Circulating Pro-Vascular Progenitor Cell Depletion During Type 2 Diabetes: Translational Insights Into the Prevention of Ischemic Complications in Diabetes

Daniella C Terenzi, Mohammed Al-Omran, Adrian Quan, Hwee Teoh, Subodh Verma, David A Hess, Daniella C Terenzi, Mohammed Al-Omran, Adrian Quan, Hwee Teoh, Subodh Verma, David A Hess

Abstract

Detection of vascular regenerative cell exhaustion is required to combat ischemic complications during type 2 diabetes mellitus (T2D). We used high aldehyde dehydrogenase (ALDH) activity and surface marker co-expression to develop a high-throughput flow cytometry-based assay to quantify circulating proangiogenic and proinflammatory cell content in the peripheral blood of individuals with T2D. Circulating proangiogenic monocytes expressing anti-inflammatory M2 markers were decreased in patients with T2D. Individuals with longer duration of T2D exhibited reduced frequencies of circulating proangiogenic ALDHhiCD34+ progenitor cells with primitive (CD133) and migratory (CXCR4) phenotypes. This approach consistently detected increased inflammatory cell burden and decreased provascular progenitor content in individuals with T2D.

Keywords: ALDH, aldehyde dehydrogenase; BM, bone marrow; HbA1c, glycosylated hemoglobin; ROS, reactive oxygen species; SSC, side scatter; T2D, type 2 diabetes mellitus; Wnt, wingless related integration site; aldehyde dehydrogenase; angiogenesis; ischemia; progenitor cells; type 2 diabetes.

Figures

Graphical abstract
Graphical abstract
Figure 1
Figure 1
Circulating Progenitor Cell Subpopulations Are Discerned According to High ALDH-Activity and SSC Properties (A and B) Representative flow cytometry plots using N,N-diethylaminobenzaldehyde (DEAB) to inhibit aldehyde dehydrogenase (ALDH) activity establishing gates for low versus high ALDH-activity. Without inhibition, cells with high ALDH-activity exhibit increased fluorescence intensity (right shift) and detect primitive cells with a self-protective progenitor cell phenotype. High ALDH-activity combined with side scatter (SSC) properties selects for a progenitor cell subpopulation with low intracellular complexity (R1 = ALDHhiSSClow cells), a monocyte subpopulation with intermediate intracellular complexity (R2 = ALDHhiSSCmid cells), and a granulocytic subpopulation with high intracellular complexity (R3 = ALDHhiSSChi cells). (C and D) Representative flow cytometry plots showing the frequencies of each population in control subjects and patients with type 2 diabetes mellitus (T2D). Compared with control subjects, patients with T2D exhibited an increased frequency of ALDHhi cells within the granulocyte subpopulation (R3) and an equal frequency of circulating ALDHhi cells with low (R1) and intermediate (R2) complexity. Values are mean ± SEM. ***p < 0.001 with the Student's t-test.
Figure 2
Figure 2
Circulating ALDHhiSSClow Cells With Primitive, Myeloid, and Migratory Phenotypes Are Decreased in Patients with T2D (A–C) The frequency of circulating ALDHhiSSClow progenitor cells with primitive cell phenotype (CD34+CD133+) was reduced in patients with T2D compared with control subjects. (D–F) The frequency of circulating ALDHhiSSClow progenitor cells with early myeloid cell phenotype (CD34+CD33+) was reduced in patients with T2D compared with control subjects. (G–I) The frequency of circulating ALDHhiSSClow progenitor cells with migratory phenotype (CD34+CXCR4+) was reduced in patients with T2D compared with control subjects. Values are mean ± SEM. ***p < 0.01 with the Student's t-test. Abbreviations as in Figure 1.
Figure 3
Figure 3
Circulating ALDHhiSSClow Cells With Endothelial Cell and Pericyte-associated Adhesive Phenotypes Are Decreased in Patients With T2D (A–C) The frequency of circulating ALDHhiSSClow progenitor cells co-expressing CD34 with hematopoietic/endothelial cell marker platelet endothelial cell adhesion molecule (PECAM) (CD31) was equivalent in patients with T2D compared with control subjects. (D–F) The frequency of circulating ALDHhiSSClow cells in progenitor cells co-expressing CD34 with the endothelial cell–associated marker vascular endothelial (VE)-cadherin (CD144) was decreased in patients with T2D compared with control subjects. (G–I) The frequency of circulating ALDHhiSSClow progenitor cells co-expressing CD34 with the endothelial/pericytes marker melanoma cell adhesion molecule (MCAM) (CD146) was decreased in patients with T2D compared with control subjects. Values are mean ± SEM. ∗p < 0.001 with the Student's t-test. Other abbreviations as in Figure 1.
Figure 4
Figure 4
Circulating ALDHhiSSCmid Cells With M2 Phenotype Are Decreased in Patients With T2D (A to C) The frequency of circulating ALDHhiSSCmid cells co-expressing CD34 with the macrophage scavenger receptor (CD68) was decreased in patients with T2D compared with control subjects. (D to F) The frequency of circulating ALDHhiSSCmid cells co-expressing CD68 with the M1 macrophage–associated marker CD80 was equal in patients with T2D compared with control subjects. (G to I) The frequency of circulating ALDHhiSSCmid progenitor cells co-expressing CD68 with the M2 macrophage–associated marker CD163 was decreased in patients with T2D compared with control subjects. Values are mean ± SEM. *p < 0.05 with the Student's t-test. Other abbreviations as in Figure 1.
Figure 5
Figure 5
Circulating ALDHhiSSChi Inflammatory Cells Are Decreased in Patients Taking Insulin (A and B) The frequency of cells with high ALDH-activity and high SSC properties was decreased in patients with T2D taking insulin. (C and D) The frequency of primitive and migratory progenitor cells was equivalent in patients with T2D despite insulin therapy. Values are mean ± SEM. *p < 0.05 with the Student's t-test. Abbreviations as in Figure 1.
Figure 6
Figure 6
Circulating ALDHhiSSClow Cells With Primitive and Migratory Progenitor Cell Phenotypes Decreased With Longer Duration of Diabetes (A and B) In patients with T2D, the frequency of cells with high ALDH-activity was equivalent in patients ≤70 years of age compared with patients >70 years of age with T2D. (C and D) The frequency of circulating primitive progenitor cells (CD34+/CD133+) and migratory progenitor cells (CD34+/CXCR4+) was equivalent in patients ≤70 years of age compared with patients >70 years of age. (E and F) In patients with T2D, the frequency of cells with high ALDH-activity was equivalent in patients with diabetes duration ≤13 years compared with patients with diabetes duration >13 years. (G and H) However, the frequency of circulating primitive progenitor cells (CD34+/CD133+) and migratory progenitor cells was decreased in patients with diabetes duration ≤13 years compared with patients with diabetes duration >13 years. Values are mean ± SEM. *p < 0.05 with the Student's t-test. Abbreviations as in Figure 1.

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