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Ischemic cardiovascular complications remain a major cause of mortality in people with type 2 diabetes (T2D). Individuals with T2D may have a reduced ability to revascularize ischemic tissues due to abnormal production of circulating provascular progenitor cells. This 'regenerative cell exhaustion' process is intensified by increasing oxidative stress and inflammation and during T2D progression. Chronic exhaustion may be mediated by changes in the bone marrow microenvironment that dysregulate the wingless related integration site network, a central pathway maintaining the progenitor cell pool. Restoration of vascular regenerative cell production by reducing glucotoxicity with contemporary antihyperglycemic agents, by reducing systemic inflammation postbariatric surgery, or by modulating progenitor cell provascular functions using exosomal manipulation, may provide unique approaches for mitigating ischemic disease.

Highlights •This study combined ALDH activity with cell surface marker expression to develop a multiparametric flow cytometry assay to assess proangiogenic progenitor and proinflammatory cell content in the peripheral blood of patients with T2D compared with age-matched control subjects. •Patients with T2D exhibited an increased frequency of proinflammatory ALDHhi cells with granulocyte side scatter properties and a decreased frequency of circulating monocytes with an M2 phenotype that is associated with proangiogenic and anti-inflammatory functions. •Patients with T2D exhibited significant depletion of circulating provascular ALDHhiCD34+ progenitor cells with primitive, migratory, endothelial, and pericyte phenotypes. •Subgroup analyses that stratified patients with T2D according to age, duration of T2D, insulin requirement, and glycosylated hemoglobin levels revealed that only the duration of T2D correlated with vascular progenitor cell depletion. •Flow cytometric assessment of circulating ALDHhi cell subsets represents a promising translational approach for identifying patients with T2D at increased risk for cardiovascular comorbidities.

Summary The examination of circulating pro-vascular progenitor cell frequency and function is integral in understanding aberrant blood vessel homeostasis in individuals with cardiometabolic disease. Here, we outline the characterization of progenitor cell subsets from peripheral blood using high aldehyde dehydrogenase (ALDH) activity, an intracellular detoxification enzyme previously associated with pro-vascular progenitor cell status. Using this protocol, cells can be examined by flow cytometry for ALDH activity and lineage restricted cell surface markers simultaneously. For complete details on the use and execution of this protocol, please refer to Terenzi et al. (2019) and Hess et al. (2019, 2020)., Graphical Abstract, Highlights • Aldehyde dehydrogenase is superior in the isolation of progenitor cells • Flow cytometry is an effective method to characterize pro-vascular cells • Aggressive gating strategies allows for in-depth progenitor cell characterization • The use of fresh blood samples will yield most accurate cell prevalence, The examination of circulating pro-vascular progenitor cell frequency and function is integral in understanding aberrant blood vessel homeostasis in individuals with cardiometabolic disease. Here, we outline the characterization of progenitor cell subsets from peripheral blood using high aldehyde dehydrogenase (ALDH) activity, an intracellular detoxification enzyme previously associated with pro-vascular progenitor cell status. Using this protocol, cells can be examined by flow cytometry for ALDH activity and lineage restricted cell surface markers simultaneously.

Summary Bariatric surgery, in addition to the benefit of sustained weight loss, can also reduce cardiometabolic risk and mortality. Lifelong vessel maintenance is integral to the prevention of cardiovascular disease. Using aldehyde dehydrogenase activity, an intracellular detoxifying enzyme present at high levels within pro-vascular progenitor cells, we observed an association between chronic obesity and “regenerative cell exhaustion” (RCE), a pathology whereby chronic assault on circulating regenerative cell types can result in adverse inflammation and diminished vessel repair. We also describe that, at 3 months following bariatric surgery, systemic inflammatory burden was reduced and pro-angiogenic macrophage precursor content was improved in subjects with severe obesity, suggesting the restoration of a microenvironment to support vessel homeostasis. These data suggest that bariatric surgery may reverse deleterious events that predispose patients with morbid obesity to cardiovascular risk., Graphical Abstract, Highlights Obesity features a low frequency of ALDH and CD133 co-expressing cells Bariatric surgery results in lower granulocyte precursors expressing ALDH Macrophage balance favors M2 polarization following bariatric surgery Cellular changes after bariatric surgery give insight into reducing CV risk, In Hess et al., the quantification of circulating progenitor cells in individuals with obesity, before and 3 months after bariatric surgery, demonstrates reduced granulocyte precursors and increased pro-angiogenic monocytes with M2 polarization. These alterations are consistent with improved vessel homeostasis and recovered cardiovascular health observed following bariatric surgery.

In response to the Letter by Fadini, Hess et al. discuss the interpretation of their data and the details of the multiparametric analyses employed to measure the changes in circulating provascular cell content in patients with type 2 diabetes receiving empagliflozin compared to placebo treatment.

Critical limb ischemia (CLI), the most severe form of peripheral artery disease, is characterized by pain at rest and non-healing ulcers in the lower extremities. For patients with CLI, where the extent of atherosclerotic artery occlusion is too severe for surgical bypass or percutaneous interventions, limb amputation remains the only treatment option. Thus, cell-based therapy to restore perfusion and promote wound healing in patients with CLI is under intense investigation. Despite promising preclinical studies in animal models, transplantation of bone marrow (BM)-derived cell populations in patients with CLI has shown limited benefit preventing limb amputation. Early trials injected heterogenous mononuclear cells containing a low frequency of cells with pro-vascular regenerative functions. Most trials transferred autologous cells damaged by chronic disease that demonstrated poor survival in the ischemic environment and impaired function conferred by atherosclerotic or diabetic co-morbidities. Finally, recent preclinical studies suggest optimized blood vessel formation may require paracrine and/or structural contributions from multiple progenitor cell lineages, angiocrine-secretory myeloid cells derived from hematopoietic progenitor cells, tubule-forming endothelial cells generated by circulating or vessel-resident endothelial precursors, and vessel-stabilizing perivascular cells derived from mesenchymal stem cells. Understanding how stem cells co-ordinate the myriad of cells and signals required for stable revascularization remains the key to translating the potential of stem cells into curative therapies for CLI. Thus, combination delivery of multiple cell types within supportive bioengineered matricies may represent a new direction to improve cell therapy strategies for CLI.

Hess et al. quantified circulating aldehyde dehydrogenase-expressing (ALDHhi) cell subsets in people with T2DM given either empagliflozin (EMPA) or placebo. EMPA treatment increased circulating pro-angiogenic CD133+ progenitor cells, decreased pro-inflammatory ALDHhi granulocyte precursors, and increased ALDHhi monocytes with M2 polarization. EMPA treatment improved T2DM-associated "regenerative cell depletion" contributing to enhanced vascular health.