High Glucose Impairs Early and Late Endothelial Progenitor Cells by Modifying Nitric Oxide–Related but Not Oxidative Stress–Mediated Mechanisms
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Figures
- FIG. 1.
Morphology and characterization of EPCs from peripheral blood. MNCs were isolated and plated on fibronectin-coated culture dish on the first day (A). Four days after plating, adherent early EPCs with spindle shape were shown (B). Twenty-one days after plating, late EPCs with cobblestone-like morphology were selected, reseeded, and grown to confluence (C). Phenotyping of endothelial characteristics of colony-forming units of EPCs (D–F). A colony of EPCs was defined as a central core of round cells with elongated sprouting cells at the periphery (D). Most cells were shown to simultaneously endocytose DiI-acLDL (red) (E) and bind fluorescein isothiocyanate UEA-1 (lectin) (green) (F). Immunofluorescence detection (green) of VE-cadherin (G), von Willebrand factor (H), PECAM-1 (platelet/endothelial cell adhesion molecule-1) (CD31) (I), CD34 (J), KDR (K), and AC133 (L) for late EPCs. Cells were counterstained with propidium iodide for nucleus (red). Scale bar: 50 μm.
- FIG. 2.
High glucose decreases EPC number, proliferation, and colony-forming capacity. Cells were isolated and incubated with different concentrations of glucose or mannitol (osmotic control) for 4 days. Early EPCs were characterized as adherent cells that were dual positive for lactin staining and DiI-acLDL uptake. The numbers of EPCs were counted under microscope, and data are expressed as mean numbers of EPCs per high-power field (HPF) ± SE (A). MTT assay was also performed for late EPC proliferation activity (B), normalized to cells incubated in control medium (5 mmol/l glucose). Colony-forming assay for control, mannitol- (20 mmol/l), and glucose- (25 mmol/l) incubated EPCs (C). Data are expressed as means ± SE; n = 5, *P < 0.05 vs. control.
- FIG. 3.
High glucose increases senescence and impairs the migratory and tube formation function of EPCs. Cellular aging of control, mannitol- (20 mmol/l), or glucose- (25 mmol/l) incubated early and late EPCs were analyzed by senescence-associated acidic β-galactosidase activity assay (A). A modified Boyden chamber assay was used with VEGF as chemoattractive factor for late EPC migratory function. Representative photos are shown; the small dots are holes in the barrier membrane. The migrated cells were stained with hematoxylin and counted under microscope (B). An in vitro angiogenesis assay for late EPCs was used with ECMatrix gel. Representative photos for in vitro angiogenesis are shown. The cells were stained with crystal violet, and the averages of the total area of complete tubes formed by cells were compared by computer software (C). Data are mean ± SE; n = 6, *P < 0.05 vs. control (5 mmol/l glucose).
- FIG. 4.
High glucose decreases eNOS phosphorytion and bioavailable NO in EPCs. After incubation of EPCs with glucose or mannitol for 4 days, NOS protein levels, NO content, and cGMP formation were analyzed. A representative immunoblot shows the p-eNOS, eNOS, iNOS, and α-tubulin amounts of early and late EPCs in response to high glucose for 4 days. Each bar graph shows the summarized data from four separate experiments by densitometry after normalization to α-tubulin (A). Nitrite production (as NO content) in early/late EPC culture medium was measured by Griess reagent (B). Intracellular cGMP (pmol/mg protein) in early/late EPCs was measured by using enzyme-linked immunosorbent assay kits (C). Data are means ± SE; n = 5, *P < 0.05 vs. control (5 mmol/l glucose).
- FIG. 5.
Roles of Akt/FoxO1, NO, p38 MAPK, and oxidative stress signalings in high glucose–downregulated EPCs. A representative immunoblot shows the p-FoxO1/FoxO, and p-Akt/Akt amounts of early/late EPCs in response to high glucose for 4 days. Each bar graph shows the summarized data from four separate experiments by densitometry after normalization (A). EPC number and proliferation (for early and late EPCs, respectively) were anylyzed after for 4 days of culture in control or glucose/mannitol medium in the absence or presence of SNP (NO donor, 25 μmol/l), l-NAME (NOS inhibitor, 100 μmol/l), LY294002 (PI3K/Akt inhibitor, 5 μmol/l), and SB203580 (p38 MAPK inhibitor, 5 μmol/l) (B) or various concentrations of diverse antioxidants (PEG-SOD, PEG-catalase, vitamin C, NAC, PDTC, DPI, apocynin, and rotenone) (D). Data are means ± SE; n = 5, *P < 0.05 vs. control (5 mmol/l glucose), #P < 0.05 vs. high glucose–treated group.
- FIG. 6.
Schematic representation of hyperglycemia-induced early and late EPC downregulation. High glucose impaired the proliferation and function of early and late EPCs. NO donor but not antioxidants reversed the impairments, suggesting the role of NO-related rather than ROS-mediated mechanisms in hyperglycemia-caused EPC downregulation. DM, diabetic.
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