Expression and functional analysis of flotillins in Dugesia japonica.

FLOTILLIN-1 and FLOTILLIN-2 are membrane rafts associated proteins that have been implicated in insulin and growth factor signaling, endocytosis, cell migration, proliferation, differentiation, cytoskeleton remodeling and membrane trafficking. Furthermore, FLOTILLINs also play important roles in the progression of cancer and neurodegenerative diseases. In this study, the roles of flotillins are investigated in planarian Dugesia japonica. The results show that Djflotillin-1 and Djflotillin-2 play a key role in homeostasis maintenance and regeneration process by regulating the proliferation of the neoblast cells, they are not involved in the maintenance and regeneration of the central nervous system in planarians.

Effects of linagliptin and liraglutide on glucose- and angiotensin II-induced collagen formation and cytoskeleton degradation in cardiac fibroblasts in vitro.

AIM: Glucagon-like peptide-1 (GLP-1) agonists and dipeptidyl peptidase-4 (DPP-4) inhibitors can not only lower blood glucose levels, but also alleviate cardiac remodeling after myocardial ischemia and hypertension. In the present study, we investigated the effects of a DPP-4 inhibitor (linagliptin) and a GLP-1 activator (liraglutide) on glucose- and angiotensin II (Ang II)-induced collagen formation and cytoskeleton reorganization in cardiac fibroblasts in vitro, and elucidated the related mechanisms. METHODS: Cardiac fibroblasts were isolated from the hearts of 6-week-old C57BL/6 mice, and then exposed to different concentrations of glucose or Ang II for 24 h. The expression of fibrotic signals (fibronectin, collagen-1, -3 and -4), as well as ERK1/2 and NF-κB-p65 in the fibroblasts was examined using Western blotting assays. F-actin degradation was detected under inverted laser confocal microscope in fibroblasts stained with Rhodamine phalloidin. RESULTS: Glucose (1-40 mmol/L) and Ang II (10-8-10-5 mol/L) dose-dependently increased the expression of fibronectin, collagens, phospho-ERK1/2 and phospho-NF-κB-p65 in cardiac fibroblasts. High concentrations of glucose (≥40 mmol/L) and Ang II (≥10-6 mol/L) caused a significant degradation of F-actin (less assembly F-actin fibers and more disassembly fibers). ERK1/2 inhibitor U0126 (10 µmol/L) and NF-κB inhibitor JSH-23 (10 µmol/L) both markedly suppressed glucose- and angiotensin II-induced fibronectin and collagen expressions in cardiac fibroblasts. Furthermore, pretreatment with liraglutide (10-100 nmol/L) or linagliptin (3 and 30 nmol/L) significantly decreased glucose- and Ang II-induced expression of fibrotic signals, phospho-ERK1/2 and phospho-NF-κB-p65 in cardiac fibroblasts. Moreover, pretreatment with liraglutide (30 nmol/L) or liraglutide (100 nmol/L) markedly inhibited glucose-induced F-actin degradation, however, only liraglutide inhibited Ang II-induced F-actin degradation. CONCLUSION: Linagliptin and liraglutide inhibit glucose- and Ang II-induced collagen formation in cardiac fibroblasts via activation of the ERK/NF-κB/pathway. Linagliptin and liraglutide also markedly inhibit glucose-induced F-actin degradation in cardiac fibroblasts, but only liraglutide inhibits Ang II-induced F-actin degradation.

Sertoli cells promote proliferation of bone marrow-derived mesenchymal stem cells in co-culture.

Bone marrow-derived mesenchymal stem cells (BMSCs) are a major source for cell transplantation. The proliferative ability of BMSCs is an important determinant of the efficiency of transplant therapy. Sertoli cells are "nurse" cells for development of sperm cells. Our recent study showed that Sertoli cells promoted proliferation of human umbilical cord mesenchymal stem cells (hUCMSCs) in co-culture. Studies by other groups also showed that Sertoli cells promoted growth of endothelial cells and neural stem cells. In this study, we investigated the effect of Sertoli cells on proliferation of BMSCs. Our results showed that Sertoli cells in co-culture significantly enhanced proliferation of BMSCs (P < 0.01). Moreover, co-culture with Sertoli cells also markedly increased mRNA and/or protein expressions of Mdm2, p-Akt and Cyclin D1, and decreased p53 expression in BMSCs (P < 0.01 or < 0.05). These findings indicate that Sertoli cells have the potential to enhance proliferation of BMSCs.

Small concentrations of TGF-ß1 promote proliferation of bone marrow-derived mesenchymal stem cells via activation of Wnt/ß-catenin pathway.

Bone marrow-derived mesenchymal stem cells (BMSCs) are the most promising seed cells for cell transplant. The proliferation of BMSCs is one of the most important determinants of the efficiency of MSC-based transplant therapy. It has been reported that transforming growth factor-ß1 (TGF-ß1) activates Wnt/ß-catenin signaling and regulates cell proliferation. In this study, we investigated the effect of low concentrations of TGF-ß1 on proliferation of BMSCs and the related mechanisms. BMSCs were treated with 0, 1, 5 and 10 ng/L recombinant mouse TGF-ß1 for 12 h. Cell proliferation was measured by cell counting and MTT assay, and the proliferation-related signals p53, Mdm2, Aktl, Wnt3, phospho-Akt and ß-catenin were measured by quantitative polymerase chain reaction (qPCR) and/or Western blot. Our results showed that TGF-ß1 at low concentrations induced BMSC proliferation and expression of Mdm2, Aktl, phospho-Akt, Wnt3 and ß-catenin, and inhibited p53 expression in dose dependent manner. Importantly, ß-catenin siRNA significantly inhibited TGF-ß1-induced BMSC proliferation. These findings suggest that low concentrations of TGF-ß1 can stimulate proliferation of BMSCs, which is at least partially dependent on the activation of Wnt/ß-catenin pathway.

TGF-ß1 induces senescence of bone marrow mesenchymal stem cells via increase of mitochondrial ROS production.

BACKGROUND: Bone marrow derived mesenchymal stem cells (bmMSCs) are multipotent cells that can differentiate into diverse cell types, including cardiomyocytes. BmMSC-based transplantation is capable of repairing acute and chronic myocardial infarction. Prior to the transplantation, MSCs are usually induced in vitro by biological reagents and chemicals for directional differentiation. Transforming growth factor beta (TGF-ß) is one of the most commonly used biological reagents for induction of cardiomyocyte differentiation of bmMSCs. Previous studies have shown that TGF-ß induces senescence in several cell types. However, whether TGF-ß affects senescence of bmMSCs has not been elucidated. The goal of this study was to investigate the effect of TGF-ß1 on senescence of bmMSCs and the underlying mechanisms. RESULTS: We found that TGF-ß1 increased activity of senescence-associated-galactosidase (SA-Gal) and production of mitochondrial reactive oxygen species (mtROS) in bmMSCs in a dose-dependent manner. TGF-ß1 also significantly decreased expression of superoxide dismutase 2 (SOD2) and Id1, and increased expression of 4-Hydroxynonenal (4-HNE) subunits and p16 in bmMSCs in a dose-dependent manner. Pre-treatment with mtROS inhibitor acetyl-L-carnitine (ALCAR, 0.1 mM) significantly inhibited TGF-ß1-induced mtROS production and SA-Gal activity. CONCLUSION: TGF-ß1 can induce senescence of bmMSCs, which at least partially depends on mtROS production.

Hypoxia induces autophagy of bone marrow-derived mesenchymal stem cells via activation of ERK1/2.

BACKGROUND: Bone marrow-derived mesenchymal stem cells (bmMSCs) are the most promising seed cells for cell transplant therapy. Hypoxia is a known stimulus of autophagy. Recent studies showed that hypoxia promotes autophagy of human placental chorionic plate-derived mesenchymal stem cells (CP-MSCs). However, whether hypoxia affects autophagy of bmMSCs has not been examined. The goal of this study was to investigate the effect of hypoxia on autophagy of mouse bmMSCs and the underlying mechanisms. METHODS: BmMSCs from mouse bone marrow were randomly divided into three groups: control (C), hypoxia (H) and hypoxia + reoxygenation (H+R) groups. Subsequent autophagic signals were analyzed by immunostaining and Western blot assays. RESULTS: The expression of autophagic signals LC-3, Atg5 and Beclin-1, as well as the conversion of LC3B-I to LC3B-II in bmMSCs were significantly increased in H group as compared with control (p<0.05). These autophagic signals were also higher in H+R group than in H group (p<0.05). Also, the expression of phospho-ERK1/2 was significantly increased in H and H+R groups as compared with control (p<0.05). Notably, application of ERK1/2 inhibitor U0126 (5µM) significantly repressed hypoxia-induced expression of LC-3 and Atg5, as well as conversion of LC3B-I to LC3B-II (p<0.05). CONCLUSION: Hypoxia can induce autophagy of bmMSCs, which depends on activation of ERK1/2 pathway.

Lectin-like oxidized LDL receptor-1 expresses in mouse bone marrow-derived mesenchymal stem cells and stimulates their proliferation.

The bone marrow-derived mesenchymal stem cells (bmMSCs) have been widely used in cell transplant therapy, and the proliferative ability of bmMSCs is one of the determinants of the therapy efficiency. Lectin-like oxidized low density lipoprotein receptor-1 (LOX-1) as a transmembrane protein is responsible for binding, internalizing and degrading oxidized low density lipoprotein (ox-LDL). It has been identified that LOX-1 is expressed in endothelial cells, vascular smooth muscle cells, cardiomyocytes, fibroblasts and monocytes. In these cells, low concentration of ox-LDL (<40 µg/mL) stimulates their proliferation via LOX-1 activation. However, it is poor understood that whether LOX-1 is expressed in bmMSCs and which role it plays. In this study, we investigated the status of LOX-1 expression in bmMSCs and its function on bmMSC proliferation. Our results showed that primary bmMSCs exhibiting a typical fibroblast-like morphology are positive for CD44 and CD90, but negative for CD34 and CD45. LOX-1 in both mRNA and protein levels is highly expressed in bmMSCs. Meanwhile, bmMSCs exhibit a strong potential to take up ox-LDL. Moreover, LOX-1 expression in bmMSCs is upregulated by ox-LDL with a dose- and time-dependent manner. Presence of ox-LDL also enhances the proliferation of bmMSCs. Knockdown of LOX-1 expression significantly inhibits ox-LDL-induced bmMSC proliferation. These findings indicate that LOX-1 plays a role in bmMSC proliferation.

Ox-LDL promotes migration and adhesion of bone marrow-derived mesenchymal stem cells via regulation of MCP-1 expression.

Bone marrow-derived mesenchymal stem cells (bmMSCs) are the most important cell source for stem cell transplant therapy. The migration capacity of MSCs is one of the determinants of the efficiency of MSC-based transplant therapy. Our recent study has shown that low concentrations of oxidized low-density lipoprotein (ox-LDL) can stimulate proliferation of bmMSCs. In this study, we investigated the effects of ox-LDL on bmMSC migration and adhesion, as well as the related mechanisms. Our results show that transmigration rates of bmMSCs and cell-cell adhesion between bmMSCs and monocytes are significantly increased by treatments with ox-LDL in a dose- and time-dependent manner. Expressions of ICAM-1, PECAM-1, and VCAM-1 as well as the levels of intracellular Ca(2+) are also markedly increased by ox-LDL in a dose-dependent manner. Cytoskeleton analysis shows that ox-LDL treatment benefits to spreading of bmMSCs and organization of F-actin fibers after being plated for 6 hours. More interestingly, treatments with ox-LDL also markedly increase expressions of LOX-1, MCP-1, and TGF- ß ; however, LOX-1 antibody and MCP-1 shRNA markedly inhibit ox-LDL-induced migration and adhesion of bmMSCs, which suggests that ox-LDL-induced bmMSC migration and adhesion are dependent on LOX-1 activation and MCP-1 expression.

Co-culture with Sertoli cells promotes proliferation and migration of umbilical cord mesenchymal stem cells.

Human umbilical cord mesenchymal stem cells (hUCMSCs) have been recently used in transplant therapy. The proliferation and migration of MSCs are the determinants of the efficiency of MSC transplant therapy. Sertoli cells are a kind of "nurse" cells that support the development of sperm cells. Recent studies show that Sertoli cells promote proliferation of endothelial cells and neural stem cells in co-culture. We hypothesized that co-culture of UCMSCs with Sertoli cells may also promote proliferation and migration of UCMSCs. To examine this hypothesis, we isolated UCMSCs from human cords and Sertoli cells from mouse testes, and co-cultured them using a Transwell system. We found that UCMSCs exhibited strong proliferation ability and potential to differentiate to other cell lineages such as osteocytes and adipocytes. The presence of Sertoli cells in co-culture significantly enhanced the proliferation and migration potential of UCMSCs (P<0.01). Moreover, these phenotypic changes were accompanied with upregulation of multiple genes involved in cell proliferation and migration including phospho-Akt, Mdm2, phospho-CDC2, Cyclin D1, Cyclin D3 as well as CXCR4, phospho-p44 MAPK and phospho-p38 MAPK. These findings indicate that Sertoli cells boost UCMSC proliferation and migration potential.

AT1R-Mediated Apoptosis of Bone Marrow Mesenchymal Stem Cells Is Associated with mtROS Production and mtDNA Reduction.

Angiotensin II (Ang II) is used as an inducer for the differentiation of mesenchymal stem cells (MSCs). Whether the commonly used doses of Ang II for MSC differentiation affect cell apoptosis has not been elucidated. In this study, we investigated the effect of Ang II on the apoptosis of bone marrow MSCs (BMMSCs), and its relations to the activation of Ang II receptor-1- (AT1R-) signaling, mitochondrial ROS (mtROS) generation, and mitochondrial DNA (mtDNA) leakage. AT1R expression in BMMSCs was identified by immunostaining and Western-blotting assays. BMMSC viability was measured by MTT assay following exposure to 1 nM~1 mM Ang II for 12 hours. Cell apoptosis, mtROS, and mtDNA levels were detected by FAM-FLICA® Poly Caspase, MitoSOX™ superoxide, and PicoGreen staining, respectively. The expressions of Bcl2 and Bax were measured by Western-blotting assays. Next, we used losartan to block AT1R-signaling and subsequently measured apoptosis, mtROS, and mtDNA levels, again. The maximum viability of BMMSCs was in response to 100 nM Ang II, after that it began to decrease with the increase of Ang II doses, indicating that Ang II (≧1 µM) may cause apoptosis of BMMSCs. As expected, 1 µM and 10 µM Ang II both caused BMMSC apoptosis. Furthermore, 1 µM and 10 µM Ang II could also induce mtROS generation and cause a marked mtDNA leakage. The application of losartan markedly inhibited Ang II-induced mtROS production, mtDNA leakage, and BMMSC apoptosis. In conclusion, the activation of AT1R-signaling stimulates apoptosis of BMMSCs, which is associated mtROS production and mtDNA reduction.

Blocking of autocrine IGF-1 reduces viability of human umbilical cord mesenchymal stem cells via inhibition of the Akt/Gsk-3ß signaling pathway.

Human umbilical cord mesenchymal stem cells (hUCMSCs) are able to secrete growth factors, such as hepatocyte growth factor, vascular endothelial growth factor and insulin­like growth factor­1 (IGF­1). The secretion of these growth factors by transplanted hUCMSCs have been identified to stimulate the growth of the host cells in the target organs or tissues. The aim of the present study was to investigate the effect of autocrine IGF­1 on cell viability of hUCMSCs. The expression levels of IGF­1 and the IGF­1 receptor (IGF­1R) in hUCMSCs were identified using immunocytochemistry staining. In order to block autocrine IGF­1, hUCMSCs were treated with 5 µg/ml αIR­3, a specific IGF­1R antibody, for 24 h. The cells cultured in medium without αIR­3 were used as the control group. Cell viability, apoptosis, cell cycle and the proliferation­associated proteins were quantified using an MTT assay, flow cytometry and western blotting. The findings of the present study revealed that IGF­1 and IGF­1R were positively expressed in hUCMSCs. Treatment with αIR­3 significantly reduced cell viability and increased apoptosis of hUCMSCs (P<0.01). Cell cycle analysis indicated that the number of cells in the G2/M phase was reduced in the αIR­3­treated group compared with the control group. Western blotting revealed that the expression levels of phosphorylated (p)­protein kinase B (Akt), p­glycogen synthase kinase 3ß (GSK­3ß), p­p70 S6 kinase and cyclin D1 were markedly reduced and p21 expression was markedly increased in the αIR­3­treated group as compared with the control group (P<0.05). However, no significant difference was identified in the p­extracellular­signal regulated kinase 1/2 expression when the αIR­3 treatment group was compared with the control group. (P>0.05). The findings of the present study suggested that the autocrine IGF­1 from hUCMSCs may be capable of influencing cell viability of hUCMSCs, which may be associated with activation of Akt/GSK­3ß signaling pathway.

Oxidized low-density lipoprotein (ox-LDL) promotes cardiac differentiation of bone marrow mesenchymal stem cells via activating ERK1/2 signaling.

BACKGROUND/AIMS: The differentiation efficiency of bone marrow mesenchymal stem cells (BM-MSCs) is low in vivo after transplantation. Therefore, it is necessary to look for effective reagents for enhancing cardiac differentiation of BM-MSCs. It has been reported that cardiac differentiation of stem cells depends on the activation of extracellular signal-regulated protein 1/2 (ERK1/2) signaling. Oxidized low-density lipoprotein (ox-LDL) is a potent reagent for ERK1/2 activation. This indicates that ox-LDL may be a potential reagent to stimulate cardiac differentiation of stem cells. In this study, we investigated the effect of ox-LDL on cardiac differentiation of BM-MSCs and its relationship with ERK1/2 signaling. METHODS: BM-MSCs were isolated from mouse bone marrow, cultured in DMEM supplemented with 15% FBS, and passaged up to the 3rd passage. Following culture with 5 µg/mL ox-LDL for 3 weeks, the cardiac differentiation of the 3rd passage BM-MSCs was identified by immunostaining, Western blotting, and RT-PCR assays for measuring the expression of cardiac-specific markers. To further explore the role of ERK1/2 signaling in cardiac differentiation of BM-MSCs, we simultaneously exposed BM-MSCs to ERK1/2 inhibitor (U0126) and ox-LDL, and identified the cardiac differentiation again. RESULTS: The expressions of cardiac-specific markers including α-cardiac actin, α-MHC, ß-MHC, ANP, and BNP were markedly increased in BM-MSCs following treatment with ox-LDL (P < .05), which indicates a directional differentiation of BM-MSCs to cardiac cells. Further, ox-LDL could also activate ERK1/2 in BM-MSCs, and application of U0126 markedly inhibited ox-LDL-induced cardiac transformation of BM-MSCs. CONCLUSIONS: Ox-LDL induces cardiac differentiation of BM-MSCs via activation of ERK1/2 signaling.

TGF-ß1 induces apoptosis of bone marrow-derived mesenchymal stem cells via regulation of mitochondrial reactive oxygen species production.

Bone marrow-derived mesenchymal stem cells (BMSCs) are the most promising seed cells in regenerative medicine. Our previous study demonstrated that transforming growth factor (TGF)-ß1 induced BMSC senescence in vitro. Whether TGF-ß1 affects the apoptosis of BMSCs has not been examined; therefore the aim of the present study was to investigate this effect. BMSCs were isolated from mouse bone marrow, and the third-passage cells were exposed to 0, 10 and 20 ng/ml TGF-ß1 for 24 h. Cell proliferation was measured by MTT assay; apoptosis was assessed using DAPI staining; and the apoptotic signals Annexin V, B-cell lymphoma (Bcl)-2 and Bcl-2-associated X protein (Bax) were measured using western blotting. Mitochondrial reactive oxygen species (ROS) were measured by flow cytometry following staining with MitoSOX™ Red mitochondrial superoxide indicator. The MTT assay showed that 10 and 20 ng/ml TGF-ß1 inhibited BMSC proliferation. DAPI staining demonstrated that 10 and 20 ng/ml TGF-ß1 promoted BMSC apoptosis, which was further confirmed by a western blotting assay showing a significant increase in the pro-apoptotic signals Annexin V and Bax but a decrease in the anti-apoptotic signal Bcl-2. It was also found that TGF-ß1 markedly increased the mitochondrial ROS levels in BMSCs. It is well known that mitochondrial ROS are strong stimulators of cell apoptosis. These findings indicate that TGF-ß1 can induce BMSC apoptosis, and the mechanism may involve mitochondrial ROS generation.

Association between endothelin-1/endothelin receptor A and inflammation in mouse kidneys following acute ischemia/reperfusion.

Renal ischemia/reperfusion (I/R) is a common risk factor for renal failure. Expression of endothelin­1 (ET­1) and its receptor ETA were also reported to be involved in the development of acute and chronic renal disease. The present study was designed to investigate the association between inflammation and ET­1/ETA expression in mouse kidneys following acute I/R. The results demonstrated that acute renal I/R caused a significant increase in ET­1 and ETA gene and transcriptional levels compared with those of the sham group (P<0.01). Ischemia alone also resulted in a marked increase of ET­1 and ETA expression compared with that of the sham group (P<0.05). In addition, ET­1 and ETA expression was significantly increased in the I/R group compared with that of the ischemia group (P<0.05 or P<0.01). Of note, the altered expression levels of inflammatory cytokines tumor necrosis factor (TNF)­α and interleukin (IL)­6 in kidneys following I/R and ischemia alone were correlated with the expression of ET­1 and ETA. Hypoxia is the most important stimulus of I/R for tissue injury. In kidneys, ET­1 is primarily produced by renal glomerular endothelial cells (RGECs). In the present study, treatment with hypoxia alone or hypoxia/reoxygenation were found to increase ET­1 and ETA expression in human RGECs (P<0.05 or P<0.01). In order to elucidate the role of inflammation in the ischemia­ and hypoxia­induced upregulation of ET­1 and ETA, human RGECs were exposed to different concentrations of TNF­α. As expected, TNF­α increased ET­1 and ETA expression in a dose­dependent manner; furthermore, application of the TNF­α inhibitor CAY10500 partially inhibited hypoxia­induced ET­1 and ETA expression. In conclusion, these results indicated that I/R induced upregulation of ET­1 and ETA in the kidneys, which was, at least in part, dependent on the production of inflammatory cytokines.

Downregulation of ß-catenin and Akt signaling is responsible for poor proliferation of the late passage of bone marrow mesenchymal stem cells.

It is well established that mesenchymal stem cells (MSCs) will partially lose their proliferative ability with continuous expansion. However, the specific mechanisms underlying this effect remain unclear. In the present study, it was identified that ß-catenin was downregulated in the late passage (passage 8) of bone marrow mesenchymal stem cells (bmMSCs). Following ß-catenin expression, the expression of phospho-Akt was also significantly decreased in the late passage of bmMSCs. More notably, overexpression of ß-catenin in passage 8 of bmMSCs by transfection with pMXs-ß-catenin plasmids, significantly increased cell proliferation and Akt expression. These results indicate that the downregulation of ß-catenin and Akt signaling may be a critical factor for the proliferation of the late passage of bmMSCs.

Overexpression of CyclinA2 ameliorates hypoxia-impaired proliferation of cardiomyocytes.

Hypoxia is a primary mediator for cell survival, and has been reported to inhibit cardiomyocyte proliferation in fetal and neonatal hearts. CyclinA2 is a key regulator of cell proliferation. Whether CyclinA2 affects cardiomyocyte proliferation in hypoxic conditions remains unexamined. This study was designed to investigate the roles of CyclinA2 expression on hypoxia-impaired cardiomyocyte proliferation. Cardiomyocytes were isolated from neonatal rats and randomly separated into six groups: Control, hypoxia, enhanced green fluorescent protein (EGFP)-Adv, EGFP-Ccna2, EGFP-Adv + hypoxia and EGFP-Ccna2 + hypoxia. The cells in the control group were cultured in a general cell incubator; the cells in the hypoxia group were placed in a hypoxic chamber for 12 h; the cells in the EGFP-Adv and EGFP-Ccna2 groups were separately transfected with EGFP-adenovirus capsids or EGFP-adenovirus capsids with CyclinA2 cDNA for 18 h, and then placed in a general incubator for an additional 12 h; the cells in the EGFP-Adv + hypoxia and EGFP-Ccna2 + hypoxia groups were separately transfected with EGFP-adenovirus capsids or EGFP-adenovirus capsids with CyclinA2 cDNA for 18 h, and then placed in a hypoxia chamber for an additional 12 h. CyclinA2 expression was measured using immunochemical staining and western blot analysis, and cardiomyocyte proliferation was measured using the cell counting kit 8. GFP fluorescence indicated a high transfection efficiency (>80%), and immunochemical staining showed that CyclinA2 was mainly distributed in the nucleus. CyclinA2 expression was downregulated following exposure to hypoxia for 12 h. Cardiomyocyte proliferation was also significantly decreased following exposure to hypoxia for 12 h. However, compared with the EGFP-Adv group, CyclinA2 expression and cardiomyocyte proliferation was markedly increased in the EGFP-Ccna2 group. Furthermore, compared with the EGFP-Adv + hypoxia group, CyclinA2 expression and cell proliferation were markedly increased in the EGFP-Ccna2 + hypoxia group. These findings indicate that CyclinA2 upregulation improves cardiomyocyte proliferation in hypoxic conditions.

Aspirin attenuates angiotensin II-induced inflammation in bone marrow mesenchymal stem cells via the inhibition of ERK1/2 and NF-κB activation.

Angiotensin II (Ang II) is a peptide hormone that plays a critical role in numerous physiological and pathophysiological processes. It is also commonly used as an inducer for the directional differentiation of bone marrow mesenchymal stem cells (bmMSCs). Previous studies demonstrated that Ang II induces inflammatory responses in endothelial cells, smooth muscle cells and fibroblasts. Aspirin is generally used as analgesic, antipyretic and occasionally anti-inflammatory medication. Whether aspirin suppresses inflammatory responses in bmMSCs has not been elucidated. In this study, we investigated the effect of aspirin on Ang II-induced inflammation in bmMSCs. Our results demonstrated that Ang II (10 nM-10 µM) increased the secretion of tumor necrosis factor (TNF)-α and interleukin (IL)-6 from bmMSCs in a dose-dependent manner. This result was further confirmed by a reverse transcription-polymerase chain reaction (RT-PCR) assay, which demonstrated a dose-dependent increase in the mRNA expression of TNF-α, IL-6, IL-1ß and monocyte chemotactic protein-1 (MCP-1) in bmMSCs following exposure to Ang II. Furthermore, it was also observed that Ang II increased the expression of phospho-extracellular signal-regulated kinase 1/2 (ERK1/2) and phospho-nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB)-p65 in bmMSCs. The application of aspirin (0.1 mM) significantly inhibited the activation of ERK1/2 and NF-κB, the expression of TNF-α, IL-6, IL-1ß and MCP-1 genes and the secretion of TNF-α and IL-6. Our findings indicated that aspirin may attenuate Ang II-induced inflammation in bmMSCs via the inhibition of ERK1/2 and NF-κB activation.

MicroRNA-10b promotes the migration of mouse bone marrow-derived mesenchymal stem cells and downregulates the expression of E-cadherin.

The ability of mesenchymal stem cells (MSCs) to migrate is an important determinant of the efficiency of MSC transplant therapy. MicroRNA-10b (miR-10b) has been positively involved in the migration of a number of tumor cells lineages. To date, it remains unknown whether miR-10b affects the migration of MSCs. In the current study, the effect of miR-10b on the migration of mouse bone marrow-derived MSCs (bmMSCs) was investigated. Third-passage bmMSCs were transfected with miR-10b mimic and negative control precursor miRNA using Lipofectamine™ 2000. miR-10b and E-cadherin expression and bmMSC migration were determined. The present results showed that primary bmMSCs exhibit a spindled or triangular morphology and that third­passage bmMSCs present a typical fibroblast-like morphology, exhibiting CD90-positive and CD45-negative expression. Compared with the transfection of negative control miRNA, transfection of miR-10b mimic markedly upregulated miR-10b expression in bmMSCs, increased their migration and downregulated E-cadherin expression. The current observations indicate that the upregulation of miR-10b increases bmMSC migration ability, which may be involved in the downregulation of E-cadherin.