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.

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.

The investigators 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, the investigators 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 exhibits 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.

The investigators 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, the investigators 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. The investigators 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.

Inclusion Criteria:

• Adults ≥18 years of age.
• Willing to provide written informed consent.
• Documented history of T2D
• No documented history of diabetes

Exclusion Criteria:

• Unable or unwilling to provide written informed consent or provide a peripheral blood sample.
• Any life-threatening disease expected to result in death within two years of consent.
• 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.
• Known severe liver disease.
• White blood cell count ≥15 x 10^9/L.
• Active infectious disease requiring systemic antibiotic or anti-viral agents.
• Known acquired immunodeficiency syndrome such as HIV.
• Treated autoimmune disorders (e.g. T1D and LADA).
• On oral steroid therapy (e.g. prednisone or other corticosteroids) or other immunosuppressive agents (e.g. methotrexate).

Individuals will be asked their self-representation of their gender identity.

Participants will be identified from primary care clinics in the Greater Toronto Area using paper-based and electronic medical records.