Dennis Jine Yuan Hsieh

17β-Estradiol and/or Estrogen Receptor β Attenuate the Autophagic and Apoptotic Effects Induced by Prolonged Hypoxia Through HIF-1α-Mediated BNIP3 and IGFBP-3 Signaling Blockage

Background/Aims: The risk of heart disease is higher in males than in females. However, this advantage of females declines with increasing age, presumably a consequence of decreased estrogen secretion and malfunctioning of the estrogen receptor. We previously demonstrated that 17β-estradiol (E2) prevents cardiomyocyte hypertrophy, autophagy and apoptosis via estrogen receptor α (ERα), but the effects of ERβ on myocardial injury remained elusive. The present paper thus, investigated the cardioprotective effects of estrogen (E2) and ERβ against hypoxia-induced cell death. Methods: Transient transfection of Tet-On ERβ gene construct was used to overexpress ERβ in hypoxia-treated H9c2 cardiomyoblast cells. Results: Our data revealed that IGF1R, Akt phosphorylation and Bcl-2 expression are enhanced by ERβ in H9c2 cells. Moreover, ERβ overexpression reduced accumulation of hypoxia-related proteins, autophagy-related proteins and mitochondria-apoptotic proteins and enhanced the protein levels of Bcl-2, pAkt and Bad under hypoxic condition. In neonatal rat ventricular myocytes (NRVMs), we observed that hypoxia induced cell apoptosis as measured by TUNEL staining, and E2 and/or ERβ could totally abolish hypoxia-induced apoptosis. The suppressive effects of E2 and/or ERβ in hypoxia-treated NRVMs were totally reversed by ER antagonist, ICI. Taken together, E2 and/or ERβ exert the protective effect through repressed hypoxia-inducible HIF-1α, BNIP3 and IGFBP-3 levels to restrain the hypoxia-induced autophagy and apoptosis effects in H9c2 cardiomyoblast cells. Conclusion: The results suggest that females probably could tolerate better prolonged hypoxia condition than males, and E2/ERβ treatment could be a potential therapy to prevent hypoxia-induced heart damage.”

Tanshinone IIA Prevents Leu27IGF-II-Induced Cardiomyocyte Hypertrophy Mediated by Estrogen Receptor and Subsequent Akt Activation

IGF-IIR plays important roles as a key regulator in myocardial pathological hypertrophy and apoptosis, which subsequently lead to heart failure. Salvia miltiorrhiza Bunge (Danshen) is a traditional Chinese medicinal herb used to treat cardiovascular diseases. Tanshinone IIA is an active compound in Danshen and is structurally similar to 17[Formula: see text]-estradiol (E[Formula: see text]. However, whether tanshinone IIA improves cardiomyocyte survival in pathological hypertrophy through estrogen receptor (ER) regulation remains unclear. This study investigates the role of ER signaling in mediating the protective effects of tanshinone IIA on IGF-IIR-induced myocardial hypertrophy. Leu27IGF-II (IGF-II analog) was shown in this study to specifically activate IGF-IIR expression and ICI 182,780 (ICI), an ER antagonist used to investigate tanshinone IIA estrogenic activity. We demonstrated that tanshinone IIA significantly enhanced Akt phosphorylation through ER activation to inhibit Leu27IGF-II-induced calcineurin expression and subsequent NFATc3 nuclear translocation to suppress myocardial hypertrophy. Tanshinone IIA reduced the cell size and suppressed ANP and BNP, inhibiting antihypertrophic effects induced by Leu27IGF-II. The cardioprotective properties of tanshinone IIA that inhibit Leu27IGF-II-induced cell hypertrophy and promote cell survival were reversed by ICI. Furthermore, ICI significantly reduced phospho-Akt, Ly294002 (PI3K inhibitor), and PI3K siRNA significantly reduced the tanshinone IIA-induced protective effect. The above results suggest that tanshinone IIA inhibited cardiomyocyte hypertrophy, which was mediated through ER, by activating the PI3K/Akt pathway and inhibiting Leu27IGF-II-induced calcineurin and NFATC3. Tanshinone IIA exerted strong estrogenic activity and therefore represented a novel selective ER modulator that inhibits IGF-IIR signaling to block cardiac hypertrophy.

E4BP4 is a cardiac survival factor and essential for embryonic heart development

The bZIP transcription factor E4BP4, has been demonstrated to be a survival factor in pro-B lymphocytes. GATA factors play important roles in transducing the IL-3 survival signal and transactivating the downstream survival gene, E4BP4. In heart, GATA sites are essential for proper transcription of several cardiac genes, and GATA-4 is a mediator of cardiomyocyte survival. However, the role E4BP4 plays in heart is still poorly understood. In this study, Dot-blot hybridization assays using Dig-labeled RNA probes revealed that the E4BP4 gene was expressed in cardiac tissue from several species including, monkey, dog, rabbit, and human. Western blot analysis showed that the E4BP4 protein was consistently present in all of these four species. Furthermore, immunohistochemistry revealed that the E4BP4 protein was overexpressed in diseased heart tissue in comparison with normal heart tissue. In addition, the overexpression of E4BP4 in vitro activated cell survival signaling pathway of cardiomyocytes. At last, siRNA-mediated knock down of E4BP4 in zebrafish resulted in malformed looping of the embryonic heart tube and decreased heart beating. Based on these results, we conclude that E4BP4 plays as a survival factor in heart and E4BP4 is essential for proper embryonic heart development.

Thymoquinone Induces Caspase-Independent, Autophagic Cell Death in CPT-11-Resistant LoVo Colon Cancer via Mitochondrial Dysfunction and Activation of JNK and p38

Chemotherapy causes unwanted side effects and chemoresistance, limiting its effectiveness. Therefore, phytochemicals are now used as alternative treatments. Thymoquinone (TQ) is used to treat different cancers, including colon cancer. The irinotecan-resistant (CPT-11-R) LoVo colon cancer cell line was previously constructed by stepwise CPT-11 challenges to untreated parental LoVo cells. TQ dose-dependently increased the total cell death index and activated apoptosis at 2 μM, which then diminished at increasing doses. The possibility of autophagic cell death was then investigated. TQ caused mitochondrial outer membrane permeability (MOMP) and activated autophagic cell death. JNK and p38 inhibitors (SP600125 and SB203580, respectively) reversed TQ autophagic cell death. TQ was also found to activate apoptosis before autophagy, and the direction of cell death was switched toward autophagic cell death at initiation of autophagosome formation. Therefore, TQ resulted in caspase-independent, autophagic cell death via MOMP and activation of JNK and p38 in CPT-11-R LoVo colon cancer cells.

Hyperphosphate-Induced Myocardial Hypertrophy through the GATA-4/NFAT-3 Signaling Pathway Is Attenuated by ERK Inhibitor Treatment

Background/Aims: Numerous epidemiological studies have associated elevated serum phosphorus levels with cardiovascular disease and the risk of death in the general population as well as in chronic kidney disease (CKD) and dialysis patients. In this study, we explored whether elevated phosphate conditions induce cardiac hypertrophy and attempted to identify the molecular and cellular mechanisms in the hypertrophic response. Methods: H9c2 myocardial cells were incubated in high-phosphate conditions to induce hypertrophy. Pathological hypertrophic responses were measured in terms of cell size, arrangement of actin filaments, and hypertrophy markers such as atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) in myocardial cells. Several transcriptional factors involved in cardiac hypertrophy development were measured to investigate the molecular pathways involved in elevated phosphate-induced cardiac hypertrophy. Results: High-phosphate conditions induced cellular hypertrophy, marked by increased cell size, reorganization of actin filaments, and upregulation of both ANP and BNP in H9c2 cells. Both upstream calcineurin and downstream transcription factors, including GATA-4 and NFAT-3, were significantly increased under hyperphosphate conditions. Moreover, both MEK1/2 and ERK1/2 expression increased significantly, and cellular hypertrophy was markedly attenuated by U0126, an ERK1/2 inhibitor. Conclusions: These results suggest that hyperphosphate conditions induce myocardial hypertrophy through the ERK signaling pathway in H9c2 cells. Our findings provide a link between the hyperphosphate-induced response and the ERK/NFAT-3 signaling pathway that mediates the development of cardiac hypertrophy. In view of the potent and selective activity of the ERK inhibitor U0126, this agent warrants further investigation as a candidate for preventing hyperphosphate-induced cardiac hypertrophy in CKD and dialysis patients.

BNIP3 induces IL6 and calcineurin/NFAT3 hypertrophic-related pathways in H9c2 cardiomyoblast cells

Ischemia/reperfusion injury causes cardiomyocyte apoptosis, ventricular remodeling, leading to a dilated heart. Hypoxia is one of the causes involved in ischemia damage, and BNIP3 is a hypoxia-inducible marker and also a sensor to induce mitochondria-dependent apoptosis. Recent reports discussed ablating BNIP3 can restrain cardiomyocytes apoptosis and post-infarction remodeling. BNIP3 is a crucial therapeutic target. However, the BNIP3-induced hypertrophy aspect is rarely investigated. Here, we transiently transfected BNIP3 plasmids into H9c2 cardiomyoblast cells to evaluate the molecular signaling and hypertrophy markers using Western blot. We measured the cell size change using actin staining. We disclose that BNIP3 overexpression induced an increase in cell size, activated the pathological-related hypertrophy signaling pathways, such as IL6-MEK5-ERK5, IL6-JAK2-STAT1/3, calcineurin/NFAT3 and p38β MAPK resulting in the fetal genes, ANP and BNP expressing. Concluding above, BNIP3 acts as a pathological hypertrophy inducer, which might be a potential therapeutic target for heart damage prevention.

Heat Killed Lactobacillus reuteri GMNL-263 Reduces Fibrosis Effects on the Liver and Heart in High Fat Diet-Hamsters via TGF-β Suppression.

Obesity is one of the major risk factors for nonalcoholic fatty liver disease (NAFLD), and NAFLD is highly associated with an increased risk of cardiovascular disease (CVD). Scholars have suggested that certain probiotics may significantly impact cardiovascular health, particularly certain Lactobacillus species, such as Lactobacillus reuteri GMNL-263 (Lr263) probiotics, which have been shown to reduce obesity and arteriosclerosis in vivo. In the present study, we examined the potential of heat-killed bacteria to attenuate high fat diet (HFD)-induced hepatic and cardiac damages and the possible underlying mechanism of the positive effects of heat-killed Lr263 oral supplements. Heat-killed Lr263 treatments (625 and 3125 mg/kg-hamster/day) were provided as a daily supplement by oral gavage to HFD-fed hamsters for eight weeks. The results show that heat-killed Lr263 treatments reduce fatty liver syndrome. Moreover, heat-killed Lactobacillus reuteri GMNL-263 supplementation in HFD hamsters also reduced fibrosis in the liver and heart by reducing transforming growth factor β (TGF-β) expression levels. In conclusion, heat-killed Lr263 can reduce lipid metabolic stress in HFD hamsters and decrease the risk of fatty liver and cardiovascular disease.

Long-term hypoxia exposure enhanced IGFBP-3 protein synthesis and secretion resulting in cell apoptosis in H9c2 myocardial cells.

Abstract Myocardial infarction (MI) usually results in myocardial ischemia, remodeling and hypoxia that lead to cell death. To date, the insulin-like growth factor binding protein-3 (IGFBP3) is known to play an important role in insulin growth factor (IGF) bioavailability. Previous studies have found that hypoxia results in cell apoptosis. However, the detailed mechanism and roles of IGFBP3 in long-term hypoxia (LTH) regulated heart cell apoptosis remains unknown. In this study H9c2 cardiomyoblast cells were treated with investigated long-term hypoxic exposure with the possible mechanisms involved. The results showed that LTH enhanced IGFBP3 protein synthesis and induced its secretion. The accumulated IGFBP3 sequestered Insulin growth factor 1 (IGF-1) away from the type I IGF receptor (IGF-1 R), which blocked the IGF1R/PI3K/Akt survival signaling pathway, resulting in cell apoptosis. According to our findings, IGFBP3 could be a valuable target for developing treatments for cardiac diseases in long-term hypoxia exposure patients.

Danshen mediates through estrogen receptors to activate Akt and inhibit apoptosis effect of Leu27IGF-II-induced IGF-II receptor signaling activation in cardiomyoblasts.

Post-menopausal women show dramatically increased cardiovascular disease morbidity (CVD). Danshen is used widely in China for the treatment of cardiovascular disorders, including coronary heart disease. Danshen possesses lipid-soluble biologically active components with a structure similar to 17β-estrodiol (E2). This study assesses whether the cardio-protection exerted by Danshen is mediated through the ERs within H9c2 cardiomyoblast cells. Cardiomyoblast cells pretreated with Fulvestrant (ICI 182,780), an estrogen receptor antagonist was applied to investigate the estrogenic activity of Danshen. The Danshen extract preventive effects on Leu27IGF-II-induced IGF-IIR signaling activator and H9c2 cell apoptosis were identified using TUNEL assay, JC-1 staining and Western blot assay. We found that Danshen extract treatments significantly enhanced phosphorylated Akt through estrogen receptor activation to inhibit Leu27IGF-II-induced calcineurin activation and block H9c2 cell apoptosis. Danshen extracts suppressed the IGF-IIR signaling proteins, pro-apoptotic proteins and reversed the mitochondrial membrane instability induced by Leu27IGF-II. However, the cardioprotective properties of Danshen to inhibit Leu27IGF-II-induced cell apoptosis and promote cell survival were attenuated by applying ICI, which suggests that the Danshen cardioprotective effect is mediated through estrogen receptors. All our data indicated that Danshen exerts strong estrogenic activity which can be considered a novel selective estrogen receptor modulator (SERM) against IGF2R signaling that blocks cardiac apoptosis.

Mesenchymal stem cell insights: Prospects in hematological transplantation

Adult stem cells have been proven to possess tremendous potential in the treatment of hematological disorders, possibly in transplantation. Mesenchymal stem cells (MSCs) are a heterogeneous group of cells in culture, with hypoimmunogenic character to avoid alloreactive T-cell recognition as well as inhibition of T-cell proliferation. Numerous experimental findings have shown that MSCs also possess the ability to promote engraftment of donor cells and to accelerate the speed of hematological recovery. Despite that the exact mechanism remains unclear, the therapeutic ability of MSCs on hematologic transplantation have been tested in preclinical trials. Based on encouraging preliminary findings, MSCs might become a potentially efficacious tool in the therapeutic options available to treat and cure hematological malignancies and nonmalignant disorders. The molecular mechanisms behind the real efficacy of MSCs on promoting engraftment and accelerating hematological recovery are awaiting clarification. It is hypothesized that direct cell-to-cell contact, paracrine factors, extracellular matrix scaffold, BM homing capability, and endogenous metabolites of immunologic and nonimmunologic elements are involved in the interactions between MSCs and HSCs. This review focuses on recent experimental and clinical findings related to MSCs, highlighting their roles in promoting engraftment, hematopoietic recovery, and GvHD/graft rejection prevention after HSCT, discussing the potential clinical applications of MSC-based treatment strategies in the context of hematological transplantation.