZE ion beams (16O, 28Si, and 56Fe) accelerated to the same velocity (600 MeV/amu) and having similar track structure dimensions, but different ionization densities will be compared; (ii) to study how high LET radiation concurrently exploits eNOS, Hsp-90, and IKK# signaling to cause endothelial dysfunction, while impairing the repair capacity of bone-marrow derived endothelial progenitor cells (EPCs); and (iii) to examine whether the findings, whilst allowing us to gain knowledge on the mechanism of cardiovascular alterations by high LET radiation exposure, would lead us to develop and quantitatively assess biological countermeasures for cardiovascular risks.

This study emphasizes a multi-stage approach (in vitro, ex vivo, and in vivo) to understand the underlying mechanism of functional alteration of flow-adapted endothelial cells in response to space radiation. We have initially carried out parallel plate flow system at NSRL with a minimal facility in the absence of dedicated reach-in incubator and absence of CO2 atmosphere. At that time the medium were flushed with CO2 before assembled into the shear system at NSRL. In the current year we established a dedicated facility at NSRL that can be used to run four shear systems in parallel at-a-time under humidified CO2 atmosphere. The beam shape, beam uniformity, and other staging logistics at the beam path were optimized.

Second, we progressed through the in vivo animal study to validate the results obtained from in vitro flow shear system. One wild type and two genetically modified (eNOS-/- and Tie2–eNOS) mice colonies with C57Bl/6 background were developed by standard breeding methods. The genotypes were confirmed with tail snip DNA analysis. When reached 18 months, the animals (n=8/group) were exposed to 0.8 Gy 56Fe (600 MeV/u) to (i) determine the radiation-altered regulation of vascular contractile and relaxation function and (ii) whether impaired eNOS/NO signaling after irradiation is responsible for those altered vasomotor function. The readouts comparing with the mock irradiated wild type, eNOS-/-, and Tie2–eNOS mice were carried out after 30 days post irradiation.

Third, wild type C57Bl/6 mice were either mock irradiated or exposed to 0.8 Gy 56Fe (600 MeV/u) and used to determine the radiation-mediated impairment of endothelial progenitor cells in the bone marrow versus circulating blood. This approach was carried out to examine whether the back-up repair mechanism mediated through endothelial progenitor cells is also negated by the radiation and therefore unavailable to rescue the damaged cells on the vascular bed.

Fourth, a pre-atherosclerotic model in wild type C57Bl/6 mice was developed by modulating the fluid dynamics of the artery. Carotid artery ligation was carried out on the right carotid artery near to bifurcation. The left carotid artery was used as control. The inflicted low shear region at the ligation site was anticipated to cause atherosclerotic lesion in three to four week. A total of 8 mice were irradiated (56Fe (600 MeV/u) and the sections of the carotid arteries were examined for lesions after 4 weeks post radiation.

Fifth, in order to examine the IKK versus eNOS competition for Hsp-90 that was established through in vitro studies will also occur at the physiological condition, in vivo inhibition of IKK was carried out. Since the global blocking of IKK will result in an embryo with lethal phenotype, we developed IKK knockouts site-specifically at the vessel bed. Enriched endothelial cells from isolated arteries from IKK floxed mouse (IKKbf+/f+) and then transfected with cre-GFP were used to determine the influence of IKK in inhibiting eNOS binding to Hsp-90. Sixth, confirmatory experiments and additional data on polarization studies to biophysically prove the in vitro competitive binding of eNOS versus IKK were completed and submitted as a manuscript to Journal of Biological Chemistry (JBC).

Finally, as a collaborative effort with another related cardiovascular project funded by NSBRI, the regulation of transcription factors NF-kB, STAT-3, and GATA were examined in cardiac tissue in mice exposed to 90 cGy of 1 GeV proton or 15 cGy of 1 GeV/u iron particle and sacrificed after 1, 3, 7, 14, and 28 days. From year two study we demonstrated the impaired regulation of nitric oxide signaling alters the physiological functioning of endothelium that leads to vascular abnormalities including activation of inflammatory responses, vasomotor function. Also, we confirmed the in vitro mechanistic studies and proved the proof of principal hypothesized in the original project (a full-length manuscript was submitted to JBC).

Research Impact/Earth Benefits: In the clinical set-up an increased risk of cardiovascular disease after radiotherapy is a major concern. Results from epidemiological studies clearly suggested that the cause of radiotherapy-induced vasculopathy leads to the induction or acceleration of atherosclerosis in conduit arteries located in the irradiated field. Second, therapeutic radiation while alleviating cancer burden can simultaneously be involved in the re-development of the disease at the treatment site. This may account for the tumor recurrence and raise the risk of metastasis at distant site. The approaches proposed in this current NSBRI project, once established will add significant advancement to the understanding of the mechanisms involved in two different diverse fields, i.e., cardiovascular disease and cancer after radiotherapy. Understanding the mechanism is important to develop targeted countermeasure approaches.

Task Progress & Bibliography Information FY2015 
Task Progress: This study addresses the concerns about whether space radiation affects endothelial cells which might initiate or accelerate coronary heart disease. In year 2 we progressed through the in vivo animal study. One wild type and two genetically modified (eNOS-/- and Tie2–eNOS) mice colonies with C57Bl/6 background were developed by standard breeding method. The genotypes were confirmed with tail snip DNA analysis. When reached 18 months, the animals (n=8/group) were exposed to 0.8 Gy 56Fe (600 MeV/u) to (i) determine the radiation-altered regulation of vascular contractile and relaxation function and (ii) whether dysfunctional endothelium with impaired eNOS/NO signaling after irradiation is responsible for those altered vasomotor function. The readouts comparing with the mock irradiated wild type, eNOS-/-, and Tie2–eNOS mice (n=8/group) were carried out after 30 days post irradiation. Second, wild type C57Bl/6 mice were used to determine the radiation (0.8 Gy 56Fe (600 MeV/u)-mediated impairment of endothelial progenitor cells in the bone marrow versus circulating blood. This approach was carried out to examine whether the back-up repair mechanism mediated through endothelial progenitor cells is also negated by the radiation and therefore unavailable to rescue the damaged cells on the vascular bed. Third, a pre-atherosclerotic model in wild type C57Bl/6 mice was developed by modulating the fluid dynamics of the artery. Carotid artery ligation was carried out on the right carotid artery near to bifurcation. The left carotid artery was used as control. The inflicted low shear region at the ligation site has been known to cause atherosclerotic lesion in three to four week in C57Bl/6 mice. A total of 8 mice were irradiated (56Fe (600 MeV/u) and compared to the mock irradiated controls (n=8). The sections of the carotid arteries were examined for lesions after 2-4 weeks post radiation. Fourth, in order to examine the IKK versus eNOS competition for Hsp-90 that was established through in vitro studies, will also occur at the physiological condition, in vivo inhibition of IKK was carried out. Since the global blocking of IKK will result in an embryo with lethal phenotype, we developed IKK knockouts site-specifically at the vessel bed. Isolated endothelial cells from IKK floxed mouse (IKKbf+/f+) transfected with cre-GFP were used to determine the influence of IKK in inhibiting eNOS binding to Hsp-90. Fifth, confirmatory experiments and additional data on polarization studies to biophysically prove the competitive binding of eNOS versus IKK were completed and submitted as a manuscript to Journal of Biological Chemistry (JBC). Finally, as a collaborative effort with another related cardiovascular project funded by NSBRI, the regulation of transcription factors NF-kB, STAT-3, and GATA were examined in cardiac tissue in mice exposed to 90 cGy of 1 GeV proton or 15 cGy of 1 GeV/u iron particle and sacrificed after 1, 3, 7, 14, and 28 days.

Bibliography Type: Description: (Last Updated: 04/11/2021) 

Show Cumulative Bibliography Listing
 
Awards Manickam K. "Radiation Research Society Travel Award, May 2014." May-2014
Project Title:  Targeting NO/IKK Signaling to Counteract Hemodynamic Flow-Dependent Endothelial Dysfunction and Vascular Damage after Space Radiation Reduce
Fiscal Year: FY 2014 
Division: Human Research 
Research Discipline/Element:
HRP HHC:Human Health Countermeasures
Start Date: 11/01/2012  
End Date: 10/31/2015  
Task Last Updated: 12/24/2013 
Download report in PDF pdf
Principal Investigator/Affiliation:   Natarajan, Mohan  Ph.D. / The University of Texas Health Science Center at San Antonio 
Address:  Pathology 
7703 Floyd Curl Dr 
San Antonio , TX 78229-3901 
Email: natarajan@uthscsa.edu 
Phone: 210-567-5663  
Congressional District: 21 
Web:  
Organization Type: UNIVERSITY 
Organization Name: The University of Texas Health Science Center at San Antonio 
Joint Agency:  
Comments:  
Co-Investigator(s)
Affiliation: 
Prihoda, Tom  The University of Texas Health Science Center at San Antonio 
Mohan, Sumathy  The University of Texas Health Science Center at San Antonio 
Blakely, Eleanor  Lawrence Berkeley National Laboratory 
Project Information: Grant/Contract No. NCC 9-58-CA02802 
Responsible Center: NSBRI 
Grant Monitor:  
Center Contact:   
Solicitation / Funding Source: 2011 Crew Health NNJ11ZSA002NA 
Grant/Contract No.: NCC 9-58-CA02802 
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) HHC:Human Health Countermeasures
Human Research Program Risks: (1) Arrhythmia:Risk of Cardiac Rhythm Problems
Human Research Program Gaps: (1) CV08:Can manifestations of sub-clinical or environmentally induced cardiovascular diseases during spaceflight be predicted?
Flight Assignment/Project Notes: NOTE: Period of performance change per NSBRI; previous POP was 9/1/2012-8/31/2015 (Ed., 11/13/12)

Task Description: Space travelers are irradiated with cosmic rays at a dose rate considerably higher than that received on Earth. At the dose and dose rates encountered in space, one of the important risks is the induction of subclinical vascular abnormalities during the mission that leads to cardiovascular disease. Endothelial dysfunction is regarded as a primary sub-clinical condition that could progress into cardiovascular diseases over the life of the astronauts. The proposed hypothesis is that space radiation at low doses may impair the interplay between three key proteins (eNOS, Hsp-90, and IKK-ß), and cause functional alterations of endothelial cells. This dysfunctional endothelium fails to regulate vascular healing processes and negate cell migration/motility. When unchecked, this may predispose the vascular bed to become a sustained pro-inflammatory milieu for the initiation of cardiovascular abnormalities. We proposed to address these above concerns by investigating how these molecular mediators are functionally interrelated and how they coordinately provide a niche for the development of cardiovascular abnormalities upon high LET radiation exposure. The proposed specific aims are: Aim 1: To study the significance of low doses (0, 0.2, 0.4, or 0.8 Gy) of high-LET radiation exposure on vascular bed damage, negation of cell migration/motility, and impairment of vascular healing processes. Aim 2: To determine the mechanism of high LET radiation-initiated functional alterations of endothelial cells that are sustained to cause progressive cardiovascular diseases. Aim 3: To assess in vitro, ex vivo and in vivo the potential of targeting eNOS and IKK-ß independently and in combination as a promising countermeasure to limit endothelial dysfunction. The specific aims originally proposed were not altered.

The key findings are: (a) In year 1, we established the flow shear system at NSRL and cultured human primary endothelial cells under simulated high (16 dynes/cm2) hemodynamic shear stress - a local environment in which the endothelial cells are in constant physiological shear stress due to continuous blood flow. Experiments were performed to determine the response of endothelial cells to radiation after parallel plate flow versus static culture. This was carried out first at low LET radiation exposures using gamma irradiator at the P.I.'s institution. This allowed us to optimize the conditions and prepared for the heavy ion exposure at NSRL. Also, this approach allowed us to generate data from low LET radiation for comparison with heavy ion exposures. A significant difference, both in cell morphology and radiation-induced gene expression profile examined by real-time Q-PCR micro-array, were observed in cells grown on matrigel (simulated extra cellular matrix) and experiencing physiological hemodynamic flow shear stress (that occurs in normal blood vessels) compared to static culture. Next, we confirmed the differential regulation of key proteins involved in NO signaling pathway after both high and low LET radiation exposures at doses ranging from 0.1 to 1.6 Gy. Sustained activation of IKK-beta/NF-kB pathway was validated in cells exposed to 56Fe (600 MeV/amu) and 137Cs gamma radiation. Second, time- and dose-dependent impaired regulation of nitric oxide was measured in terms of intracellular accumulation of nitric oxide by FACS analysis. Third, to determine whether radiation-induced IKK binds with heat shock protein (Hsp-90), a Mammalian two hybrid system was established. This approach proved the favorable binding of IKK with Hsp-90. Next, to examine whether this binding of IKK competitively uncoupled eNOS binding to Hsp-90, both cell-free system and intra-cellular competitive binding assays were performed. To further confirm any increase in IKK activation after radiation exposure is responsible for decreased bioavailability of nitric oxide, a blocking assay was performed to block IKK and measured a time-dependent reactivation of nitric oxide in the exposed cells. Cells treated with sodium salicylate, a specific inhibitor of IKK, showed an increased bioavailability of nitric oxide even after 3, 6, 12, and 24 h post-exposure.

From year 1 study we concluded that: (i) for in vitro mechanistic studies in response to radiation the endothelial cells should be cultured under their micro-environmental niche -hemodynamic flow shear, and (ii) space radiation can alter nitric oxide signaling through sustained activation of IKK signaling pathway. The activation of IKK competitively binds to Hsp-90 and prevents eNOS binding and subsequent phosphorylation of eNOS by AKT signaling pathway. These sequential events after radiation are responsible for decreased bioavailability of nitric oxide that could lead to abnormal vascular homeostasis.

In the next year study during year 2, we will be investigating whether the impaired regulation of nitric oxide signaling alters the physiological functioning of the endothelium that leads to vascular abnormalities including inflammation, vascular permeability, and vasomotor function. This will be carried out both in vitro system under hemodynamic flow shear stress and in vivo animal models.

Research Impact/Earth Benefits: Therapeutic radiation, while alleviating cancer burden can simultaneously be involved in redevelopment of the disease at the treatment site, which may account for the tumor recurrence and risk of metastasis at distant site. The relationship linking the altered responses that could occur simultaneously in cancer cells versus surrounding normal tissue and also the molecular alterations occur after radiation exposure and re-growth of tumor remain elusive and unknown. Ascertaining those altered responses and selectively targeting the responsible mediators might significantly improve radiotherapy outcome. The approach proposed to NSBRI once established will help to test whether in remnant cancer cells that survive treatment where key proteins are de-nitrosylated due to aberrant regulation of altered eNOS/NO. The functionally redefined proteins are responsible for tumor re-growth, invasion, and metastasis. The overall outcome from this proposed study will add a significant advancement towards achieving disease-free survival of cancer patients.

Task Progress & Bibliography Information FY2014 
Task Progress: The development of subclinical vascular abnormalities, which have been known to occur during space missions, is largely due to functional alterations of endothelial cells (inner lining of the vessels). Endothelial dysfunction is regarded as a primary sub-clinical condition that could progress into cardiovascular diseases over the life of the astronauts. Our hypothesis is that space radiation at low doses may impair the interplay between three key proteins (eNOS, Hsp-90, and IKK-ß), and cause functional alterations of endothelial cells. When unchecked, this may predispose the vascular bed to become a sustained pro-inflammatory milieu for the initiation of cardiovascular abnormalities. We proposed to address these above concerns by investigating how these molecular mediators are functionally interrelated and how they coordinately provide a niche for the development of cardiovascular abnormalities upon high LET radiation exposure. The results are compared with low LET radiation at the same total dose and dose rate specified for high LET radiation.

(a) In year 1, we established the flow shear system at NSRL and cultured human primary endothelial cells under high (16 dynes/cm2) hemodynamic shear stress. Experiments were performed to determine the response of endothelial cells to radiation after parallel plate flow versus static culture.

(b) Next we confirmed the differential regulation of key proteins involved in NO signaling pathway after both high and low LET radiation exposures at doses ranging from 0.1 to 1.6 Gy. Sustained activation of IKK-beta/NF-kB pathway was validated in cells exposed to 56Fe (600 MeV/amu) and 137Cs gamma radiation. Second, time- and dose-dependent impaired regulation of nitric oxide was measured in-terms of intracellular accumulation of nitric oxide by FACS analysis. Third, to determine whether radiation-induced IKK binds with heat shock protein (Hsp-90), a Mammalian two hybrid system was established. This approach proved the favorable binding of IKK with Hsp-90. Next, to examine whether this binding of IKK competitively uncoupled eNOS binding to Hsp-90, both cell-free system and intra-cellular competitive binding assays were performed. To further confirm any increase in IKK activation after radiation exposure is responsible for decreased bioavailability of nitric oxide, a blocking assay was performed to block IKK and measured a time-dependent reactivation of nitric oxide in the exposed cells. From year 1 study we concluded that space radiation can alter nitric oxide signaling through sustained activation of IKK signaling pathway.

In year 2, we will be investigating whether the impaired regulation of nitric oxide signaling alters the physiological functioning of the endothelium that leads to vascular abnormalities including inflammation, vascular permeability, and vasomotor function. This will be carried out both in vitro system and in vivo animal models.

Bibliography Type: Description: (Last Updated: 04/11/2021) 

Show Cumulative Bibliography Listing
 
 None in FY 2014
Project Title:  Targeting NO/IKK Signaling to Counteract Hemodynamic Flow-Dependent Endothelial Dysfunction and Vascular Damage after Space Radiation Reduce
Fiscal Year: FY 2013 
Division: Human Research 
Research Discipline/Element:
HRP HHC:Human Health Countermeasures
Start Date: 11/01/2012  
End Date: 10/31/2015  
Task Last Updated: 07/24/2012 
Download report in PDF pdf
Principal Investigator/Affiliation:   Natarajan, Mohan  Ph.D. / The University of Texas Health Science Center at San Antonio 
Address:  Pathology 
7703 Floyd Curl Dr 
San Antonio , TX 78229-3901 
Email: natarajan@uthscsa.edu 
Phone: 210-567-5663  
Congressional District: 21 
Web:  
Organization Type: UNIVERSITY 
Organization Name: The University of Texas Health Science Center at San Antonio 
Joint Agency:  
Comments:  
Co-Investigator(s)
Affiliation: 
Blakely, Eleanor  Lawrence Berkeley National Laboratory 
Mohan, Sumathy  University of Texas, San Antonio 
Prihoda, Tom  University of Texas, San Antonio 
Project Information: Grant/Contract No. NCC 9-58-CA02802 
Responsible Center: NSBRI 
Grant Monitor:  
Center Contact:   
Solicitation / Funding Source: 2011 Crew Health NNJ11ZSA002NA 
Grant/Contract No.: NCC 9-58-CA02802 
Project Type: GROUND 
Flight Program:  
TechPort: No 
No. of Post Docs:  
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Human Research Program Elements: (1) HHC:Human Health Countermeasures
Human Research Program Risks: (1) Arrhythmia:Risk of Cardiac Rhythm Problems
Human Research Program Gaps: (1) CV08:Can manifestations of sub-clinical or environmentally induced cardiovascular diseases during spaceflight be predicted?
Flight Assignment/Project Notes: NOTE: Period of performance change per NSBRI; previous POP was 9/1/2012-8/31/2015 (Ed., 11/13/12)

Task Description: The development of subclinical vascular abnormalities, which have been known to occur during space mission, is largely due to functional alterations of endothelial cells (inner lining of the vessels). Endothelial dysfunction is regarded as a primary sub-clinical condition that could progress into cardiovascular diseases over the life of the astronauts. Our hypothesis is that space radiation at low doses may impair the interplay between three key proteins (eNOS, Hsp-90 and IKK-#) and cause functional alterations of endothelial cells. This dysfunctional endothelium fails to regulate vascular healing processes and negates cell migration/motility. When unchecked, this may predispose the vascular bed to become a sustained pro-inflammatory milieu for the initiation of cardiovascular abnormalities. Radiation exposure can simultaneously also have an impact on endothelial progenitor cells (EPCs) and thereby attenuate EPC-dependent repair and reendothelializqation. The objectives are: (i) to investigate the significance of high LET radiation on causing endothelial dysfunction and associated damages on vascular bed, impairment of cell migration/motility and inhibition of vascular healing processes. Three different HZE ion beams (16O, 28Si, and 56Fe) accelerated to the same velocity (600 MeV/amu) and having similar track structure dimensions, but different ionization densities will be compared; (ii) to study how high LET radiation concurrently exploits eNOS, Hsp-90, and IKK# signaling to cause endothelial dysfunction, while impairing the repair capacity of bone-marrow derived endothelial progenitor cells (EPCs); and (iii) to examine whether the findings, whilst allowing us to gain knowledge on the mechanism of cardiovascular alterations by high LET radiation exposure, would lead us to develop and quantitatively assess biological countermeasures for cardiovascular risks.

This study emphasizes a multi-stage approach (in vitro, ex vivo and in vivo) to understand the underlying mechanism of functional alteration of flow-adapted endothelial cells in response to space radiation. This study fits-in very well with HRP-Integrated Research Program road map.

Research Impact/Earth Benefits: 0

Task Progress & Bibliography Information FY2013 
Task Progress: New project for FY2012.

Bibliography Type: Description: (Last Updated: 04/11/2021) 

Show Cumulative Bibliography Listing
 
 None in FY 2013