| Angiogenesis plays an essential role during the whole lifespan and is progressively impaired with increasing age. Endothelial progenitor cells (EPCs) play an important role in angiogenesis, but have reduced proliferation, survival rates and migration in older individuals. Recent studies have reported the involvement of microRNAs (miRNAs) in angiogenesis. miR-34a, identified as a tumor suppressor, has been found to target silent information regulator 1 (SIRT1), leading to cell cycle arrest or apoptosis. However, the role of miR-34a in EPC-mediated angiogenesis is unknown. miR-34a was recently reported to be up-regulated in heart and spleen of aged mice, suggesting that aging may up-regulate miR-34a level. EPCs are critically regulated by eNOS, which function is reduced with aging. Tetrahydrobiopterin (BH4) is an essential cofactor for eNOS. When BH4 is limited, eNOS becomes uncoupled, which reduces EPC number and migration. GTP cyclohydrolase I (GTPCH I) is the rate-limiting enzyme for BH4 synthesis physiologically. The decreased activity and expression of GTPCH I may diminish BH4 level, leading to eNOS uncoupling and subsequent elevated reactive oxygen species (ROS) level. However, whether GTPCH I regulates miR-34a expression related to ROS production in EPCs is unknown.The present study is to explore whether and how miR-34a regulates EPC-mediated angiogenesis and the upstream regulator of miR-34a under normal and aging conditions. Firstly, we hypothesized that miR-34a impairs EPC-mediated angiogenesis through inhibition of SIRT1. The effect of miR-34a on angiogenic function and SIRT1-FoxO1 expression in EPCs overexpressed with miR-34a was observed. The role of SIRT1 in EPC-mediated angiogenesis was also examined. Furthermore, we tested the hypothesis that impaired angiogenesis in aged mice was related to miR-34a. We detected the level of miR-34a in EPCs from aged mice and observed the effect of miR-34a down-regulation on angiogenic activity of EPCs and expression of SIRT1 in aged EPCs. Finally, we hypothesized that GTPCH I may regulate miR-34a expression in EPCs. The levels of miR-34a and SIRT1 were detected in EPCs from aged eNOS/GCH double transgenic mice, GTPCH I transgenic (Tg-GCH) mice and GTPCH I-deficient Hph-1 mice.Methods:Bone marrow-derived EPCs recovered from adult male Spraque-Dawley rats (201-225 g), young C57BL/6 (wild-type, WT) mice, Tg-GCH (endothelial-specific overexpression of GTPCH I) mice, Hph-1 (with constitutively reduced expression of GTPCH I) (8 weeks) mice, aged C57BL/6 and eNOS/GCH double-transgenic mice (18-20 months, all males) were characterized by flow cytometry with the combination of stem cell and endothelial cell markers. The levels of miR-34a and SIRT1 protein were analyzed by Real-time PCR and Western blot, respectively. miR-34a was up-regulated or down-regulated in EPCs by transfection with miR-34a mimic or inhibitor. SIRT1 siRNA was also transfected into EPCs to knockdown SIRT1 level. Matrigel tube formation assay was used to determine EPC angiogenesis in vitro, while Matrigel plug assay was performed to detect EPC angiogenic activity in vivo. The senescence of EPCs was determined by in situ staining for senescence-associated β-galactosidase (SA-β-gal). ROS levels were evaluated via measuring DCF fluorescence by flow cytometry. Moreover, hind-limb ischemia in mice was performed and rate of blood flow recovery was assessed by laser Doppler imaging.Results:1) miR-34a regulated EPC-mediated angiogenesis. EPCs were recovered from bone marrow of adult male Spraque-Dawley rats. Results of flow cytometry showed that EPCs cultured for 7 days expressed both stem cell markers such as CD34 and CD133 and endothelial cell markers including VEGFR-2 (flk-1) and VE-Cadherin. The expression of miR-34a was confirmed in normal EPCs by Real-time PCR. Overexpression of miR-34a in EPCs by transfection of miR-34a mimic significantly impaired EPC angiogenesis in vitro, obviously induced senescence of EPCs, paralleled with a-40% SIRT1 reduction. Furthermore, knockdown of SIRT1 by transfection of siRNA resulted in diminished angiogenesis and increased senescence of EPCs. Finally, overexpression of miR-34a increased the level of acetylated Forkhead box O transcription factors 1 (FoxO1), a SIRT1 effector. Similarly, SIRT1 knockdown also elevated the level of acetylated FoxO1. These results indicate that SIRT1-FoxO1 pathway may mediate the inhibitory effect of miR-34a on EPC angiogenesis in vitro.2) miR-34a was involved in age-induced impaired EPC angiogenesis. Aged mice displayed a slower recovery rate of hind-limb blood flow in comparison with young mice. In consistence with delayed recovery of blood flow, EPCs from aged mice also showed impaired in vitro angiogenesis by 56% vs. young mice. Expression of miR-34a in EPCs was increased by～1 fold in aged vs. young WT mice, paralleled with 46% reduction of SIRT1 expression. Suppression of miR-34a in aged EPCs rescued impaired angiogenesis by 151%, while overexpression of miR-34a in young EPCs impaired in vitro angiogenesis. The effect of miR-34a on angiogenesis of EPCs in vivo was further investigated by Matrigel plug assay. The results showed that administration of EPCs with miR-34a overexpression induced formation of disorganized cell clusters, while administration of EPCs with transfection of scramble miRNA mimic induced formation of vessel-like structures and the presence of erythrocytes was evidenced in the lumen, indicating that miR-34a also inhibited angiogenic function of EPC in vivo. Moreover, SIRT1 was increased by 127% in aged EPCs with miR-34a inhibitor transfection, but reduced by 40% in young EPCs transfected with miR-34a mimic.3) GTPCH I regulated miR-34a and SIRT1 expression. ROS level was significantly elevated in EPCs from aged vs. young WT mice, accompanied by reduced GTPCH I protein level. miR-34a level in EPCs from aged eNOS/GCH double-transgenic mice was attenuated vs. aged WT mice, with a concomitant augmentation of Sirtl level. There was no significant difference in miR-34a and SIRT1 expression between aged eNOS/GCH double-transgenic EPCs and young WT EPCs. Interestingly, miR-34a was down-regulated in EPCs from Tg-GCH mice with an increase of SIRT1 level. On the contrary, miR-34a was up-regulated in EPCs from Hph-1 mice with a decrease in SIRT1 level vs. WT mice.Conclusions:In the present study, we found that 1) miR-34a inhibited EPC-mediated angiogenesis both in vitro and in vivo; 2) SIRT1-FoxOl pathway mediated the inhibitory effect of miR-34a on EPC-mediated angiogenesis; 3) up-regulation of miR-34a and down-regulation of SIRT1 in EPCs from aged mice were involved in impaired angiogenic function of EPCs; 4) GTPCH I regulated expression of miR-34a and its target SIRT1. These findings may provide a mechanistic basis for targeting miR-34a to rejuvenate angiogenesis of EPCs in aging. |
PDF Full Text Request