Resveratrol reduces endothelial progenitor cells senescence through augmentation of telomerase activity by Akt-dependent mechanisms

Abstract

Background and purpose: Recent studies have shown that resveratrol increased endothelial progenitor cells (EPCs) numbers and functional activity. However, the mechanisms remain to be determined. Previous studies have demonstrated that increased EPC numbers and activity were associated with the inhibition of EPC senescence, which involves activation of telomerase. Therefore, we investigated whether resveratrol inhibits the onset of EPC senescence through telomerase activation, leading to potentiation of cellular activity.

Experimental approach: After prolonged in vitro cultivation, EPCs were incubated with or without resveratrol. The senescence of EPCs were determined by acidic beta-galactosidase staining. The bromo-deoxyuridine incorporation assay or a modified Boyden chamber assay were employed to assess proliferative or migratory capacity, respectively. To further examine the underlying mechanisms of these effects, we measured telomerase activity and the phosphorylation of Akt by western blotting.

Key results: Resveratrol dose dependently prevented the onset of EPCs senescence and increased the proliferation and migration of EPCs. The effect of resveratrol on senescence could not be abolished by eNOS inhibitor or by an oestrogenic receptor antagonist. Resveratrol significantly increased telomerase activity and Akt phosphorylation. Pre-treatment with the PI3K inhibitor, LY294002, significantly attenuated resveratrol-induced telomerase activity.

Conclusions and implications: Resveratrol delayed the onset of EPC senescence and this effect was accompanied by activation of telomerase through the PI3K-Akt signalling pathway. The inhibition of EPCs senescence by resveratrol might protect EPCs against dysfunction induced by pathological factors in vivo and improve EPC functional activities in a way that may be important for cell therapy.

Figure 1

Characterization of endothelial progenitor cells (EPCs). (a, left panel) Mononuclear cells (MNCs) were plated on culture dishes just after isolation from peripheral blood ( × 200); (right panel) after 7 days in culture, attached cells exhibited a spindle-shaped, endothelial cells-like morphology ( × 200). (b) Adherent cells stained for uptake of acetylated low-density lipoprotein (acLDL; left panel, red, exciting wavelength 543 nm) or Ulex europaeus agglutinin (UEA-1) binding (right panel, green, exciting wavelength 477 nm) were assessed under a laser scanning confocal microscope (LSCM, × 400). (c) Flow cytometry analyses of adherent cells at day 7 of culture (n=6). Adherent cells were positive for KDR (82.1±6.4%), CD34 (29.0±5.7%), AC133 (19.1±4.8%), VE-cadherin (69.6±6.4%) and CD45 (17.8±4.0%). Data are percentage of positive cells.

Figure 2

Resveratrol prevents endothelial progenitor cell (EPC) senescence. Freshly isolated mononuclear cells were cultivated in Medium 199 supplemented with 20% foetal-calf serum and vascular endothelial growth factor (VEGF). At day 4, cells were seeded onto methylcellulose plates and cultured with resveratrol in the indicated concentrations for 7 days. In some experiments cells treated with resveratrol were also exposed to N^G-mono-methyl-L-arginine (L-NAME, 1 mM) or ICI182780 (1 μM). (a) Representative micrographs of senescence-associated β-galactosidase (SA-β-gal)-positive cells (senescent cells) in EPCs treated with 50 μM resveratrol (left) or without resveratrol (right) are shown. (b) The number of blue cells was counted manually from a total of 200 cells. Data are means±s.d., n=6; ^*P<0.05, ^#P<0.01 vs control.

Figure 3

Effects of resveratrol on proliferation and migration of endothelial progenitor cells (EPCs). Cultures of EPCs were treated with the indicated concentrations of resveratrol for 7 days before cells were harvested. (a) Cell proliferation was estimated from the incorporation of 5-bromo-2′-deoxyuridine (BrdU) and calculated as a percentage of the control value. (b) Cell migration was assessed by a modified Boyden chamber assay. The data shown are mean±s.d., n=6; ^*P<0.05, ^#P<0.01 vs control.

Figure 4

Resveratrol induces the telomerase activity in endothelial progenitor cells (EPCs) through the PI3K-Akt pathway. Freshly isolated mononuclear cells (MNCs) were cultivated in Medium 199 supplemented with 20% foetal-calf serum and vascular endothelial growth factor (VEGF). After 4 days of cultivation, cells were treated with indicated doses of resveratrol for 24 h with or without pre-treatment with LY294002 (10 μM). Telomerase activity was measured by the telomeric repeat-amplification protocol (TRAP) assay. A representative polyacrylamide gel electrophoresis gel (PAGE) figure of TRAP-PCR is shown on the top panel. Data are means±s.d., n=6. ^*P<0.05, ^#P<0.01 vs control.

Figure 5

Effect of resveratrol on Akt phosphorylation at Ser⁴⁷³. Mononuclear cells at day 4 were stimulated for 24 h with indicated concentrations of resveratrol, and phosphorylation of Akt was determined with a phospho-specific Akt antibody. A representative blot from six independent experiments is shown on the top panel. Data are expressed as a ratio of phospho-Akt over total Akt. Bars represent means±s.d. (n=6). ^*P<0.05, ^#P<0.01 vs control.

Figure 6

Effect of resveratrol on endothelial progenitor cell (EPC) apoptosis. (A) Representative dot plots of apoptotic cells from group of 0 (a), 50 (b) and 100 (c) μM resveratrol. (B) The levels of apoptotic rate of EPCs in different groups. Resveratrol (50 μM ) had no effect on apoptosis in EPCs. Values represent mean±s.d. (n=6). ^# P<0.05 vs control (0 μM resveratrol).