Estrogen reduces endothelial progenitor cell senescence through augmentation of telomerase activity.
BACKGROUND
Recent studies have demonstrated that aging or senescence constitutes a potential limitation to the ability of endothelial progenitor cells (EPCs) to sustain ischemic tissue and repair. Conversely, estrogens have been shown to accelerate recovery of the endothelium after vascular injury.


OBJECTIVE
To investigate whether estrogens are able to prevent senescence of EPCs.


RESULTS
Human EPCs were isolated from peripheral blood and characterized. After ex-vivo cultivation, the cells became senescent as determined by acidic beta-galactosidase staining. 17beta-estradiol dose-dependently inhibited the onset of EPC senescence in culture. Because cellular senescence is critically influenced by telomerase, which elongates telomeres, we measured telomerase activity using a polymerase chain reaction (PCR)-enzyme-linked immunosorbent assay (ELISA) technique. 17beta-estradiol significantly increased telomerase activity. Interestingly, reverse transcriptase-PCR analysis demonstrated that 17beta-estradiol dose-dependently increased the catalytic subunit, telomerase reverse transcriptase (TERT) - an effect that was significantly inhibited by pharmacological phosphatidylinositol 3-kinase (PI3-K) blockers (either wortmannin or LY294002). Because the expression of TERT is regulated by the PI3-K/Akt pathway, we examined the effect of 17beta-estradiol on Akt activity in EPCs. Immunoblotting analysis revealed that 17beta-estradiol dose-dependently led to phosphorylation and, thus, to activation of Akt in EPCs. We also examined whether the protective effect of 17beta-estradiol on EPC senescence translates into the augmentation of mitogenic activity in EPCs. A [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenol)-2-(4-sulfophenyl)-2H-tetrazolium] (MTS) assay demonstrated that the mitogenic potential in EPCs treated with 17beta-estradiol exceeded that in untreated (control) EPCs (P < 0.01). In addition, EPCs released vascular endothelial growth factor (VEGF) protein--an effect that was significantly augmented by 17beta-estradiol. Finally, in a Matrigel assay, EPCs treated with both 17beta-estradiol and VEGF were shown to be more likely to integrate into the network formation than those treated with VEGF alone.


CONCLUSIONS
The inhibition of EPC senescence by estrogen in vitro may improve the functional activity of EPCs in a way that is important for potential cell therapy.
Imanishi
Toshio
T
Department of Cardiovascular Medicine, Wakayama Medical University, 811-1 Kimidera, Wakayama City, Wakayama 641-8510, Japan. t-imani@wakayama-med.ac.jp
Hano
Takuzo
T
Nishio
Ichiro
I
D000730:Androstadienes / Q000494:pharmacology; D015153:Blotting, Western; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D016922:Cellular Senescence / Q000187:drug effects*; D002867:Chromones / Q000494:pharmacology; D003094:Collagen / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004338:Drug Combinations; D004730:Endothelium, Vascular / Q000166:cytology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D004967:Estrogens / Q000494:pharmacology*; D006801:Humans; D007797:Laminin / Q000378:metabolism; D009025:Morpholines / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011509:Proteoglycans / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013234:Stem Cells / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism; D019098:Telomerase / Q000378:metabolism*; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism / Q000494:pharmacology; D000077191:Wortmannin; D001616:beta-Galactosidase / Q000378:metabolism
D000730:Androstadienes; D002867:Chromones; D004268:DNA-Binding Proteins; D004338:Drug Combinations; D004791:Enzyme Inhibitors; D004967:Estrogens; D007797:Laminin; D009025:Morpholines; D011509:Proteoglycans; D011518:Proto-Oncogene Proteins; D042461:Vascular Endothelial Growth Factor A; C059485:matrigel; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D003094:Collagen; C494918:AKT1 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; C509186:TERT protein, human; D019098:Telomerase; D001616:beta-Galactosidase; D000077191:Wortmannin