Bmi‐1 high‐expressing cells enrich cardiac stem/progenitor cells and respond to heart injury

Bmi‐1 gene is well recognized as an oncogene, but has been recently demonstrated to play a role in the self‐renewal of tissue‐specific stem cells. By using Bmi‐1^GFP/+ mice, we investigated the role of Bmi‐1 in cardiac stem/progenitor cells and myocardial repair. RT‐PCR and flow cytometry analysis indicated that the expression of Bmi‐1 was significantly higher in cardiac side population than the main population from CD45^?Ter119^?CD31^? heart cells. More Sca‐1⁺ cardiac stem/progenitor cells were found in Bmi‐1 GFP^hi subpopulation, and these Bmi‐1 GFP^hi heart cells showed the potential of differentiation into SMM⁺ smooth muscle‐like cells and TnT⁺ cardiomyocyte‐like cells in vitro. The silencing of Bmi‐1 significantly inhibited the proliferation and differentiation of heart cells. Otherwise, myocardial infarction induced a significantly increase (2.7‐folds) of Bmi‐1 GFP^hi population, mainly within the infarction and border zones. These preliminary data suggest that Bmi‐1^hi heart cells are enriched in cardiac stem/progenitor cells and may play a role in myocardial repair.     

Interleukin‐8 and CXCL10 expression in oral keratinocytes and fibroblasts via Toll‐like receptors

Oral keratinocytes and fibroblasts may be the first line of host defense against oral microorganisms. Here, the contention that oral keratinocytes and fibroblasts recognize microbial components via Toll‐like receptors (TLRs) and participate in development of oral inflammation was examined. It was found that immortalized oral keratinocytes (RT7), fibroblasts (GT1) and primary cells express mRNA of TLRs 1–10. Interleukin‐8 (IL‐8) production by RT7 cells was induced by treatment with TLRs 1–9 with the exception of TLR7 agonist, whereas GT1 cells were induced to produce IL‐8 by all TLR agonists tested except for TLR7 and TLR9. GT1 cells showed increased CXCL10 production following treatment with agonists for TLR1/2, TLR3, TLR4, and TLR5, whereas only those for TLR3 and TLR5 increased CXCL10 production in RT7 cells. Moreover, TLR agonists differentially regulated tumor necrosis factor‐alpha‐induced IL‐8 and CXCL10 production by the tested cell types. These findings suggest that recognition of pathogenic microorganisms in oral keratinocytes and fibroblasts by TLRs may have important roles in orchestrating host immune responses via production of various chemokines.     

Fucoxanthin inhibits tumour‐related lymphangiogenesis and growth of breast cancer

Tumour lymphangiogenesis plays an important role in promoting the growth and lymphatic metastasis of tumours. The process is associated with cell proliferation, migration and tube‐like structure formation in lymphatic endothelial cells (LEC), but no antilymphangiogenic agent is currently used in clinical practice. Fucoxanthin is a material found in brown algae that holds promise in the context of drug development. Fucoxanthin is a carotenoid with variety of pharmacological functions, including antitumour and anti‐inflammatory effects. The ability of fucoxanthin to inhibit lymphangiogenesis remains unclear. The results of experiments performed as part of this study show that fucoxanthin, extracted from Undaria pinnatifida (Wakame), inhibits proliferation, migration and formation of tube‐like structures in human LEC (HLEC). In this study, fucoxanthin also suppressed the malignant phenotype in human breast cancer MDA‐MB‐231 cells and decreased tumour‐induced lymphangiogenesis when used in combination with a conditional medium culture system. Fucoxanthin significantly decreased levels of vascular endothelial growth factor (VEGF)‐C, VEGF receptor‐3, nuclear factor kappa B, phospho‐Akt and phospho‐PI3K in HLEC. Fucoxanthin also decreased micro‐lymphatic vascular density (micro‐LVD) in a MDA‐MB‐231 nude mouse model of breast cancer. These findings suggest that fucoxanthin inhibits tumour‐induced lymphangiogenesis in vitro and in vivo, highlighting its potential use as an antilymphangiogenic agent for antitumour metastatic comprehensive therapy in patients with breast cancer.     

Targeting miR‐193a‐AML1‐ETO‐β‐catenin axis by melatonin suppresses the self‐renewal of leukaemia stem cells in leukaemia with t (8;21) translocation

AML1‐ETO, the most common fusion oncoprotein by t (8;21) in acute myeloid leukaemia (AML), enhances hematopoietic self‐renewal and leukemogenesis. However, currently no specific therapies have been reported for t (8;21) AML patients as AML1‐ETO is still intractable as a pharmacological target. For this purpose, leukaemia cells and AML1‐ETO‐induced murine leukaemia model were used to investigate the degradation of AML1‐ETO by melatonin (MLT), synthesized and secreted by the pineal gland. MLT remarkedly decreased AML1‐ETO protein in leukemic cells. Meanwhile, MLT induced apoptosis, decreased proliferation and reduced colony formation. Furthermore, MLT reduced the expansion of human leukemic cells and extended the overall survival in U937T‐AML1‐ETO‐xenografted NSG mice. Most importantly, MLT reduced the infiltration of leukaemia blasts, decreased the frequency of leukaemia stem cells (LSCs) and prolonged the overall survival in AML1‐ETO‐induced murine leukaemia. Mechanistically, MLT increased the expression of miR‐193a, which inhibited AML1‐ETO expression via targeting its putative binding sites. Furthermore, MLT decreased the expression of β‐catenin, which is required for the self‐renewal of LSC and is the downstream of AML1‐ETO. Thus, MLT presents anti‐self‐renewal of LSC through miR‐193a‐AML1‐ETO‐β‐catenin axis. In conclusion, MLT might be a potential treatment for t (8;21) leukaemia by targeting AML1‐ETO oncoprotein.     

Intragraft gene expression profile associated with the induction of tolerance by allochimeric MHC I in the rat heart transplantation model

The MHC class I allochimeric protein containing donor‐type epitopes on recipient‐type heavy chains induces indefinite survival of heterotopic cardiac allografts in rats. We analyzed gene expression profile of heart allograft tissue. Mutated peptide [α1h1/u]‐RT1.Aa that contains donor‐type (Wistar Furth, WF; RT1u) immunogenic epitopes displayed on recipient‐type (ACI, RT1a) was delivered into ACI recipients of WF hearts at the time of transplantation in addition to a 3 days course of oral cyclosporine. Microarray analysis was performed using Affymetrix Rat 230 2.0 Microarray. Allochimeric molecule treatment caused upregulation of genes involved in structural integrity of heart muscle, downregulation of IL‐1β a key modulator of the immune response, and downregulation of partitioning defective six homolog gamma PAR6, which is involved in T cell polarity, motility, and ability to scan dendritic cells (DC). These indicate that the immunosuppressive function of allochimeric molecule and/or the establishment of allograft tolerance depend on the induction of genes responsible for the heart tissue integrity, the suppression of cytokine pathway(s), and possibly the impairment of T cells mobility and their DC scanning ability. These novel findings may have important clinical implications for inhibition of chronic rejection in transplant recipients. genesis 48:8–19, 2010. © 2009 Wiley‐Liss, Inc.     

Deregulation of XBP1 expression contributes to myocardial vascular endothelial growth factor‐A expression and angiogenesis during cardiac hypertrophy in vivo

Endoplasmic reticulum (ER) stress has been reported to be involved in many cardiovascular diseases such as atherosclerosis, diabetes, myocardial ischemia, and hypertension that ultimately result in heart failure. XBP1 is a key ER stress signal transducer and an important pro‐survival factor of the unfolded protein response (UPR) in mammalian cells. The aim of this study was to establish a role for XBP1 in the deregulation of pro‐angiogenic factor VEGF expression and potential regulatory mechanisms in hypertrophic and failing heart. Western blots showed that myocardial XBP1s protein was significantly increased in both isoproterenol (ISO)‐induced and pressure‐overload‐induced hypertrophic and failing heart compared to normal control. Furthermore, XBP1 silencing exacerbates ISO‐induced cardiac dysfunction along with a reduction of myocardial capillary density and cardiac expression of pro‐angiogenic factor VEGF‐A in vivo. Consistently, experiments in cultured cardiomyocytes H9c2 (2‐1) cells showed that UPR‐induced VEGF‐A upregulation was determined by XBP1 expression level. Importantly, VEGF‐A expression was increased in failing human heart tissue and blood samples and was correlated with the levels of XBP1. These results suggest that XBP1 regulates VEGF‐mediated cardiac angiogenesis, which contributes to the progression of adaptive hypertrophy, and might provide novel targets for prevention and treatment of heart failure.     

Erythroid 5‐aminolevulinate synthase mediates the upregulation of membrane band 3 protein expression by iron

Iron deficiency leads to abnormal expression and function of band 3 protein in erythrocytes, but the underlying mechanisms remain elusive. The mRNA of erythroid‐specific 5‐aminolevulinate synthase (eALAS) contains an iron response element and the eALAS protein is an important mediator of iron utilization by erythrocytes. In this study, we investigated the effect of short hairpin RNA (shRNA) mediated silencing of eALAS on the expression of band 3 protein induced by iron. By real‐time RT‐PCR and Western blot we showed that at mRNA and protein level iron‐induced expression of band 3 protein was lower in eALAS‐shRNA transfected K562 cells than in control cells. Of note, the lowest expression was detected in K562 cells cultured in iron deficiency condition (p < 0.01). Thus either iron deficiency or depletion of eALAS could suppress the expression of erythroid band 3 protein. These results demonstrated for the first time that iron and the iron‐regulatory system regulate the expression of the erythrocyte membrane proteins. Copyright © 2010 John Wiley & Sons, Ltd.     

Glycyrrhizin protects against sodium iodate‐induced RPE and retinal injury though activation of AKT and Nrf2/HO‐1 pathway

Glycyrrhizin is a bioactive triterpenoid saponin extracted from a traditional Chinese medicinal herb, glycyrrhiza, and has been reported to protect the organs such as liver and heart from injuries. However, there is no report about the effects of glycyrrhizin on atrophic age‐related macular degeneration (AMD). This study investigated the effects of glycyrrhizin on retinal pigment epithelium (RPE) in vitro and retina of mice in vivo treated with sodium iodate (SI). Glycyrrhizin significantly inhibited SI‐induced reactive oxygen species (ROS), and decreased apoptosis of RPE in vitro. The underlying mechanisms included increased phosphorylation of Akt, and increased expression of nuclear factor erythroid 2‐related factor2 (Nrf‐2) and HO‐1, thereby protecting RPE from SI‐induced ROS and apoptosis. Furthermore, glycyrrhizin significantly decreased the apoptosis of retinal cells in vivo, resulting in the inhibition of thinning of retina, decreasing the number of drusen and improving the function of retina. These findings suggested that glycyrrhizin may be a potential candidate for the treatment of atrophic AMD in clinical practice.     

miR‐34b‐5p inhibition attenuates lung inflammation and apoptosis in an LPS‐induced acute lung injury mouse model by targeting progranulin

Inflammation and apoptosis play important roles in the initiation and progression of acute lung injury (ALI). Our previous study has shown that progranulin (PGRN) exerts lung protective effects during LPS‐induced ALI. Here, we have investigated the potential roles of PGRN‐targeting microRNAs (miRNAs) in regulating inflammation and apoptosis in ALI and have highlighted the important role of PGRN. LPS‐induced lung injury and the protective roles of PGRN in ALI were first confirmed. The function of miR‐34b‐5p in ALI was determined by transfection of a miR‐34b‐5p mimic or inhibitor in intro and in vivo. The PGRN level gradually increased and subsequently significantly decreased, reaching its lowest value by 24 hr; PGRN was still elevated compared to the control. The change was accompanied by a release of inflammatory mediators and accumulation of inflammatory cells in the lungs. Using bioinformatics analysis and RT‐PCR, we demonstrated that, among 12 putative miRNAs, the kinetics of the miR‐34b‐5p levels were closely associated with PGRN expression in the lung homogenates. The gain‐ and loss‐of‐function analysis, dual‐luciferase reporter assays, and rescue experiments confirmed that PGRN was the functional target of miR‐34b‐5p. Intravenous injection of miR‐34b‐5p antagomir in vivo significantly inhibited miR‐34b‐5p up‐regulation, reduced inflammatory cytokine release, decreased alveolar epithelial cell apoptosis, attenuated lung inflammation, and improved survival by targeting PGRN during ALI. miR‐34b‐5p knockdown attenuates lung inflammation and apoptosis in an LPS‐induced ALI mouse model by targeting PGRN. This study shows that miR‐34b‐5p and PGRN may be potential targets for ALI treatments.     

Morphology Controlling of All‐Inorganic Perovskite at Low Temperature for Efficient Rigid and Flexible Solar Cells

All‐inorganic cesium lead halide (CsPbX₃) perovskites have emerged as promising photovoltaic materials owing to their superior thermal stability compared to traditional organic–inorganic hybrid counterparts. However, the CsPbX₃ perovskites generally need to be prepared at high‐temperature, which restricts their application in multilayer or flexible solar cells. Herein, the formation of CsPbX₃ perovskites at room‐temperature (RT) induced by dimethylsulphoxide (DMSO) coordination is reported. It is further found that a RT solvent (DMSO) annealing (RTSA) treatment is valid to control the perovskite crystallization dynamics, leading to uniform and void‐free films, and consequently a maximum power conversion efficiency (PCE) of 6.4% in the device indium tin oxide (ITO)/NiO _x /RT‐CsPbI₂Br/C₆₀/Bathocuproine (BCP)/Ag, which is, as far as it is known, the first report of RT solution‐processed CsPbX₃‐based perovskite solar cells (PSCs). Moreover, the efficiency can be boosted up to 10.4% by postannealing the RTSA‐treated perovskite film at an optimal temperature of 120 °C. Profiting from the moderate temperature, flexible PSCs are also demonstrated with a maximum PCE of 7.3% for the first time. These results may stimulate further development of all‐inorganic CsPbX₃ perovskites and their application in flexible electronics.     

MicroRNA-18 and microRNA-19 regulate CTGF and TSP-1 expression in age-related heart failure

To understand the process of cardiac aging, it is of crucial importance to gain insight into the age‐related changes in gene expression in the senescent failing heart. Age‐related cardiac remodeling is known to be accompanied by changes in extracellular matrix (ECM) gene and protein levels. Small noncoding microRNAs regulate gene expression in cardiac development and disease and have been implicated in the aging process and in the regulation of ECM proteins. However, their role in age‐related cardiac remodeling and heart failure is unknown. In this study, we investigated the aging‐associated microRNA cluster 17–92, which targets the ECM proteins connective tissue growth factor (CTGF) and thrombospondin‐1 (TSP‐1). We employed aged mice with a failure‐resistant (C57Bl6) and failure‐prone (C57Bl6 × 129Sv) genetic background and extrapolated our findings to human age‐associated heart failure. In aging‐associated heart failure, we linked an aging‐induced increase in the ECM proteins CTGF and TSP‐1 to a decreased expression of their targeting microRNAs 18a, 19a, and 19b, all members of the miR‐17–92 cluster. Failure‐resistant mice showed an opposite expression pattern for both the ECM proteins and the microRNAs. We showed that these expression changes are specific for cardiomyocytes and are absent in cardiac fibroblasts. In cardiomyocytes, modulation of miR‐18/19 changes the levels of ECM proteins CTGF and TSP‐1 and collagens type 1 and 3. Together, our data support a role for cardiomyocyte‐derived miR‐18/19 during cardiac aging, in the fine‐tuning of cardiac ECM protein levels. During aging, decreased miR‐18/19 and increased CTGF and TSP‐1 levels identify the failure‐prone heart.     

Involvement of Yes‐associated protein 1 (YAP1) in doxorubicin‐induced cytotoxicity in H9c2 cardiac cells

Cardiac cell death is one of the major events implicated in doxorubicin‐induced cardiotoxicity, which leads to heart failure. We recently reported that Yes‐associated protein 1 (YAP1) regulates cell survival and apoptosis. However, it is unclear whether YAP1 regulates doxorubicin‐induced cell death in cardiomyocytes. We investigated whether YAP1 is involved in doxorubicin‐induced cell death using H9c2 cardiac cells and mouse heart. In an in vivo study, YAP1 protein expression was significantly decreased in hearts of doxorubicin‐treated mice with increased caspase‐3 activation. Doxorubicin also caused cell death by increasing caspase‐3 activation in H9c2 cells. Doxorubicin reduced YAP1 protein expression and messenger RNA expression accompanied by increased phosphorylation of YAP1 at Ser127. Doxorubicin further increased cell death with increased caspase‐3/7 activation in the absence of YAP1 when compared with doxorubicin or siYAP1 treatment alone. Overexpression of constitutively active YAP1 (YAP1–5SA) using an adenovirus gene transfer technique significantly reversed doxorubicin‐induced cell death by decreasing caspase‐3/7 activation in H9c2 cells. Akt, a potential prosurvival factor, decreased in doxorubicin‐ and YAP1 short interfering RNA (siRNA)‐treated cells. Doxorubicin further significantly decreased Akt protein expression when YAP1 was silenced. Overexpression of YAP1 canceled decreased Akt protein expression induced by doxorubicin treatment in H9c2 cells. In conclusion, these results suggest that doxorubicin‐induced cardiac cell death is mediated in part by down‐regulation of YAP1 and YAP1‐targeted gene, Akt. Modulating YAP1 and its related Hippo pathway on local cardiomyocytes may be a promising therapeutic approach for doxorubicin‐induced cardiotoxicity.     

Effects of d‐galactose‐induced ageing on the heart and its potential interventions

Ageing is a strong independent risk factor for disability, morbidity and mortality. Post‐mitotic cells including those in the heart are a particular risk to age‐related deterioration. As the occurrence of heart disease is increasing rapidly with an ageing population, knowledge regarding the mechanisms of age‐related cardiac susceptibility and possible therapeutic interventions needs to be acquired to prevent advancing levels of heart disease. To understand more about the ageing heart, numerous aged animal models are being used to explore the underlying mechanisms. Due to time‐consuming for investigations involving naturally aged animals, mimetic ageing models are being utilized to assess the related effects of ageing on disease occurrence. d ‐galactose is one of the substances used to instigate ageing in various models, and techniques involving this have been widely used since 1991. However, the mechanism through which d ‐galactose induces ageing in the heart remains unclear. The aim of this review was to comprehensively summarize the current findings from in vitro and in vivo studies on the effects of d ‐galactose‐induced ageing on the heart, and possible therapeutic interventions against ageing heart models. From this review, we hope to provide invaluable information for future studies and based on the findings from experiments involving animals, we can inform possible therapeutic strategies for the prevention of age‐related heart diseases in clinical settings.     

Down‐regulation of mitogen‐activated protein kinases and nuclear factor‐κB signaling is involved in rapamycin suppression of TLR2‐induced inflammatory response in monocytic THP‐1 cells

Tripalmitoyl‐S‐glycero‐Cys‐(Lys) 4 (Pam3CSK4) interacted with TLR2 induces inflammatory responses through the mitogen‐activated protein kinases (MAPKs) and nuclear factor‐κB (NF‐κB) signal pathway. Rapamycin can suppress TLR‐induced inflammatory responses; however, the detailed molecular mechanism is not fully understood. Here, the mechanism by which rapamycin suppresses TLR2‐induced inflammatory responses was investigated. It was found that Pam3CSK4‐induced pro‐inflammatory cytokines were significantly down‐regulated at both the mRNA and protein levels in THP‐1 cells pre‐treated with various concentrations of rapamycin. Inhibition of phosphatidylinositol 3‐kinase/protein kinase‐B (PI3K/AKT) signaling did not suppress the expression of pro‐inflammatory cytokines, indicating that the immunosuppression mediated by rapamycin in THP1 cells is independent of the PI3K/AKT pathway. RT‐PCR showed that Erk and NF‐κB signal pathways are related to the production of pro‐inflammatory cytokines. Inhibition of Erk or NF‐κB signaling significantly down‐regulated production of pro‐inflammatory cytokines. Additionally, western blot showed that pre‐treatment of THP‐1 cells with rapamycin down‐regulates MAPKs and NF‐κB signaling induced by Pam3CSK4 stimulation, suggesting that rapamycin suppresses Pam3CSK4‐induced pro‐inflammatory cytokines via inhibition of TLR2 signaling. It was concluded that rapamycin suppresses TLR2‐induced inflammatory responses by down‐regulation of Erk and NF‐κB signaling.