Jingjin Liu

Angiopoietin-like 4 confers resistance to hypoxia/serum deprivation-induced apoptosis through PI3K/Akt and ERK1/2 signaling pathways in mesenchymal stem cells.

Angiopoietin-like 4 (ANGPTL4) is a potential anti-apoptotic agent for various cells. We examined the protective effect of ANGPTL4 on hypoxia/serum deprivation (SD)-induced apoptosis of MSCs, as well as the possible mechanisms. MSCs were obtained from rat bone marrow and cultured in vitro. Apoptosis was induced by hypoxia/SD for up to 24 hr, and assessed by flow cytometry and TUNEL assay. Expression levels of Akt, ERK1/2, focal adhesion kinase (FAK), Src, Bcl-2, Bax, cytochrome C and cleaved caspase-3 were detected by Western blotting. Integrin β1 mRNA was detected by qRT-PCR. Mitochondrial membrane potential was assayed using a membrane-permeable dye. Hypoxia/SD-induced apoptosis was significantly attenuated by recombinant rat ANGPTL4 in a concentration dependent manner. Moreover, ANGPTL4 decreased the hypoxia/SD-induced caspase-3 cleavage and the cytochrome C release, but increased the Bcl-2/Bax ratio and the mitochondrial membrane potential. Decreased expression of integrin β1, the ANGPTL4 receptor was observed during hypoxia/SD conditions, however, such decrease was reversed by ANGPTL4. In addition, ANGPTL4 induced integrin β1-associated FAK and Src phosphorylation, which was blocked by anti-integrin β1 antibody. ANGPTL4 also reversed the hypoxia/SD-induced decrease of Akt and ERK 1/2 phosphorylation, and the effect of ANGPTL4 was abolished by inhibitors of either integrins, ERK1/2, or phosphatidylinositol 3-kinase (PI3K). Blocking integrinβ1, Akt or ERK largely attenuated anti-apoptotic effect of ANGPTL4. ANGPTL4 protects MSCs from hypoxia/SD-induced apoptosis by interacting with integrins to stimulate FAK complex, leading to downstream ERK1/2 and PI3K/Akt signaling pathways and mimicking the pathway in which MSCs contact with the extracellular matrix.

C1q tumor necrosis factor-related protein-3 protects mesenchymal stem cells against hypoxia- and serum deprivation-induced apoptosis through the phosphoinositide 3-kinase/Akt pathway

Bone marrow (BM)-derived mesenchymal stem cells (MSCs) represent the leading candidate cell for tissue regeneration in the ischemic myocardium. However, the poor survival of stem cells transplanted into the ischemic myocardium presents a major obstacle in stem cell-based therapy. C1q tumor necrosis factor-related protein 3 (CTRP3) is a newly identified adipokine, similar to adiponectin, with beneficial effects on metabolic regulation. It has been shown to enhance the survival of cardiomyocytes during ischemia, while its expression is reduced following ischemia. In the present study, we examined the hypothesis that CTRP3 may enhance the survival of MSCs during exposure to hypoxia/serum deprivation (SD), and attempted to elucidate the underlying mechanisms. MSCs were obtained from rat bone marrow and cultured. Apoptosis was induced by hypoxia/SD for up to 24 h and the apoptotic rates were assessed by flow cytometry. MSC proliferation was measured using a Cell Counting kit-8 assay. The expression levels of Akt, Bcl-2, Bax, cytochrome c and cleaved caspase-3 were detected by western blot analysis. Mitochondrial membrane potential was examined using a membrane-permeable dye. CTRP3 significantly reduced hypoxia/SD-induced apoptosis in a concentration-dependent manner. The hypoxia/SD-induced decrease in the Bcl-2/Bax ratio and the mitochondrial membrane potential, and the increase in cytochrome c and caspase-3 levels were largely reversed by CTRP3. The anti-apoptotic effects of CTRP3 were blocked by inhibiting the activation of phosphoinositide 3-kinase (PI3K)/Akt signaling pathway with the PI3K inhibitor, LY294002. In conclusion, CTRP3 is a novel anti-apoptotic adipokine that protects MSCs from hypoxia/SD-induced apoptosis through the PI3K/Akt signaling pathway.

Wnt1 Inhibits Hydrogen Peroxide-Induced Apoptosis in Mouse Cardiac Stem Cells

Background Because of their regenerative and paracrine abilities, cardiac stem cells (CSCs) are the most appropriate, optimal and promising candidates for the development of cardiac regenerative medicine strategies. However, native and exogenous CSCs in ischemic hearts are exposed to various pro-apoptotic or cytotoxic factors preventing their regenerative and paracrine abilities. Methods and Results We examined the effects of H2O2 on mouse CSCs (mCSCs), and observed that hydrogen peroxide (H2O2) treatment induces mCSCs apoptosis via the caspase 3 pathway, in a dose-dependent manner. We then examined the effects of Wnt1 over-expression on H2O2-induced apoptosis in mCSCs and observed that Wnt1 significantly decreased H2O2-induced apoptosis in mCSCs. On the other hand, inhibition of the canonical Wnt pathway by the secreted frizzled related protein 2 (SFRP2) or knockdown of β-catenin in mCSCs reduced cells resistance to H2O2-induced apoptosis, suggesting that Wnt1 predominantly prevents H2O2-induced apoptosis through the canonical Wnt pathway. Conclusions Our results provide the first evidences that Wnt1 plays an important role in CSCs’ defenses against H2O2-induced apoptosis through the canonical Wnt1/GSK3β/β-catenin signaling pathway.

MiR218 Modulates Wnt Signaling in Mouse Cardiac Stem Cells by Promoting Proliferation and Inhibiting Differentiation through a Positive Feedback Loop.

MiRNA expression was determined in both proliferating and differentiated cardiac stem cells (CSCs) through a comprehensive miRNA microarray analysis. We selected miR218 for functional follow-up studies to examine its significance in CSCs. First, we observed that the expression of miR218 was altered in CSCs during differentiation into cardiomyocytes, and transfection of an miR218 mimic or miR218 inhibitor affected the myocardial differentiation of CSCs. Furthermore, we observed that a negative regulator of Wnt signaling, sFRP2, was a direct target of miR218, and the protein levels of sFRP2 were increased in cells transfected with the synthetic miR218 inhibitor. In contrast, transfection with the miR218 mimic decreased the expression of sFRP2 and potentiated Wnt signaling. The subsequent down-regulation of sFRP2 by shRNA potentiated Wnt signaling, contributing to a gene expression program that is important for CSC proliferation and cardiac differentiation. Specifically, canonical Wnt signaling induced miR218 transcription. Thus, miR218 and Wnt signaling were coupled through a feed-forward positive feedback loop, forming a biological regulatory circuit. Together, these results provide the first evidence that miR218 plays an important role in CSC proliferation and differentiation through the canonical Wnt signaling pathway.

Macrophage migration inhibitory factor promotes cardiac stem cell proliferation and endothelial differentiation through the activation of the PI3K/Akt/mTOR and AMPK pathways

Macrophage migration inhibitory factor (MIF) has pleiotropic immune functions in a number of inflammatory diseases. Recent evidence from expression and functional studies has indicated that MIF is involved in various aspects of cardiovascular disease. In this study, we aimed to determine whether MIF supports in vitro c-kit+CD45− cardiac stem cell (CSC) survival, proliferation and differentiation into endothelial cells, as well as the possible mechanisms involved. We observed MIF receptor (CD74) expression in mouse CSCs (mCSCs) using PCR and immunofluorescence staining, and MIF secretion by mCSCs using PCR and ELISA in vitro. Increasing amounts of exogenous MIF did not affect CD74 expression, but promoted mCSC survival, proliferation and endothelial differentiation. By contrast, treatment with an MIF inhibitor (ISO-1) or siRNA targeting CD74 (CD74-siRNA) suppressed the biological changes induced by MIF in the mCSCs. Increasing amounts of MIF increased the phosphorylation of Akt and mammalian target of rapamycin (mTOR), which are known to support cell survival, proliferation and differentiation. These effects of MIF on the mCSCs were abolished by LY294002 [a phosphoinositide 3-kinase (PI3K) inhibitor] and MK-2206 (an Akt inhibitor). Moreover, adenosine monophosphate-activated protein kinase (AMPK) phosphorylation increased following treatment with MIF. The AMPK inhibitor, compound C, partly blocked the pro-proliferative effects of MIF on the mCSCs. In conclusion, our results suggest that MIF promotes mCSC survival, proliferation and endothelial differentiation through the activation of the PI3K/Akt/mTOR and AMPK signaling pathways. Thus, MIF may prove to be a potential therapeutic factor in the treatment of heart failure and myocardial infarction by activating CSCs.

Cables1 Inhibits Proliferation and Induces Senescence by Angiotensin II via a p21-Dependent Pathway in Human Umbilical Vein Endothelial Cells

Cables1 (Cdk5 and Abl enzyme substrate 1) is a vital cell cycle regulator and a candidate tumor suppressor that negatively regulates cell growth by inhibiting cyclin-dependent kinases. Here, we report on the critical role of the Cables1/p21 pathway, which inhibits cell proliferation and induces cell senescence in human umbilical vein endothelial cells. Moreover, we confirmed that silencing of Cables1 promoted cell proliferation as well as increased resistance to angiotensin II-induced senescence, at least in part, by altering Cables1 activation. We further demonstrated that knockdown of p21 reverses Cables1-mediated cell growth inhibition and cell senescence. Taken together, these results suggest that the Cables1/p21 pathway has a strong effect on the induction of cell senescence and inhibition of cell growth, and acts as a novel regulatory mechanism in which p21 is probably one of several downstream effector molecules to mediate Cables1.

THE CROSSTALK BETWEEN ERK1/2 AND STAT3 IN THE REGULATION OF CAR EXPRESSION DURING CVB3 INFECTION IN CARDIOMYOCYTE.

Objectives We performed a novel analysis of cardiac expression of receptors for several adenovirus serotypes with a focus on expression of CAR, as adenoviruses targeting receptor has been used in various applications, that more than the viral receptor which is significantly induced in the heart tissue of CVB3 infected and considered to be the dominant aetiology of viral myocarditis. Methods We treated cardiomyocytes with culture medium in the uninfected or infected of CVB3 (MOI=100). The lentiviral vector system derived from HIV-1 was used to express short hairpin RNAs (shRNA) directed against STAT3 and ERK1/2 activation was blocked with U0126, an ERK1/2 inhibitor. Western blot was used to observe the level of CAR, ERK and STAT3. The degrees of cells injury were judged by LDH levels in cells supernatant. Results Up-regulation activities of ERK1/2 after CVB3 infected with cardiomyocytes, accompanied by positive correlation of the expression of CAR. Treatment of cardiomyocytes with Pharmacological inhibition of ERK1/2 phosphorylation with U0126 resulted in a dramatic increase in the expression of CAR.U0126 induced the JNK/STAT3 pathway activity to prevent cells from injury. Lentivector-based short hairpin RNAs provide efficient and stable knock down of STAT3. Treatment of cardiomyocytes with shERK resulted in ERK1/2 phosphorylation and a decrease in the expression of CAR accompanied by the elevation of LDH levels in infected with CVB3 cells. Conclusions In this study we investigated the effect of signalling through the Raf-MEK-ERK1/2 pathway on CAR expression in cardiac myocytes that are potential targets for adenovirus-based therapies. Our findings in modulation of CAR expression, may lead to new strategies in the gene therapy of DCM.

ERK1/2 pathway regulates coxsackie and adenovirus receptor expression in mouse cardiac stem cells

Cardiac stem cells (CSCs) are the most promising and effective candidates for the therapy of cardiac regenerative diseases; however, they have marked limitations. For instance, the implantation of CSCs is hampered by factors such as their sustainability and long-term durability. Gene modification appears to be the most effective method of optimizing CSCs and gene therapy trials have demonstrated that efficient gene transfer is key to achieving therapeutic efficacy. However, the transduction ability of adenovirus (Ad) is limited. Previous studies have reported that low expression of coxsackie and adenovirus receptor (CAR) in target cells decreases the transduction efficiency. A promising method for improving Ad-mediated gene transfer is to increase CAR expression in target cells. The present study investigated the effect of the Raf-mitogen-associated protein kinase (MAPK) kinase (MEK)-extracellular signal-associated protein kinase (ERK) signaling pathway on the expression of CAR on CSCs, as this pathway decreases cell-cell adhesion via cell surface molecules. The results demonstrated that interference with the Raf-MEK-ERK signaling pathway by knockdown of ERK1/2 upregulated the expression of CAR. The entry of the Ad into the cells was increased following inhibition of ERK1/2. Moreover, following knockdown of CAR, the entry of Ad into cells was decreased. However, knockdown of c-Jun N-terminal kinase and p38 as other components of the MAPK pathway did not affect CAR expression. Therefore, CAR expression in CSCs may be mediated via the Raf-MEK-ERK signaling pathway. Upregulation of CAR by knockdown of ERK1/2 may significantly improve Ad-mediated genetic modification of CSCs in the treatment of cardiovascular diseases.