Xianbao Liu

Angiopoietin-1 protects mesenchymal stem cells against serum deprivation and hypoxia-induced apoptosis through the PI3K/Akt pathway

AbstractAim:The angiopoietin-1 (Ang1)/Tie-2 signaling system not only plays a pivotal role in vessel growth, remodeling, and maturation, but also reduces apoptosis of endothelial cells, neurons, and cardiomyocytes. However, relatively little is known as to whether Ang1 has a protective effect on mesenchymal stem cells (MSC). The aim of the present study was to investigate the protective effect of Ang1/Tie-2 signaling on MSC against serum deprivation and hypoxia-induced apoptosis, and to determine the possible mechanisms.Methods:Hoechst 33342 and terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling staining were used to assess the apoptosis of MSC. The expression of Tie-2, Akt, Bcl-2, Bax, and cleaved caspase-9 and -3 was detected by Western blot analysis.Results:This study showed that MSC expressed Tie-2 receptor, and Ang1 induced Tie-2 receptor phosphorylation. The protective effect of Ang1 on MSC was dose-dependent and peaked at 50 μg/L; however, the soluble Tie-2/Fc fusion protein, which acts as an inhibitor by sequestering Ang1, abrogated the anti-apoptotic effect. Ang1 induced Akt phosphorylation, increased the Bcl-2/Bax ratio, and decreased the activation of caspase-9 and -3. All these effects were attenuated by Tie-2/Fc and a phosphatidylinositol 3 kinase (PI3K) inhibitor, wortmannin.Conclusion:These results demonstrate that Ang1 can protect MSC against serum deprivation and hypoxia-induced apoptosis; Ang1/Tie-2 signaling and its downstream PI3K/Akt messenger pathway are crucial in the processes leading to MSC survival.

Prolyl hydroxylase inhibitor dimethyloxalylglycine enhances mesenchymal stem cell survival

Mesenchymal stem cell (MSC) transplantation is a promising approach in the therapy of ischemic heart or CNS diseases; however, the poor viability of MSCs after transplantation critically limits the efficacy of this new strategy. Prolyl hydroxylase inhibition followed by HIF‐1α up‐regulation participates in the regulation of apoptosis and cell survival, which have been shown in cancer cells and neurons. The role of prolyl hydroxylase inhibition by dimethyloxalylglycine (DMOG) in regulation of cell survival has not been investigated in MSCs. In the present investigation with MSCs, apoptosis and cell death induced by serum deprivation were assessed by caspase‐3 activation and trypan blue staining, respectively. The mitochondrial apoptotic pathway and PI3K/Akt cell survival pathway were evaluated. DMOG significantly attenuated apoptosis and cell death of MSCs, stabilized HIF‐1α and induced downstream glucose transport 1 (Glut‐1) synthesis. DMOG treatment reduced mitochondrial cytochrome c release, nuclear translocation of apoptosis inducing factor (AIF), and promoted Akt phosphorylation. A specific PI3K inhibitor, wortmannin, blocked Akt phosphorylation and abrogated the beneficial effect of DMOG. These data suggest that the DMOG protection of MSCs may provide a novel approach to promote cell survival during cell stress. J. Cell. Biochem. 106: 903–911, 2009.

SIRT1 ameliorates age-related senescence of mesenchymal stem cells via modulating telomere shelterin

Mesenchymal stem cells (MSCs) senescence is an age-related process that impairs the capacity for tissue repair and compromises the clinical use of autologous MSCs for tissue regeneration. Here, we describe the effects of SIRT1, a NAD+-dependent deacetylase, on age-related MSCs senescence. Knockdown of SIRT1 in young MSCs induced cellular senescence and inhibited cell proliferation whereas overexpression of SIRT1 in aged MSCs reversed the senescence phenotype and stimulated cell proliferation. These results suggest that SIRT1 plays a key role in modulating age-induced MSCs senescence. Aging-related proteins, P16 and P21 may be downstream effectors of the SIRT1-mediated anti-aging effects. SIRT1 protected MSCs from age-related DNA damage, induced telomerase reverse transcriptase (TERT) expression and enhanced telomerase activity but did not affect telomere length. SIRT1 positively regulated the expression of tripeptidyl peptidase 1 (TPP1), a component of the shelterin pathway that protects chromosome ends from DNA damage. Together, the results demonstrate that SIRT1 quenches age-related MSCs senescence by mechanisms that include enhanced TPP1 expression, increased telomerase activity and reduced DNA damage.

Role of SIRT1 and AMPK in mesenchymal stem cells differentiation.

The differentiation capabilities of mesenchymal stem cells (MSCs) compromise with age and with in vitro passages which could impair the efficacy of cell therapy and tissue engineering. However, how to maintain these capabilities is not fully understood. Calorie restriction (CR, decreasing caloric intake by 30-40%) could extend longevity and reduce aging-related diseases. Recent studies revealed that CR could influence the lineage determination of stem cells including MSCs. Two important mediators of CR might be silent mating type information regulation 2 homolog 1 (SIRT1), a NAD(+)-dependent deacetylase, and AMP-activated protein kinase (AMPK), an energy-sensing kinase. Evidences are mounting that both SIRT1 and AMPK play important roles in cell fate determination of MSCs. Herein, we intend to sum up our understanding about the role of SIRT1 and AMPK in osteogenic and adipogenic potential of MSCs. Metabolic process of MSCs differentiation and the putative interplay of SIRT1 and AMPK in this process was also discussed.

Heat shock protein 90 protects rat mesenchymal stem cells against hypoxia and serum deprivation-induced apoptosis via the PI3K/Akt and ERK1/2 pathways

Mesenchymal stem cell (MSC) transplantation has shown a therapeutic potential to repair the ischemic and infracted myocardium, but the effects are limited by the apoptosis and loss of donor cells in host cardiac microenvironment. The aim of this study is to explore the cytoprotection of heat shock protein 90 (Hsp90) against hypoxia and serum deprivation-induced apoptosis and the possible mechanisms in rat MSCs. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis was assessed by Hoechst 33258 nuclear staining and flow cytometric analysis with annexin V/PI staining. The gene expression of Toll-like receptor-4 (TLR-4) and V-erb-b2 erythroblastic leukemia viral oncogene homolog 2 (ErbB2) was detected by real-time polymerase chain reaction (PCR). The protein levels of cleaved caspase-3, Bcl-2, Bcl-xL, Bax, total-ERK, phospho-ERK, total-Akt, phospho-Akt, and Hsp90 were detected by Western blot. The production of nitric oxide was measured by spectrophotometric assay. Hsp90 improves MSC viability and protects MSCs against apoptosis induced by serum deprivation and hypoxia. The protective role of Hsp90 not only elevates Bcl-2/Bax and Bcl-xL/Bax expression and attenuates cleaved caspase-3 expression via down-regulating membrane TLR-4 and ErbB2 receptors and then activating their downstream PI3K/Akt and ERK1/2 pathways, but also enhances the paracrine effect of MSCs. These findings demonstrated a novel and effective treatment strategy against MSC apoptosis in cell transplantation.

Hypercholesterolemia blocked sevoflurane-induced cardioprotection against ischemia–reperfusion injury by alteration of the MG53/RISK/GSK3β signaling

BACKGROUND Recent studies have demonstrated that volatile anesthetic preconditioning confers myocardial protection against ischemia-reperfusion (IR) injury through activation of the reperfusion injury salvage kinase (RISK) pathway. As RISK has been shown to be impaired in hypercholesterolemia, we investigate whether anesthetic-induced cardiac protection was maintained in hypercholesterolemic rats. METHODS Normocholesteolemic or hypercholesterolemic rat hearts were subjected to 30 min of ischemia and 2 h of reperfusion. Animals received 2.4% sevoflurane during three 5 min periods with and without PI3K antagonist wortmannin (10 μg/kg, Wort) or the ERK inhibitor PD 98059 (1 mg/kg, PD). The infarct size, apoptosis, p-Akt, p-ERK1/2, p-GSK3β were determined. RESULTS Two hundred and six rats were analyzed in the study. In the healthy rats, sevoflurane significantly reduced infarct size by 42%, a phenomenon completely reversed by wortmannin and PD98059 and increased the phosphorylation of Akt, ERK1/2 and their downstream target of GSK3β. In the hypercholesterolemic rats, sevoflurane failed to reduce infarct size and increase the phosphorylated Akt, ERK1/2 and GSK3β. In contrast, GSK inhibitor SB216763 conferred cardioprotection against IR injury in healthy and hypercholesterolemic hearts. CONCLUSIONS Hyperchoesterolemia abrogated sevoflurane-induced cardioprotection against IR injury by alteration of upstream signaling of GSK3β and acute GSK inhibition may provide a novel therapeutic strategy to protect hypercholesterolemic hearts against IR injury.

Angiopoietin-1 preconditioning enhances survival and functional recovery of mesenchymal stem cell transplantation.

ObjectiveMesenchymal stem cell (MSC) transplantation is a promising therapy for ischemic heart diseases. However, poor cell survival after transplantation greatly limits the therapeutic efficacy of MSCs. The purpose of this study was to investigate the protective effect of angiopoietin-1 (Ang1) preconditioning on MSC survival and subsequent heart function improvement after transplantation.MethodsMSCs were cultured with or without 50 ng/ml Ang1 in complete medium for 24 h prior to experiments on cell survival and transplantation. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Hoechst staining were applied to evaluate MSC survival after serum deprivation in vitro, while cell survival in vivo was detected by terminal deoxynucleotidyl transferase biotin-dUPT nick end labeling (TUNEL) assay 24 and 72 h after transplantation. Heart function and infarct size were measured four weeks later by small animal echocardiography and Masson’s trichrome staining, respectively.ResultsAng1 preconditioning induced Akt phosphorylation and increased expression of Bcl-2 and the ratio of Bcl-2/Bax. In comparison with non-preconditioned MSCs, Ang1-preconditioned cell survival was significantly increased while the apoptotic rate decreased in vitro. However, the PI3K/Akt pathway inhibitor, LY294002, abrogated the protective effect of Ang1 preconditioning. After transplantation, the Ang1-preconditioned-MSC group showed a lower death rate, smaller infarct size, and better heart functional recovery compared to the non-preconditioned-MSC group.ConclusionsAng1 preconditioning enhances MSC survival, contributing to further improvement of heart function.

Hypercholesterolemic Myocardium Is Vulnerable to Ischemia-Reperfusion Injury and Refractory to Sevoflurane-Induced Protection

Recent studies have demonstrated that volatile anesthetic postconditioning confers myocardial protection against ischemia-reperfusion (IR) injury through activation of the reperfusion injury salvage kinase (RISK) pathway. As RISK has been shown to be impaired in hypercholesterolemia. Therefore, we investigate whether anesthetic-induced cardiac protection was maintained in hypercholesterolemic rats. In the present study, normocholesteolemic or hypercholesterolemic rat hearts were subjected to 30 min of ischemia and 2 h of reperfusion. Animals received 2.4% sevoflurane for 5 min or 3 cycles of 10-s ischemia/10-s reperfusion. The hemodynamic parameters, including left ventricular developed pressure, left ventricular end-diastolic pressure and heart rate, were continuously monitored. The infarct size, apoptosis, p-Akt, p-ERK1/2, p-GSK3β were determined. We found that both sevoflurane and ischemic postconditioning significantly improved heart pump function, reduced infarct size and increased the phosphorylation of Akt, ERK1/2 and their downstream target of GSK3β in the healthy rats. In the hypercholesterolemic rats, neither sevoflurane nor ischemic postconditioning improved left ventricular hemodynamics, reduced infarct size and increased the phosphorylated Akt, ERK1/2 and GSK3β. In contrast, GSK inhibitor SB216763 conferred cardioprotection against IR injury in healthy and hypercholesterolemic hearts. In conclusions, hyperchoesterolemia abrogated sevoflurane-induced cardioprotection against IR injury by alteration of upstream signaling of GSK3β and acute GSK inhibition may provide a novel therapeutic strategy to protect hypercholesterolemic hearts against IR injury.

Safety and efficacy of intracoronary hypoxia-preconditioned bone marrow mononuclear cell administration for acute myocardial infarction patients: The CHINA-AMI randomized controlled trial

BACKGROUND Pre-clinical studies have shown that hypoxia preconditioning can enhance stem cell therapeutic potential for myocardial repair. We sought to investigate the safety and feasibility of intracoronary administration of hypoxia-preconditioned bone marrow mononuclear cells (HP-BMCs) for acute ST segment elevation myocardial infarction (STEMI). METHODS We randomized 22 patients with acute STEMI to receive intracoronary administration of normoxia bone marrow mononuclear cells (N-BMCs) (n=11) or HP-BMCs (n=11) following successful reperfusion. Another 14 patients receiving standard therapy were recruited as control (n=14). RESULTS There were no differences in the occurrence of major adverse cardiovascular events at 30 days and 1 year among three groups. There were significant improvement in the change of left ventricular end-diastolic volume (LVEDV) and end-systolic volume (LVESV) in HP-BMC group both at 6 and 12 months compared with N-BMCs or control group (P<0.05). No differences were observed in the change of left ventricular ejection fraction (LVEF), or wall motion score index (WMSI) among three groups. Nevertheless, WMSI was improved in HP-BMCs and N-BMC group (P<0.05, within group), but not in control. The ratio of myocardial perfusion defect determined by SPECT was significantly decreased in HP-BMCs and N-BMC groups at 6months compared with baseline (P<0.05, within group), but no significant differences were observed among three groups. CONCLUSIONS Our results provide the first-in-man evidence that intracoronary administration of HP-BMCs following acute MI appears to be safe and feasible. These results provide the basis for future prospective randomized clinical trials in a larger patient cohort. CLINICAL TRIAL REGISTRATION INFORMATION NCT01234181 (http://clinicaltrials.gov/ct2/show/NCT01234181?term=NCT01234181&rank=1).

Evaluation of the safety and efficacy of transcatheter aortic valve implantation in patients with a severe stenotic bicuspid aortic valve in a Chinese population

ObjectiveThe purpose of this study is to evaluate the safety and efficacy of transcatheter aortic valve implantation (TAVI) in patients with a severe stenotic bicuspid aortic valve (BAV) in a Chinese population. While several groups have reported the feasibility, efficacy, and safety of TAVI for patients with a BAV, worldwide experience of the technique is still limited, especially in China.MethodsFrom March 2013 to November 2014, high surgical risk or inoperable patients with symptomatic severe aortic stenosis (AS) who had undergone TAVI at our institution were selected for inclusion in our study. Results were compared between a BAV group and a tricuspid aortic valve (TAV) group.ResultsForty patients were included in this study, 15 (37.5%) of whom were identified as having a BAV. In the BAV group, the aortic valve area was smaller ((0.47±0.13) vs. (0.59±0.14) cm2), the ascending aortic diameter was larger ((40.4±4.4) vs. (36.4±4.3) mm), and the concomitant aortic regurgitation was lower. No significant differences were found between the groups in the other baseline characteristics. No differences were observed either in the choice of access or valve size. The procedural success achieved in this study was 100%. There were no differences between groups in device success (86.7% vs. 88.0%), 30-d mortality (6.7% vs. 8.0%), or 30-d combined end point (13.3% vs. 12.0%). The incidences of new pacemaker implantation, paravalvular regurgitation and other complications, recovery of left ventricle ejection fraction and heart function were similar in both groups.ConclusionsPatients with a severely stenotic BAV can be treated with TAVI, and their condition after treatment should be similar to that of people with a TAV.摘要目的评估经导管主动脉瓣置入术在中国人群二叶式主动脉瓣重度狭窄中的安全性和有效性。创新点首次在中国人群比较经导管主动脉瓣置入术在二叶式和三叶式主动脉瓣重度狭窄中的安全性和有效性。方法选取2013年3月至2014年9月行经导管主动脉瓣置入术的40位主动脉瓣狭窄的患者, 分二叶式和三叶式主动脉瓣两组, 比较基线水平、 手术以及随访1月结果的差别。结论经导管主动脉瓣置入术在中国人群中二叶式主动脉瓣重度狭窄中的应用是安全有效的。