FIERCE: Functional Investigation of Endothelial Function and Regenerative Cell Exhaustion
Study Details
Study Description
Brief Summary
FIERCE is an observational cross-sectional study. Approximately 90 individuals living with type 2 diabetes (T2D) and/or individuals living without diabetes will be randomized (2:1).
The primary objective of this trial is to determine if there are differences in the content and function of circulating vascular regenerative (VR) progenitor cell subsets isolated from individuals living with T2D versus individuals not living with T2D. The main question this study aims to answer is: Does T2D compromise or enhance VR cell functionality?
Each participant will be asked to provide a single blood sample. Blood samples will be processed to enumerate the number of vessel-repairing cells and determine the functionality of the different subtypes of vessel-repairing cells.
| Condition or Disease | Intervention/Treatment | Phase |
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Detailed Description
Type 2 diabetes (T2D) is a significant and prevalent global health concern. Individuals diagnosed with T2D are at an elevated risk of developing atherosclerotic cardiovascular (CV) disease, a leading cause of global morbidity and mortality.
Blood vessel homeostasis plays a central role in the status of CV health. Circulating vascular regenerative (VR) progenitor cells, which mediate the endogenous processes of angiogenesis, vasculogenesis, and arteriogenesis, are critical in orchestrating vessel repair. In T2D, chronic hyperglycemia and concomitant oxidative stress create a maladaptive environment that impairs vessel repair. T2D can lead to a chronic state known as vascular regenerative cell exhaustion (VRCE), characterized by the depletion of, and dysfunction in, circulating VR progenitor cells. The available data indicate that VRCE associated with T2D can lead to VR cell dysfunction and compromised vascular repair.
We have developed a multi-parametric flow cytometry assay to measure VR progenitor cell content in blood samples. This assay utilizes the cytosolic detoxification enzyme aldehyde dehydrogenase (ALDH), which is highly expressed in progenitor cells from hematopoietic, endothelial, and mesenchymal stromal cell lineages. This enzyme protects progenitor cells from oxidative damage that is driven by reactive oxygen species. ALDH activity is reduced by up to 100-fold as progenitor cells differentiate towards more expendable effector cells. As such, we identify cells with high or low ALDH activity in combination with cell surface markers to distinguish progenitor cell subsets (ALDHhi) from more differentiated progeny (ALDHlow). Used in conjunction with 'side scatter' (SSC), a parameter that correlates with the granularity or complexity of a cell, this assay can distinguish between and quantify ALDHhiSSClow hematopoietic/endothelial precursor cells, ALDHhiSSCmid monocytes, and ALDHhiSSChi granulocyte precursors. Previously, bone marrow-derived ALDHhiSSClow cells were shown to co-express the primitive cell markers CD34 and CD133 and exhibit multipotent hematopoietic colony-forming ability in vitro. In the immunodeficient NOD/SCID mouse model of hindlimb ischemia, transplantation of ALDHhiSSClow cells into the ischemic limb led to improved muscle perfusion recovery. The potential of this cell therapy to prevent amputations in individuals with critical limb ischemia has been evaluated in clinical trial settings.
The peripheral blood of individuals living with T2D for >10-years exhibit a depletion of ALDHhiSSClow VR progenitor cells, lower frequencies of ALDHhiSSCmid monocytes with vessel reparative function, and an increased frequency of ALDHhiSSChi inflammatory granulocyte precursors compared to that from individuals not living with T2D. The VRCE phenotype was partially reversed in people living with T2D and established coronary artery disease (CAD) after they had been on the SGLT2 inhibitor empagliflozin for 6 months. Obesity-induced VR cell depletion was also reversed by 3 months post-bariatric surgery. These findings collectively provided a mechanistic link between T2D, obesity, and impaired vessel homeostasis/repair, and also established that VRCE may be therapeutically reversed in a high CV-risk diabetic milieu.
FIERCE will assess VR cell content in individuals living with T2D (<10-years duration) and age- and sex-matched individuals not living with T2D. It will also assess the function of circulating ALDHhi VR cells through: (1) Endothelial peripheral arterial tonometry (EndoPAT) to clinically analyze endothelial function through reactive hyperemic index, (2) multipotent hematopoietic colony formation assays in vitro, (3) single-cell RNA-sequencing (scRNA-seq) that is focused on mRNA expression associated with angiogenesis, and (4) quantitative, label-free secretome analyses to determine changes in pro-angiogenic protein secretion.
We hypothesize that VRCE impairs vascular repair and blood vessel regeneration during T2D and is partly caused by impaired pro-angiogenic properties of VR progenitor cell subsets. Specifically, we postulate that multipotent hematopoietic colony formation, pro-angiogenic cytokine mRNA expression, and pro-angiogenic protein release will be lower in ALDHhi progenitor cell subsets from individuals living with T2D relative to participants not living with T2D. We also predict that individuals living with T2D will exhibit a lower reactive hyperemic index compared to individuals not living with T2D.
Characterization of ALDHhi progenitor VR cell dysfunction in the setting of T2D will generate proof-of-concept to support the potential use of VR cell content as a quantifiable and functional indicator of vascular health.
Study Design
Arms and Interventions
| Arm | Intervention/Treatment |
|---|---|
| Type 2 Diabetes Participants living with type 2 diabetes for less than 10 years will be recruited. |
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| No Diabetes Participants not living with diabetes will be recruited. |
Outcome Measures
Primary Outcome Measures
- Hematopoietic colony formation in ALDHhiSSClow regenerative cell subsets [Baseline]
The capacity for total multipotent hematopoietic colony formation in ALDHhiSSClow regenerative cell subsets isolated from individuals living with T2D versus individuals not living with T2D.
Secondary Outcome Measures
- Endothelial function [Baseline]
Endothelial peripheral arterial tonometry (EndoPAT) will be utilized to clinically analyze endothelial function through reactive hyperemic index in individuals living with T2D and individuals not living with T2D.
- Frequency and absolute number of circulating ALDHhiSSClowCD133+ progenitor cells [Baseline]
The change in the frequency and absolute number of circulating ALDHhiSSClowCD133+ progenitor cells between individuals living with T2D versus age- and sex-matched individuals not living with T2D
Eligibility Criteria
Criteria
Inclusion Criteria:
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Adults ≥18 years of age.
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Willing to provide written informed consent.
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Documented history of T2D
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No documented history of diabetes
Exclusion Criteria:
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Unable or unwilling to provide written informed consent or provide a peripheral blood sample.
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Any life-threatening disease expected to result in death within two years of consent.
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Any malignancy not considered cured (except basal cell carcinoma of the skin). An individual is considered cured if there has been no evidence of cancer recurrence for the five years prior to screening.
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Known severe liver disease.
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White blood cell count ≥15 x 10^9/L.
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Active infectious disease requiring systemic antibiotic or anti-viral agents.
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Known acquired immunodeficiency syndrome such as HIV.
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Treated autoimmune disorders (e.g. T1D and LADA).
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On oral steroid therapy (e.g. prednisone or other corticosteroids) or other immunosuppressive agents (e.g. methotrexate).
Contacts and Locations
Locations
| Site | City | State | Country | Postal Code | |
|---|---|---|---|---|---|
| 1 | Diagnostic Assessment Centre | Scarborough | Ontario | Canada | M1S4N6 |
Sponsors and Collaborators
- Canadian Medical and Surgical Knowledge Translation Research Group
- Unity Health Toronto
- University of Western Ontario, Canada
Investigators
- Principal Investigator: Subodh Verma, MD, PhD, Unity Health Toronto
- Principal Investigator: David A Hess, PhD, Robarts Research Institute, London, Ontario
Study Documents (Full-Text)
None provided.More Information
Additional Information:
Publications
- Bigarella CL, Liang R, Ghaffari S. Stem cells and the impact of ROS signaling. Development. 2014 Nov;141(22):4206-18. doi: 10.1242/dev.107086.
- Dimmeler S. Regulation of bone marrow-derived vascular progenitor cell mobilization and maintenance. Arterioscler Thromb Vasc Biol. 2010 Jun;30(6):1088-93. doi: 10.1161/ATVBAHA.109.191668. Epub 2010 May 7.
- Fadini GP, Boscaro E, de Kreutzenberg S, Agostini C, Seeger F, Dimmeler S, Zeiher A, Tiengo A, Avogaro A. Time course and mechanisms of circulating progenitor cell reduction in the natural history of type 2 diabetes. Diabetes Care. 2010 May;33(5):1097-102. doi: 10.2337/dc09-1999. Epub 2010 Feb 11.
- Fadini GP, Miorin M, Facco M, Bonamico S, Baesso I, Grego F, Menegolo M, de Kreutzenberg SV, Tiengo A, Agostini C, Avogaro A. Circulating endothelial progenitor cells are reduced in peripheral vascular complications of type 2 diabetes mellitus. J Am Coll Cardiol. 2005 May 3;45(9):1449-57. doi: 10.1016/j.jacc.2004.11.067.
- Haas AV, McDonnell ME. Pathogenesis of Cardiovascular Disease in Diabetes. Endocrinol Metab Clin North Am. 2018 Mar;47(1):51-63. doi: 10.1016/j.ecl.2017.10.010.
- Hayden J, O'Donnell G, deLaunois I, O'Gorman C. Endothelial Peripheral Arterial Tonometry (Endo-PAT 2000) use in paediatric patients: a systematic review. BMJ Open. 2023 Jan 18;13(1):e062098. doi: 10.1136/bmjopen-2022-062098.
- Hess DA, Verma S, Bhatt D, Bakbak E, Terenzi DC, Puar P, Cosentino F. Vascular repair and regeneration in cardiometabolic diseases. Eur Heart J. 2022 Feb 10;43(6):450-459. doi: 10.1093/eurheartj/ehab758.
- Kehl D, Generali M, Mallone A, Heller M, Uldry AC, Cheng P, Gantenbein B, Hoerstrup SP, Weber B. Proteomic analysis of human mesenchymal stromal cell secretomes: a systematic comparison of the angiogenic potential. NPJ Regen Med. 2019 Apr 16;4:8. doi: 10.1038/s41536-019-0070-y. eCollection 2019.
- Li Q, Wang M, Zhang S, Jin M, Chen R, Luo Y, Sun X. Single-cell RNA sequencing in atherosclerosis: Mechanism and precision medicine. Front Pharmacol. 2022 Oct 4;13:977490. doi: 10.3389/fphar.2022.977490. eCollection 2022.
- Mangialardi G, Spinetti G, Reni C, Madeddu P. Reactive oxygen species adversely impacts bone marrow microenvironment in diabetes. Antioxid Redox Signal. 2014 Oct 10;21(11):1620-33. doi: 10.1089/ars.2014.5944.
- Mauch P, Hellman S. Loss of hematopoietic stem cell self-renewal after bone marrow transplantation. Blood. 1989 Aug 1;74(2):872-5.
- Moore MA. Does stem cell exhaustion result from combining hematopoietic growth factors with chemotherapy? If so, how do we prevent it? Blood. 1992 Jul 1;80(1):3-7. No abstract available.
- Rawshani A, Rawshani A, Franzen S, Sattar N, Eliasson B, Svensson AM, Zethelius B, Miftaraj M, McGuire DK, Rosengren A, Gudbjornsdottir S. Risk Factors, Mortality, and Cardiovascular Outcomes in Patients with Type 2 Diabetes. N Engl J Med. 2018 Aug 16;379(7):633-644. doi: 10.1056/NEJMoa1800256.
- Szmitko PE, Fedak PW, Weisel RD, Stewart DJ, Kutryk MJ, Verma S. Endothelial progenitor cells: new hope for a broken heart. Circulation. 2003 Jun 24;107(24):3093-100. doi: 10.1161/01.CIR.0000074242.66719.4A. No abstract available.
- Terenzi DC, Trac JZ, Teoh H, Gerstein HC, Bhatt DL, Al-Omran M, Verma S, Hess DA. Vascular Regenerative Cell Exhaustion in Diabetes: Translational Opportunities to Mitigate Cardiometabolic Risk. Trends Mol Med. 2019 Jul;25(7):640-655. doi: 10.1016/j.molmed.2019.03.006. Epub 2019 Apr 30.
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