Xiaojie Xie

Hypoxic preconditioning attenuates hypoxia/reoxygenation-induced apoptosis in mesenchymal stem cells

AbstractAim:Mesenchymal stem cells (MSC) are a promising candidate for cardiac replacement therapies. However, the majority of transplanted MSC are readily lost after transplantation because of poor blood supply, ischemia-reperfusion, and inflammatory factors. We aimed to study the effects of hypoxia preconditioning (HPC) on hypoxia/reoxygenation-induced apoptosis of MSC.Methods:Three generations of MSC were divided into 6 groups, including the normal group, hypoxia-reoxygenation (H/R) group, cyclosporine A (CsA), and the HPC 10 min, 20 min, and 30 min groups. The apoptotic index, cell viability, mitochondrial membrane potential, translocation of Bcl-2 and bax, extracellular regulated kinase (ERK), Akt, hypoxia-inducible factor 1-α, and the vascular endothelial growth factor (VEGF) were tested after H/R treatment.Results:HPC decreased the apoptotic index and increased the viability induced by H/R. Moreover, HPC markedly stabilized mitochondrial membrane potential, upregulated Bcl-2 and VEGF expressions, and increased the phosphorylation of ERK and Akt. As a positive control, CsA has the same function as HPC, except for promoting ERK and Akt phosphorylation and upregulating VEGF.Conclusion:HPC had a protective effect against MSC apoptosis induced by H/R via stabilizing mitochondrial membrane potential, upregulating Bcl-2 and VEGF, and promoting ERK and Akt phosphorylation. HPC has implications for the development of novel stem cell protective strategies.

Differentiation of bone marrow mesenchymal stem cells induced by myocardial medium under hypoxic conditions

AbstractAim:To explore whether bone marrow mesenchymal stem cells (MSC) can differentiate into myocardial-like cells induced by myocardial medium, especially the hypoxia/reoxygenation-conditioned medium of cardiomyocytes.Methods:Myocardial cells obtained from neonatal Sprague-Dawley rat ventricles were isolated and cultured in vitro and a hypoxia reoxygenation model was established. The MSC isolated from adult Sprague-Dawley rats were purified and then incubated with 3 different mediums: medium A - the conditioned medium of normal cardiomyocytes; medium B - the conditioned medium of cardiomyocytes after hypoxia reoxygenation; and the control medium - ordinary medium. The expressions of the cardiac myosin heavy chain (MHC), troponin T (TnT) and connexin 43 were investigated in the MSC after 24 h, 48 h and 72 h cultivation, respectively.Results:The MSC expressed MHC and TnT when incubated with the conditioned medium of cardiomyocytes after hypoxia reoxygenation, but did not express connexin 43. None of MHC, TnT and connexin 43 was detected in the MSC incubated with the conditioned medium of normal cardiomyocytes.Conclusion:The results indicate for the first time that myocardial medium for hypoxic preconditioning can induce MSC differentiation into myocardial-like cells.

Doxycycline Does Not Influence Established Abdominal Aortic Aneurysms in Angiotensin II-Infused Mice

Background There is no proven medical approach to attenuating expansion and rupture of abdominal aortic aneurysms (AAAs). One approach that is currently being investigated is the use of doxycycline. Despite being primarily used as an antimicrobial drug, doxycycline has been proposed to function in reducing AAA expansion. Doxycycline is effective in reducing the formation in the most commonly used mouse models of AAAs when administered prior to the initiation of the disease. The purpose of the current study was to determine the effects of doxycycline on established AAAs when it was administered at a dose that produces therapeutic serum concentrations. Methods and Results LDL receptor −/− male mice fed a saturated-fat supplemented diet were infused with AngII (1,000 ng/kg/min) via mini-osmotic pumps for 28 days. Upon verification of AAA formation by noninvasive high frequency ultrasonography, mice were stratified based on aortic lumen diameters, and continuously infused with AngII while also administered either vehicle or doxycycline (100 mg/kg/day) in drinking water for 56 days. Administration of doxycycline led to serum drug concentrations of 2.3±0.6 µg/ml. Doxycycline administration had no effect on serum cholesterol concentrations and systolic blood pressures. Doxycycline administration did not prevent progressive aortic dilation as determined by temporal measurements of lumen dimensions using high frequency ultrasound. This lack of effect on AAA regression and progression was confirmed at the termination of the study by ex vivo measurements of maximal width of suprarenal aortas and AAA volumes. Also, doxycycline did not reduce AAA rupture. Medial and adventitial remodeling was not overtly changed by doxycycline as determined by immunostaining and histological staining. Conclusions Doxycycline administration did not influence AngII-induced AAA progression and aortic rupture when administered to mice with established AAAs.

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.

Statins exert differential effects on angiotensin II-induced atherosclerosis, but no benefit for abdominal aortic aneurysms.

OBJECTIVE Statins reduce atherosclerosis, but it is controversial whether they suppress abdominal aortic aneurysm (AAA) expansion. We hypothesized that statins (rosuvastatin and atorvastatin) would attenuate angiotensin II (AngII)-induced atherosclerosis and AAA. METHODS AND RESULTS Sixty apoE-/- male mice fed a normal diet were administered with either rosuvastatin (10mg/kg/day) or atorvastatin (20mg/kg/day) through drinking water for 1 week prior to initiating 28-day AngII infusion (1000 ng/kg/min). Statins administration led to therapeutic serum concentrations of drugs. Administration of either rosuvastatin or atorvastatin exerted no significant effect on AngII-induced expansion of suprarenal diameter or area. However, atorvastatin significantly reduced AngII-augmented atherosclerotic lesion areas in intimas of both aortic arches and cross-sections of aortic roots (P<0.001). Atherosclerosis was attenuated independent of reductions in serum total cholesterol concentrations. Although serum MCP-1 and MIF concentrations were not changed by either statins, atorvastatin administration increased PPAR-α and -γ mRNA abundances and decreased NF-κB p50, p65, MCP-1 and TNF-α mRNA abundances in atherosclerotic lesions. CONCLUSIONS This study demonstrated both statins failed to suppress AngII-induced AAA. In contrast, atorvastatin reduced AngII-induced atherosclerosis associated with no change in serum inflammatory markers but a shift to upregulation of anti-inflammatory status in lesions.

Chinese red yeast rice attenuates the development of angiotensin II-induced abdominal aortic aneurysm and atherosclerosis

OBJECTIVE Abdominal aortic aneurysm (AAA) is a chronic vascular disease characterized by medial degradation and inflammation. No medical approaches have been validated for treating AAA, and therapeutic options are limited to regular surveillance leading to surgical intervention. This study aimed to investigate whether administration of Chinese red yeast rice (Monascus purpureus; RYR) suppressed angiotensin II (AngII)-induced AAA and atherosclerosis. METHODS AND RESULTS Apolipoprotein E-deficient male mice fed a normal diet were administered either RYR extract (200 mg/kg/day) or vehicle by gavage for 1 week before initiating AngII infusion (1000 ng/kg/min) via subcutaneous osmotic pumps for 28 days. Red yeast rice extract administration significantly suppressed AngII-induced expansion of suprarenal diameter and area (P<.05). Furthermore, RYR extract significantly reduced atherosclerotic lesion areas in both the intima of aortic arches and cross sections of aortic roots (P<.05). These effects were associated with reductions of serum total cholesterol, intercellular adhesion molecule 1, vascular cell adhesion molecule 1, matrix metalloproteinase (MMP) 2 and increases of serum macrophage migration inhibitory factor, but no changes in serum interleukin (IL) 1α, IL-6, monocyte chemoattractant protein 1, MMP-9 and expression of MMP-2 and MMP-9 in aortic walls. CONCLUSIONS This study demonstrated that RYR extract administration suppressed AngII-induced AAA and atherosclerosis associated with regulating inflammation responses independent of lipid-lowering effects. Red yeast rice may have preventive potential for patients with AAA.

Cyclosporin A pre‐incubation attenuates hypoxia/reoxygenation‐induced apoptosis in mesenchymal stem cells

Although mesenchymal stem cells (MSCs) are being tested for cardiac repair, the majority of transplanted cells undergo apoptosis in the ischaemic heart because of the effects of ischaemia/reperfusion, poor blood supply and other pro‐apoptotic factors. Several experimental and clinical studies have suggested that cyclosporin A (CsA) treatment reduces apoptosis in human endothelial cells and neurocytes. However, the effect of CsA on the apoptosis in MSCs is still unclear. In this study, we investigated whether CsA could inhibit hypoxia/reoxygenation (H/R)‐induced apoptosis in MSCs. MSCs pre‐incubated with or without CsA were subjected to 6 h of hypoxia followed by 12 h of reoxygenation. Our data showed that pre‐incubation with 0.5–5 µM CsA dose‐dependently protected the MSCs from H/R injury, as evidenced by decreased apoptosis and increased cell viability. CsA inhibited the H/R‐induced translocation of cytochrome c, increased bcl‐2 expression and restored mitochondrial membrane potential. CsA also increased the expression of p‐BAD. We propose that pre‐incubation MSCs with CsA inhibits MSC apoptosis through the mitochondrial and BAD pathway.

Diabetes Mellitus: Is It Protective against Aortic Root Dilatation?

Objectives: There is evidence of a negative association between diabetes and both abdominal aortic aneurysm and aortic diameter. However, little information is available on the relation between diabetes and aortic root diameter. Methods: We studied 109 patients with type 2 diabetes. Two-dimensional echocardiography was used to measure the aortic root at the aortic annulus, the sinus of Valsalva, the sinotubular junction and the proximal part of the ascending aorta. Measured mean values were compared with 218 age- and sex-matched patients without diabetes. A comparison of the prevalence of aortic regurgitation between the 2 groups was also performed. Results: In patients with diabetes, the mean aortic root dimensions were significantly smaller than in nondiabetic patients (p < 0.05). The prevalence of aortic root dilatation was significantly higher in nondiabetic subjects than in patients with diabetes (9.63 vs. 2.75%; p = 0.025). In the multivariable regression model, diabetes was a significant negative determinant of aortic root size at all measured sites. The prevalence of aortic regurgitation tended to be higher in nondiabetic subjects than in diabetic participants (11 vs. 18.8%); however, the difference did not achieve statistical significance (p = 0.071). Conclusions: In patients with diabetes, the aortic root dimension is significantly smaller than in patients without diabetes.

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).

Heat Shock Protein 90 Stimulates Rat Mesenchymal Stem Cell Migration via PI3K/Akt and ERK1/2 Pathways

The objective of this study was to determine the role of Hsp90α in regulating the migration of mesenchymal stem cells (MSCs) and to investigate the underlying mechanisms of this effect. MSCs migration was assessed by wound healing assay and transwell migration assay. Hsp90α expression was silenced in MSC by siRNA (sirHsp90α). The activity of secreted metalloproteases MMP-2 and MMP-9, and their expression levels in MSC were evaluated using gelatin zymography, Western blot analysis and real-time PCR. Gene expression of VCAM-1 and CXCR4 cytokines was evaluated by real-time PCR. Akt and ERK activity were analyzed by Western blotting using antibodies against phosphorylated forms of these proteins. Treatment with Hsp90α significantly enhanced MSC migration, and this effect was blocked by transfecting MSC with sirHsp90α. Treating the cells with recombinant human Hsp90α (rhHsp90α) enhanced gene expression and protein levels of MMP-2 and MMP-9, as well as their secretion and activity. MSC incubated with rhHsp90α exhibited increased gene expression of CXCR4 and VCAM-1. Finally, the levels of phosphorylated Akt and Erk were markedly increased by rhHsp90α treatment. These findings indicate that Hsp90α promotes MSCs migration via PI3K/Akt and ERK signaling pathways, and that this effect is possibly mediated by MMPs, SDF-1/CXCR4 pathway, and VCAM-1.