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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.

SUBMITTER: Xia L

PROVIDER: S-EPMC2567879 | biostudies-other | 2008 Oct

REPOSITORIES: biostudies-other

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Project description:BackgroundCirculating endothelial progenitor cells (EPCs) play important roles in vascular repair. However, the mechanisms of high-glucose- (HG-) induced cord blood EPC senescence and the role of B2 receptor (B2R) remain unknown.MethodsCord blood samples from 26 patients with gestational diabetes mellitus (GDM) and samples from 26 healthy controls were collected. B2R expression on circulating CD34+ cells of cord blood mononuclear cells (CBMCs) was detected using flow cytometry. The plasma concentrations of 8-isoprostaglandin F2α (8-iso-PGF2α) and nitric oxide (NO) were measured. EPCs were treated with HG (40 mM) alone or with bradykinin (BK) (1 nM). The B2R and eNOS small interfering RNAs (siRNAs) and the PI3K antagonist LY294002 were added to block B2R, eNOS, and PI3K separately. To determine the number of senescent cells, senescence-associated β-galactosidase (SA-β-gal) staining was performed. The level of mitochondrial reactive oxygen species (ROS) in EPCs was assessed by Mito-Sox staining. Cell viability was evaluated by Cell Counting Kit-8 (CCK-8) assays. Mitochondrial DNA (mtDNA) copy number and the relative length of telomeres were detected by real time-PCR. The distribution of human telomerase reverse transcriptase (hTERT) in the nucleus, cytosol, and mitochondria of EPCs was detected by immunofluorescence. The expression of B2R, p16, p21, p53, P-Ser473AKT, T-AKT, eNOS, and hTERT was demonstrated by Western blot.ResultsB2R expression on circulating CD34+ cells of CBMCs was significantly reduced in patients with GDM compared to healthy controls. Furthermore, B2R expression on circulating CD34+ cells of CBMCs was inversely correlated with plasma 8-iso-PGF2α concentrations and positively correlated with plasma NO levels. BK treatment decreased EPC senescence and ROS generation. Furthermore, BK treatment of HG-exposed cells led to elevated P-Ser473AKT and eNOS protein expression compared with HG treatment alone. BK reduced hTERT translocation in HG-induced senescent EPCs. B2R siRNA, eNOS siRNA, and antagonist of the PI3K signalling pathway blocked the protective effects of BK.ConclusionBK, acting through PI3K-AKT-eNOS signalling pathways, reduced hTERT translocation, increased the relative length of telomeres while reducing mtDNA copy number, and finally protected against EPC senescence induced by HG.

Project description:Endothelial senescence plays crucial roles in diabetic vascular complication. Recent evidence indicated that transient hyperglycaemia could potentiate persistent diabetic vascular complications, a phenomenon known as "metabolic memory." Although SIRT1 has been demonstrated to mediate high glucose-induced endothelial senescence, whether and how "metabolic memory" would affect endothelial senescence through SIRT1 signaling remains largely unknown. In this study, we investigated the involvement of SIRT1 axis as well as the protective effects of resveratrol (RSV) and metformin (MET), two potent SIRT1 activators, during the occurrence of "metabolic memory" of cellular senescence (senescent "memory"). Human umbilical vascular endothelial cells (HUVECs) were cultured in either normal glucose (NG)/high glucose (HG) media for 6 days, or 3 days of HG followed by 3 days of NG (HN), with or without RSV or MET treatment. It was shown that HN incubation triggered persistent downregulation of deacetylase SIRT1 and upregulation of acetyltransferase p300, leading to sustained hyperacetylation (at K382) and activation of p53, and subsequent p53/p21-mediated senescent "memory." In contrast, senescent "memory" was abrogated by overexpression of SIRT1 or knockdown of p300. Interestingly, we found that SIRT1 and p300 could regulate each other in response to HN stimulation, suggesting that a delicate balance between acetyltransferases and deacetylases may be particularly important for sustained acetylation and activation of non-histone proteins (such as p53), and eventually the occurrence of "metabolic memory." Furthermore, we found that RSV or MET treatment prevented senescent "memory" by modulating SIRT1/p300/p53/p21 pathway. Notably, early and continuous treatment of MET, but not RSV, was particularly important for preventing senescent "memory." In conclusion, short-term high glucose stimulation could induce sustained endothelial senescence via SIRT1/p300/p53/p21 pathway. RVS or MET treatment could enhance SIRT1-mediated signaling and thus protect against senescent "memory" independent of their glucose lowering mechanisms. Therefore, they may serve as promising therapeutic drugs against the development of "metabolic memory."

Project description:Misfolding mutations in cartilage oligomeric matrix protein (COMP) cause it to be retained within the endoplasmic reticulum (ER) of chondrocytes, stimulating a multitude of damaging cellular responses including ER stress, inflammation, and oxidative stress, which ultimately culminates in the death of growth plate chondrocytes and pseudoachondroplasia (PSACH). Previously, we demonstrated that an antioxidant, resveratrol, substantially reduces the intracellular accumulation of mutant-COMP, dampens cellular stress, and lowers the level of growth plate chondrocyte death. In addition, we showed that resveratrol reduces mammalian target of rapamycin complex 1 (mTORC1) signaling, suggesting a potential mechanism. In this work, we investigate the role of autophagy in treatment of COMPopathies. In cultured chondrocytes expressing wild-type COMP or mutant-COMP, resveratrol significantly increased the number of Microtubule-associated protein 1A/1B-light chain 3 (LC3) vesicles, directly demonstrating that resveratrol-stimulated autophagy is an important component of the resveratrol-driven mechanism responsible for the degradation of mutant-COMP. Moreover, pharmacological inhibitors of autophagy suppressed degradation of mutant-COMP in our established mouse model of PSACH. In contrast, blockage of the proteasome did not substantially alter resveratrol clearance of mutant-COMP from growth plate chondrocytes. Mechanistically, resveratrol increased SIRT1 and PP2A expression and reduced MID1 expression and activation of phosphorylated protein kinase B (pAKT) and mTORC1 signaling in growth plate chondrocytes, allowing clearance of mutant-COMP by autophagy. Importantly, we show that optimal reduction in growth plate pathology, including decreased mutant-COMP retention, decreased mTORC1 signaling, and restoration of chondrocyte proliferation was attained when treatment was initiated between birth to 1 week of age in MT-COMP mice, translating to birth to approximately 2 years of age in children with PSACH. These results clearly demonstrate that resveratrol stimulates clearance of mutant-COMP by an autophagy-centric mechanism. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.

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

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