Shuyan Chen

SIRT1 Protects Against Oxidative Stress‐Induced Endothelial Progenitor Cells Apoptosis by Inhibiting FOXO3a via FOXO3a Ubiquitination and Degradation

Cell loss due to apoptosis induced by oxidative stress is a major hurdle for endothelial progenitor cells (EPCs)‐based therapy. Sirtuin 1 (SIRT1) plays important roles in many pathophysiological processes by deacetylating various substrates, including forkhead transcription factor (FOXO). However, after deacetylation, the fate of FOXO protein remains to be explored. In the present study, we investigated whether SIRT1 exerted a protective effect on hydrogen peroxide (H2O2)‐induced EPCs apoptosis and, if so, what the underlying mechanism might be. EPCs were isolated and obtained from human umbilical cord blood by density gradient centrifugation and identified by morphology, tube formation ability, cell surface markers, and the ability to take up acetylated low‐density lipoprotein (Dil‐Ac‐LDL) and bind ulex europaeus agglutinin 1 (FITC‐UEA‐1). Immunofluorescence showed that SIRT1 is localized in the nucleus of EPCs in the presence or absence of H2O2. SIRT1 protein level in EPCs was increased by the treatment with H2O2 for 24 h. Incubation of EPCs with H2O2 dose dependently induced EPCs apoptosis. SIRT1 overexpression reduced the rate of EPCs apoptosis induced by H2O2, whereas SIRT1 downregulation and EX527, a specific SIRT1 inhibitor, exerted the opposite effect. SIRT1 overexpression decreased the total FOXO3a protein expression, whereas SIRT1 downregulation and EX527 increased the amount of FOXO3a protein. SIRT1 reduced FOXO3a transcriptional activity according to Bim expression. Co‐immunoprecipitation assay showed that SIRT1 could bind to FOXO3a, reduce its acetylation level and increase its ubiquitination level. To sum up, our work demonstrated that SIRT1 had a pivotally protective role in the regulation of EPCs apoptosis induced by H2O2 and that SIRT1 protected against apoptosis by inhibiting FOXO3a via FOXO3a ubiquitination and subsequent degradation. J. Cell. Physiol. 230: 2098–2107, 2015.

Hydrogen peroxide induced impairment of endothelial progenitor cell viability is mediated through a FoxO3a dependant mechanism.

OBJECTIVES Increased oxidative stress has been suggested to contribute to the functional impairment of endothelial progenitor cells (EPCs). The Forkhead box O transcription factors (FoxOs) are critical regulators involved in various cellular processes including cell apoptosis. Here, we investigated whether FoxOs are required in oxidative stress induced EPC apoptosis. METHODS AND RESULTS EPCs were cultured from cord blood derived mononuclear cells and treated with hydrogen peroxide (H2O2) for induction of oxidative stress. Incubation with H2O2 dose dependently reduced viability and increased apoptosis in EPCs. Western blotting showed that EPCs predominantly expressed FoxO3a and the expression was markedly increased upon H2O2 treatment. Transduction with adenoviral vectors expressing either a wide-type or a non-phosphorylatable, constitutively active mutant of FoxO3a led to further increased apoptosis of EPCs after H2O2 treatment. Conversely, FoxO3a silencing rescued EPCs from these H2O2 induced deleterious effects. Overexpression of FoxO3a also increased the level of the pro-apoptotic protein Bim, whereas FoxO3a silencing downregulated H2O2 induced Bim expression. Furthermore, Matrigel assay demonstrated that FoxO3a overexpression significantly impaired the tube forming ability of EPCs, whereas its silencing completely protected EPCs from H2O2 induced decrease of capillary formation. CONCLUSIONS These data suggest that oxidative stress induced impairment of EPC survival is mediated through a FoxO3a dependant mechanism, possibly by transcriptional regulation of Bim. Our data indicate FoxO3a as a potential therapeutic target for improvement of EPC number and function in patients with ischemic heart disease.

Hepatocyte-targeting gene transfer mediated by galactosylated poly(ethylene glycol)- graft-polyethylenimine derivative

Biscarbamate cross-linked polyethylenimine derivative (PEI-Et) has been reported as a novel nonviral vector for efficient and safe gene transfer in our previous work. However, it had no cell-specificity. To achieve specific delivery of genes to hepatocytes, galactosylated poly(ethylene glycol)-graft-polyethylenimine derivative (GPE) was prepared through modification of PEI-Et with poly(ethylene glycol) and lactobionic acid, bearing a galactose group as a hepatocyte-targeting moiety. The composition of GPE was characterized by proton nuclear magnetic resonance. The weight-average molecular weight of GPE measured with a gel permeation chromatography instrument was 9489 Da, with a polydispersity of 1.44. GPE could effectively condense plasmid DNA (pDNA) into nanoparticles. Gel retardation assay showed that GPE/pDNA complexes were completely formed at weigh ratios (w/w) over 3. The particle size of GPE/pDNA complexes was 79–100 nm and zeta potential was 6–15 mV, values which were appropriate for cellular uptake. The morphology of GPE/pDNA complexes under atomic force microscopy appeared spherical and uniform in size, with diameters of 53–65 nm. GPE displayed much higher transfection efficiency than commercially available PEI 25 kDa in BRL-3A cell lines. Importantly, GPE showed good hepatocyte specificity. Also, the polymer exhibited significantly lower cytotoxicity compared to PEI 25 kDa at the same concentration or weight ratio in BRL-3A cell lines. To sum up, our results indicated that GPE might carry great potential in safe and efficient hepatocyte-targeting gene delivery.

Delivery of Therapeutic AGT shRNA by PEG-Bu for Hypertension Therapy

Gene silencing by RNA interference (RNAi) is a promising approach for gene therapy. However, up to today, it is still a major challenge to find safe and efficient non-viral vectors. Previously, we reported PEI-Bu, a small molecular weight PEI derivative, as an efficient non-viral carrier. However, like many PEI-based polymers, PEI-Bu was too toxic. In order to reduce cytotoxicity while maintain or even enhance transfecion efficiency, we modified PEI-Bu with poly(ethylene glycol) (PEG) to obtain PEG-Bu, and used it to delivery a theraputic short hairpin RNA (shRNA) targeting angiotensinogen (AGT) to normal rat liver cells (BRL-3A), which was a key target for the treatment of hypertension. The structure of PEG-Bu was confirmed by proton nuclear magnetic resonance (1H-NMR). Gel permeation chromatography (GPC) showed that the weight average molecular weight (Mw) of PEG-Bu was 5880 Da, with a polydispersity of 1.58. PEG-Bu could condense gene cargo into spherical and uniform nanoparticles with particle size (65–88 nm) and zeta potential (7.3–9.6 mV). Interestingly and importantly, PEG-Bu displayed lower cytotoxicity and enhanced tranfection efficiency than PEI-Bu after PEGylation in both normal cells BRL-3A and tumor cells HeLa. Moreover, PEG-Bu could efficiently delivery AGT shRNA to knockdown the AGT expression. To sum up, PEG-Bu would be a promising non-viral vector for delivering AGT shRNA to BRL-3A cells for hypertension therapy.

Overexpression or Silencing of FOXO3a Affects Proliferation of Endothelial Progenitor Cells and Expression of Cell Cycle Regulatory Proteins

Endothelial dysfunction is involved in the pathogenesis of many cardiovascular diseases such as atherosclerosis. Endothelial progenitor cells (EPCs) have been considered to be of great significance in therapeutic angiogenesis. Furthermore, the Forkhead box O (FOXO) transcription factors are known to be important regulators of cell cycle. Therefore, we investigated the effects of changes in FOXO3a activity on cell proliferation and cell cycle regulatory proteins in EPCs. The constructed recombinant adenovirus vectors Ad-TM (triple mutant)-FOXO3a, Ad-shRNA-FOXO3a and the control Ad-GFP were transfected into EPCs derived from human umbilical cord blood. Assessment of transfection efficiency using an inverted fluorescence microscope and flow cytometry indicated a successful transfection. Additionally, the expression of FOXO3a was markedly increased in the Ad-TM-FOXO3a group but was inhibited in the Ad-shRNA-FOXO3a group as seen by western blotting. Overexpression of FOXO3a suppressed EPC proliferation and modulated expression of the cell cycle regulatory proteins including upregulation of the cell cycle inhibitor p27kip1 and downregulation of cyclin-dependent kinase 2 (CDK2), cyclin D1 and proliferating cell nuclear antigen (PCNA). In the Ad-shRNA-FOXO3a group, the results were counter-productive. Furthermore, flow cytometry for cell cycle analysis suggested that the active mutant of FOXO3a caused a noticeable increase in G1- and S-phase frequencies, while a decrease was observed after FOXO3a silencing. In conclusion, these data demonstrated that FOXO3a could possibly inhibit EPC proliferation via cell cycle arrest involving upregulation of p27kip1 and downregulation of CDK2, cyclin D1 and PCNA.

MeCP2-mediated epigenetic regulation in senescent endothelial progenitor cells

BackgroundCellular aging may be associated with epigenetics. Methyl-CpG-binding protein 2 (MeCP2) and sirtuin 1 (SIRT1) are two important epigenetic factors. Our former work demonstrated that MeCP2 expression increased and SIRT1 expression decreased in senescent endothelial progenitor cells (EPCs). This article aims to reveal the epigenetic regulation caused by MeCP2 in EPCs and discuss its mechanism.MethodsTube formation assay and cell apoptosis detection were used to evaluate the function of senescent EPCs induced by MeCP2 overexpression. Western blot analysis was used to testify the relative protein expression changed by MeCP2. Bisulfite sequencing methylation assay and chromatin immunoprecipitation assay were used to assess the degree of methylation and the relation of MeCP2 and SIRT1.ResultsMeCP2 reduced angiogenesis of senescent EPCs, promoted apoptosis, and caused senescent EPC dysfunction through SIRT1 promoter hypermethylation and histone modification.ConclusionsMeCP2 mediated senescent EPC dysfunction through epigenetic regulation.

Acidic Fibroblast Growth Factor Promotes Endothelial Progenitor Cells Function via Akt/FOXO3a Pathway

Acidic fibroblast growth factor (FGF1) has been suggested to enhance the functional activities of endothelial progenitor cells (EPCs). The Forkhead homeobox type O transcription factors (FOXOs), a key substrate of the survival kinase Akt, play important roles in regulation of various cellular processes. We previously have shown that FOXO3a is the main subtype of FOXOs expressed in EPCs. Here, we aim to determine whether FGF1 promotes EPC function through Akt/FOXO3a pathway. Human peripheral blood derived EPCs were transduced with adenoviral vectors either expressing a non-phosphorylable, constitutively active triple mutant of FOXO3a (Ad-TM-FOXO3a) or a GFP control (Ad-GFP). FGF1 treatment improved functional activities of Ad-GFP transduced EPCs, including cell viability, proliferation, antiapoptosis, migration and tube formation, whereas these beneficial effects disappeared by Akt inhibitor pretreatment. Moreover, EPC function was declined by Ad-TM-FOXO3a transduction and failed to be attenuated even with FGF1 treatment. FGF1 upregulated phosphorylation levels of Akt and FOXO3a in Ad-GFP transduced EPCs, which were repressed by Akt inhibitor pretreatment. However, FGF1 failed to recover Ad-TM-FOXO3a transduced EPCs from dysfunction. These data indicate that FGF1 promoting EPC function is at least in part mediated through Akt/FOXO3a pathway. Our study may provide novel ideas for enhancing EPC angiogenic ability and optimizing EPC transplantation therapy in the future.

MeCP2 mediated dysfunction in senescent EPCs

Aging endothelial progenitor cells (EPCs) exhibit functional impairment in terms of proliferation, migration and survival. SIRT1 plays an important role in improving EPCs function. MeCP2, another important epigenetic regulator, is involved in regulating many life-related activities such as cell growth, death and senescence. Here we aim to explore the effect of MeCP2 on the functional activities of senescent EPCs and the underlying mechanisms. By using western blot and real-time PCR, we found that the expression levels of MeCP2 were up-regulated and SIRT1 were down-regulated with replicative senescence and H2O2-induced senescence. Through transduction with adenoviral vectors, EPCs overexpressing MeCP2 had significantly reduced EPCs function, and silencing MeCP2 improved EPCs function. In addition, the protein and mRNA levels of SIRT1 were decreased with MeCP2 overexpression and increased with MeCP2 knockdown. Through co-transfection of EPCs with MeCP2 and SIRT1, we observed that SIRT1 could reverse the effects of MeCP2 on EPCs. In summary, our work demonstrated that MeCP2 inhibited SIRT1 in senescent EPCs.

SIRT1 inhibits TGF-β-induced endothelial-mesenchymal transition in human endothelial cells with Smad4 deacetylation: LI et al.

Endothelial‐mesenchymal transition (EndMT) plays a pivotal role in organ fibrosis. This study examined the effect of SIRT1 on transforming growth factor beta (TGF‐β)‐induced EndMT in human endothelial cells (ECs) and its probable molecular mechanism. We assessed EndMT by immunofluorescence staining, quantitative real‐time polymerase chain reaction, Western blotting, and migration and invasion assays. Adenovirus was used to overexpress or knockdown SIRT1 in ECs. The regulatory relationship between SIRT1 and Smad4 was analyzed by coimmunoprecipitation assay. We found that SIRT1 was decreased in TGF‐β‐induced EndMT, and SIRT1 inhibited TGF‐β‐induced EndMT through deacetylating Smad4. Our findings suggest that SIRT1 has an important role in inhibiting EndMT by regulating the TGF‐β/Smad4 pathway in human ECs and, thus, protecting against fibrosis.

Effects of the visual-feedback-based force platform training with functional electric stimulation on the balance and prevention of falls in older adults: a randomized controlled trial

Background Force platform training with functional electric stimulation aimed at improving balance may be effective in fall prevention for older adults. Aim of the study is to evaluate the effects of the visual-feedback-based force platform balance training with functional electric stimulation on balance and fall prevention in older adults. Methods A single-centre, unblinded, randomized controlled trial was conducted. One hundred and twenty older adults were randomly allocated to two groups: the control group (n = 60, one-leg standing balance exercise, 12 min/d) or the intervention group (n = 60, force platform training with functional electric stimulation, 12 min/d). The training was provided 15 days a month for 3 months by physical therapists. Medial–lateral and anterior–posterior maximal range of sway with eyes open and closed, the Berg Balance Scale, the Barthel Index, the Falls Efficacy scale-International were assessed at baseline and after the 3-month intervention. A fall diary was kept by each participant during the 6-month follow-up. Results On comparing the two groups, the intervention group showed significantly decreased (p < 0.01) medial–lateral and anterior–posterior maximal range of sway with eyes open and closed. There was significantly higher improvement in the Berg Balance Scale (p < 0.05), the Barthel Index (p < 0.05) and the Falls Efficacy Scale-International (p < 0.05), along with significantly lesser number of injurious fallers (p < 0.05), number of fallers (p < 0.05), and fall rates (p < 0.05) during the 6-month follow-up in the intervention group. Conclusion This study showed that the visual feedback-based force platform training with functional electric stimulation improved balance and prevented falls in older adults.