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Project Title:  Potential Role of the Endothelium in Internal Jugular Venous Thrombosis due to Abnormal Venous Flow Patterns During Spaceflight Reduce
Images: icon  Fiscal Year: FY 2024 
Division: Human Research 
Research Discipline/Element:
HRP HHC:Human Health Countermeasures
Start Date: 11/13/2020  
End Date: 11/01/2024  
Task Last Updated: 11/22/2023 
Download report in PDF pdf
Principal Investigator/Affiliation:   Taylor, W Robert  M.D., Ph.D. / Emory University 
Address:  The Wallace H. Coulter Department of Biomedical Engineering 
101 Woodruff Circle, Suite 319 WMB 
Atlanta , GA 30322 
Email: w.robert.taylor@emory.edu 
Phone: 404-727-3754  
Congressional District:
Web:  
Organization Type: UNIVERSITY 
Organization Name: Emory University 
Joint Agency:  
Comments:  
Key Personnel Changes / Previous PI: October 2021 report: None
Project Information: Grant/Contract No. 80NSSC21K0251 
Responsible Center: NASA JSC 
Grant Monitor: Stenger, Michael  
Center Contact: 281-483-1311 
michael.b.stenger@nasa.gov 
Unique ID: 14188 
Solicitation / Funding Source: 2019-2020 HERO 80JSC019N0001-HHCBPSR, OMNIBUS2: Human Health Countermeasures, Behavioral Performance, and Space Radiation-Appendix C; Omnibus2-Appendix D 
Grant/Contract No.: 80NSSC21K0251 
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) Cardiovascular:Risk of Cardiovascular Adaptations Contributing to Adverse Mission Performance and Health Outcomes
Human Research Program Gaps: (1) CV-101:Determine whether long-duration weightlessness induces cardiovascular structural and functional changes and/or oxidative stress & damage (OSaD)/inflammation, that can contribute to development of disease.
Flight Assignment/Project Notes: NOTE: End date changed to 11/01/2024 per NSSC information (Ed., 1/13/24)

NOTE: End date changed to 11/12/2023 per NSSC information (Ed., 11/16/22)

NOTE: End date changed to 11/12/2022 per NSSC information (Ed., 11/18/21)

Task Description: The recent report of complete and partial thrombosis of the internal jugular vein in crew members on the International Space Station (ISS) raises concerns for the potential of life threatening pulmonary emboli. There appear to be very abnormal blood flow patterns in the internal jugular vein in zero gravity that may predispose to local thrombus formation. Given that the endothelium is uniquely sensitive to changes in the local flow/shear stress environment, the focus of this proposal is on the potential role of the endothelium in mediating localized thrombus formation. The purpose of this proposal is to identify changes in gene expression in venous endothelium exposed to the same flow patterns as those observed in the ISS crew members using isolated vein organ culture system. These studies will help us to develop a better understanding of the basic mechanisms responsible for thrombosis formation with the ultimate goal of potentially identifying biomarkers that would enable screening and risk stratification of crew members.

Research Impact/Earth Benefits: This work will help us to better understand the fundamental physiologic impact of altered flow patterns on the venous endothelium. In addition to allowing us to better understand the differences between venous and arterial endothelial responses, the work may have direct applications to helping us to understand the pathophysiology of venous disease. Finally, venous grafts are used in peripheral and coronary artery bypass surgery and we know that they have a limited, useful lifespan. Given that these segments are placed in a flow environment that is different from the natural flow environment, the work in this proposal may provide insight into how venous endothelium responds to non-physiologic flow stimuli.

Task Progress & Bibliography Information FY2024 
Task Progress: The major focus over the past year has been trying to increase the viability and number of the cells that we are collecting from the veins. Unlike our experience with arterial tissue, this has been a very significant technical challenge. In terms of collecting cells from the rodent inferior vena cava, we first attempted an endothelial-specific isolation using a variety of collagenase-based cocktails. We were able to develop a relatively pure collection of endothelial cells (ECs), but these cell numbers were insufficient for quantitative single-cell analysis. We then moved on to whole-tissue digestion and attempted to evaluate a series of different digestion cocktails that would preserve the viability of the cells that we were collecting.

We ultimately used a cocktail that contained warmed collagenase/dispase [0.2%/0.2%] which resulted in increased cell numbers but an insufficient number of endothelial cells. This is due to the relatively low ratio of endothelial cells to total cells in the tissue. We retired the collagenase/dispase [0.2%/0.2%] perfusion approach which again gave us issues with cell viability.

We then consulted with another lab that has been using a different technique on the mouse carotid artery for endothelial cell harvest. They found that flushing the internal carotid with QAIzol lysis reagent resulted in the isolation of relatively pure mRNA from endothelial cells. We found that when we used this approach, we did get good quality mRNA. Before undertaking sequencing, we performed standard PCR comparing the isolated material from the flushing approach with the mRNA isolated from digesting the entire vessel. We specifically targeted smooth muscle and endothelial markers (alpha smooth muscle actin and CD-31/PECAM) to quantify purity. We were very disappointed to find that in contrast to republished work, we found that this technique resulted in a very high level of contamination by vascular smooth muscle cells and relatively low expression of endothelial cell markers.

Our current strategy is to focus on the human tissues with larger amounts of cells and we have applied a new digestion protocol using a cocktail of Liberaseā„¢, elastase, and DNAse to digest the entire vessel segment. To overcome the low number of ECs isolated for scRNAseq, we will use fluorescent-activated cell sorting (FACS) to increase the proportion of ECs submitted for scRNAseq. CD31 will be used to sort the cells and the final mix of cells will include 50% CD31 positive cells and 50% CD31 negative cells. This will yield an endothelial cell-enriched sample for single-cell RNA sequencing.

Bibliography: Description: (Last Updated: ) 

Show Cumulative Bibliography
 
 None in FY 2024
Project Title:  Potential Role of the Endothelium in Internal Jugular Venous Thrombosis due to Abnormal Venous Flow Patterns During Spaceflight Reduce
Images: icon  Fiscal Year: FY 2022 
Division: Human Research 
Research Discipline/Element:
HRP HHC:Human Health Countermeasures
Start Date: 11/13/2020  
End Date: 11/12/2022  
Task Last Updated: 09/15/2021 
Download report in PDF pdf
Principal Investigator/Affiliation:   Taylor, W Robert  M.D., Ph.D. / Emory University 
Address:  The Wallace H. Coulter Department of Biomedical Engineering 
101 Woodruff Circle, Suite 319 WMB 
Atlanta , GA 30322 
Email: w.robert.taylor@emory.edu 
Phone: 404-727-3754  
Congressional District:
Web:  
Organization Type: UNIVERSITY 
Organization Name: Emory University 
Joint Agency:  
Comments:  
Key Personnel Changes / Previous PI: October 2021 report: None
Project Information: Grant/Contract No. 80NSSC21K0251 
Responsible Center: NASA JSC 
Grant Monitor: Stenger, Michael  
Center Contact: 281-483-1311 
michael.b.stenger@nasa.gov 
Unique ID: 14188 
Solicitation / Funding Source: 2019-2020 HERO 80JSC019N0001-HHCBPSR, OMNIBUS2: Human Health Countermeasures, Behavioral Performance, and Space Radiation-Appendix C; Omnibus2-Appendix D 
Grant/Contract No.: 80NSSC21K0251 
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) Cardiovascular:Risk of Cardiovascular Adaptations Contributing to Adverse Mission Performance and Health Outcomes
Human Research Program Gaps: (1) CV-101:Determine whether long-duration weightlessness induces cardiovascular structural and functional changes and/or oxidative stress & damage (OSaD)/inflammation, that can contribute to development of disease.
Flight Assignment/Project Notes: NOTE: End date changed to 11/12/2022 per NSSC information (Ed., 11/18/21)

Task Description: The recent report of complete and partial thrombosis of the internal jugular vein in crew members on the International Space Station (ISS) raises concerns for the potential of life threatening pulmonary emboli. There appear to be very abnormal blood flow patterns in the internal jugular vein in zero gravity that may predispose to local thrombus formation. Given that the endothelium is uniquely sensitive to changes in the local flow/shear stress environment, the focus of this proposal is on the potential role of the endothelium in mediating localized thrombus formation. The purpose of this proposal is to identify changes in gene expression in venous endothelium exposed to the same flow patterns as those observed in the ISS crew members using isolated vein organ culture system. These studies will help us to develop a better understanding of the basic mechanisms responsible for thrombosis formation with the ultimate goal of potentially identifying biomarkers that would enable screening and risk stratification of crew members.

Research Impact/Earth Benefits: This work will help us to better understand the fundamental physiologic impact of altered flow patterns on the venous endothelium. In addition to allowing us to better understand the differences between venous and arterial endothelial responses, the work may have direct applications to helping us to understand the pathophysiology of venous disease. Finally, venous grafts are used in peripheral and coronary artery bypass surgery and we know that they have a limited, useful lifespan. Given that these segments are placed in a flow environment that is different from the natural flow environment, the work in this proposal may provide insight into how venous endothelium responds to non-physiologic flow stimuli.

Task Progress & Bibliography Information FY2022 
Task Progress: Several cases of internal jugular vein thrombosis have been reported in crew members onboard the International Space Station. While these cases were asymptomatic, the presence of a blood clot in the venous circulation poses a potentially very significant health risk for crew members. This risk may be even higher in longer, interplanetary flights.

The reason for blood clot formation in the internal jugular vein is unclear. We hypothesize that the changes in blood flow patterns that occur in zero gravity may be the reason for clot formation. In normal gravity, blood drains from the internal jugular vein and flow is more uniform and unidirectional. However, without normal gravitational effects, blood flow patterns can be significantly altered. Previous work from NASA scientists demonstrated that there are several patterns of internal jugular vein blood flow in zero gravity that included stagnant flow as well as oscillatory flow. Previous work from ours and others have shown that the endothelial cells that line the inner wall of blood vessels are uniquely sensitive to changes in flow patterns and that disturbed blood flow can induce and pro-inflammatory and pro-thrombotic phenotype. Therefore, we are determining the impact of the disturbed flow patterns observed in the jugular vein of crew members on the expression of genes in the endothelial cells that could lead to an increased risk of thrombosis.

To date, we have established an ex vivo flow system that allows us to recapitulate the flow patterns observed in crew members onboard the International Space Station. We are using this system to perfuse isolate vein segments under the various conditions that will allow us to determine if the observed abnormal flow conditions result in expression of genes and proteins that promote thrombosis. We are studying human saphenous veins as well as a segment of the mouse inferior vena cava. We have optimized techniques for endothelial cell isolation and analysis from these venous segments and we are beginning to collect RNA samples for sequencing. As we are at the beginning of these studies, no results are available at this time as we are in the process of collecting samples for analysis.

Bibliography: Description: (Last Updated: ) 

Show Cumulative Bibliography
 
 None in FY 2022
Project Title:  Potential Role of the Endothelium in Internal Jugular Venous Thrombosis due to Abnormal Venous Flow Patterns During Spaceflight Reduce
Images: icon  Fiscal Year: FY 2021 
Division: Human Research 
Research Discipline/Element:
HRP HHC:Human Health Countermeasures
Start Date: 11/13/2020  
End Date: 11/12/2021  
Task Last Updated: 12/14/2020 
Download report in PDF pdf
Principal Investigator/Affiliation:   Taylor, W Robert  M.D., Ph.D. / Emory University 
Address:  The Wallace H. Coulter Department of Biomedical Engineering 
101 Woodruff Circle, Suite 319 WMB 
Atlanta , GA 30322 
Email: w.robert.taylor@emory.edu 
Phone: 404-727-3754  
Congressional District:
Web:  
Organization Type: UNIVERSITY 
Organization Name: Emory University 
Joint Agency:  
Comments:  
Project Information: Grant/Contract No. 80NSSC21K0251 
Responsible Center: NASA JSC 
Grant Monitor: Stenger, Michael  
Center Contact: 281-483-1311 
michael.b.stenger@nasa.gov 
Unique ID: 14188 
Solicitation / Funding Source: 2019-2020 HERO 80JSC019N0001-HHCBPSR, OMNIBUS2: Human Health Countermeasures, Behavioral Performance, and Space Radiation-Appendix C; Omnibus2-Appendix D 
Grant/Contract No.: 80NSSC21K0251 
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) Cardiovascular:Risk of Cardiovascular Adaptations Contributing to Adverse Mission Performance and Health Outcomes
Human Research Program Gaps: (1) CV-101:Determine whether long-duration weightlessness induces cardiovascular structural and functional changes and/or oxidative stress & damage (OSaD)/inflammation, that can contribute to development of disease.
Task Description: The recent report of complete and partial thrombosis of the internal jugular vein in crew members on the International Space Station (ISS) raises concerns for the potential of life threatening pulmonary emboli. There appear to be very abnormal blood flow patterns in the internal jugular vein in zero gravity that may predispose to local thrombus formation. Given that the endothelium is uniquely sensitive to changes in the local flow/shear stress environment, the focus of this proposal is on the potential role of the endothelium in mediating localized thrombus formation. The purpose of this proposal is to identify changes in gene expression in venous endothelium exposed to the same flow patterns as those observed in the ISS crew members using isolated vein organ culture system. These studies will help us to develop a better understanding of the basic mechanisms responsible for thrombosis formation with the ultimate goal of potentially identifying biomarkers that would enable screening and risk stratification of crew members.

Research Impact/Earth Benefits:

Task Progress & Bibliography Information FY2021 
Task Progress: New project for FY2021.

Bibliography: Description: (Last Updated: ) 

Show Cumulative Bibliography
 
 None in FY 2021