Functional Investigation of Endothelial Function and Regenerative Cell Exhaustion in Type 2 Diabetes

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.