Wenchao Wu

Nogo‐B: A potential indicator for hepatic cirrhosis and regulator in hepatic stellate cell activation

To evaluate plasma Nogo‐B levels in liver cirrhotic patients and declare a novel molecular basis by which Nogo‐B modulates hepatic stellate cell (HSC) activation.

MicroRNA-297 promotes cardiomyocyte hypertrophy via targeting sigma-1 receptor

Aims: Sigma‐1 receptor (Sig‐1R) is a ligand‐regulated endoplasmic reticulum (ER) chaperone involved in cardiac hypertrophy, but it is not known whether Sig‐1R is regulated by microRNAs (miRNAs). According to bioinformatic analysis, miR‐297 was suggested as a potential target miRNA for Sig‐1R. Therefore, we verified whether miR‐297 could target Sig‐1R and investigated the possible mechanisms underlying the role of miR‐297 in cardiac hypertrophy. Main methods: Bioinformatic analysis combined with laboratory experiments, including quantitative RT‐PCR, Western blotting, and luciferase assay, were performed to identify the target miRNA of Sig‐1R. Transverse aortic constriction (TAC) model and neonatal rat cardiomyocytes (NCMs) stimulated with angiotensin II (AngII) were used to explore the relationship between miR‐297 and Sig‐1R. Additionally, the function of miR‐297 in cardiomyocyte hypertrophy and ER stress/unfolded protein response (UPR) signaling pathway was investigated by transfecting miR‐297 mimics/inhibitor. Key findings: miR‐297 levels were increased in both TAC‐induced hypertrophic heart tissue and AngII‐induced cardiomyocyte hypertrophy. Up‐regulation of miR‐297 by specific mimics exacerbated AngII‐induced cardiomyocyte hypertrophy, whereas inhibition of miR‐297 suppressed the process. During cardiomyocyte hypertrophy, Sig‐1R expression, which was negatively regulated by miR‐297 by directly targeting its 3′untranslated region (UTR), was decreased. Furthermore, attenuation of miR‐297 inhibited the activation of X‐box binding protein 1 (Xbp1) and activating transcriptional factor 4 (ATF4) signaling pathways in NCMs. Significance: Our data demonstrate that miR‐297 promotes cardiomyocyte hypertrophy by inhibiting the expression of Sig‐1R and activation of ER stress signaling, which provides a novel interpretation for cardiac hypertrophy.

Critical role of X-box binding protein 1 in NADPH oxidase 4-triggered cardiac hypertrophy is mediated by receptor interacting protein kinase 1

ABSTRACT NADPH oxidase 4 (NOX4) and the NOX4-related redox signaling are implicated in cardiac hypertrophy. NOX4 is interrelated with endoplasmic reticulum stress (ERS). Spliced X-box binding protein 1 (Xbp1s) is a key mediator of ERS while its role in cardiac hypertrophy is still poorly understood. Recently, receptor interacting protein kinase 1(RIPK1) has been increasingly reported to be associated with ERS. Therefore, we aimed to test the hypothesis that Xbp1s mediates NOX4-triggered cardiac hypertrophy via RIPK1 signaling. In the heart tissue of transverse aortic constriction (TAC) rats and in primary cultured neonatal cardiomyocytes(NCMs) treated with angiotensinII(AngII) or isoproterenol (ISO), NOX4 expression and reactive oxygen species (ROS) generation, and expression of Xbp1s as well as RIPK1-related phosphorylation of P65 subunit of NF-κB were elevated. Gene silencing of NOX4 by specific small interfering RNA (siRNA) significantly blocked the upregulation of NOX4, generation of ROS, splicing of Xbp1 and activation of the RIPK1-related NF-κB signaling, meanwhile attenuated cardiomyocyte hypertrophy. In addition, ROS scavenger (N-acetyl-L-cysteine, NAC) and NOX4 inhibitor GKT137831 reduced ROS generation and alleviated activation of Xbp1 and RIPK1-related NF-κB signaling. Furthermore, splicing of Xbp1 was responsible for the increase in RIPK1 expression in AngII or ISO-treated NCMs. Upregulated RIPK1 in turn activates NF-κB signaling in a kinase activity-independent manner. These findings suggest that Xbp1s plays an important role in NOX4-triggered cardiomyocyte hypertrophy via activating its downstream effector RIPK1, which may prove significant for the development of future therapeutic strategies.

Inhibition of Receptor Interacting Protein Kinases Attenuates Cardiomyocyte Hypertrophy Induced by Palmitic Acid.

Palmitic acid (PA) is known to cause cardiomyocyte dysfunction. Cardiac hypertrophy is one of the important pathological features of PA-induced lipotoxicity, but the mechanism by which PA induces cardiomyocyte hypertrophy is still unclear. Therefore, our study was to test whether necroptosis, a receptor interacting protein kinase 1 and 3 (RIPK1 and RIPK3-) dependent programmed necrosis, was involved in the PA-induced cardiomyocyte hypertrophy. We used the PA-treated primary neonatal rat cardiac myocytes (NCMs) or H9c2 cells to study lipotoxicity. Our results demonstrated that cardiomyocyte hypertrophy was induced by PA treatment, determined by upregulation of hypertrophic marker genes and cell surface area enlargement. Upon PA treatment, the expression of RIPK1 and RIPK3 was increased. Pretreatment with the RIPK1 inhibitor necrostatin-1 (Nec-1), the PA-induced cardiomyocyte hypertrophy, was attenuated. Knockdown of RIPK1 or RIPK3 by siRNA suppressed the PA-induced myocardial hypertrophy. Moreover, a crosstalk between necroptosis and endoplasmic reticulum (ER) stress was observed in PA-treated cardiomyocytes. Inhibition of RIPK1 with Nec-1, phosphorylation level of AKT (Ser473), and mTOR (Ser2481) was significantly reduced in PA-treated cardiomyocytes. In conclusion, RIPKs-dependent necroptosis might be crucial in PA-induced myocardial hypertrophy. Activation of mTOR may mediate the effect of necroptosis in cardiomyocyte hypertrophy induced by PA.

MircoRNA-145 promotes activation of hepatic stellate cells via targeting krüppel-like factor 4

Krüppel-like Factor 4 (KLF4), a target gene of miR-145, can negatively regulate lung fibrosis. However, the potential role of KLF4 and miR-145 in hepatic stellate cells (HSCs) activation or in hepatic fibrosis keeps unclear. This study aims to characterize miR-145 and KLF4 in activated HSCs and liver cirrhotic, and the underlying molecular basis. miR-145 was significantly up-regulated, while KLF4 was dramatically down-regulated during the activation of rat primary HSCs and TGF-βtreated HSCs. Furthermore, miR-145 mimics induced and inhibition of miR-145 reduced α-SMA and COL-I expression in primary HSCs. Additionally, the mRNA and protein levels of KLF4 in the liver of cirrhotic patients and rats were significantly down-regulated. α-SMA and COL-I were increased after inhibition of KLF4 by specific shRNA in primary HSCs. Forced KLF4 expression led to a reduction of α-SMA and COL-I expression in HSCs. miR-145 promotes HSC activation and liver fibrosis by targeting KLF4.

HIF-1α mediates visfatin-induced CTGF expression in vascular endothelial cells.

Visfatin is an adipokine that functions as a mediator of endothelial dysfunction and cardiovascular diseases. Connective tissue growth factor (CTGF) is a key factor in vascular remodeling and atherosclerosis. However, the association between visfatin and CTGF is unclear. Therefore the study was to test the hypothesis that visfatin could modulate the expression of CTGF in vascular endothelial cells. In our study, cultured endothelial cell line EA.Hy926 cells were treated with different concentrations of visfatin for different times. The CTGF gene expression was analyzed by real-time PCR, and the protein expression of CTGF was assessed by Western Blot. The results showed that 100ng/mL concentration of visfatin could induce CTGF mRNA expression after 6 hours treatment, which peaked at 24 hours. And 100ng/mL concentration of visfatin also increased CTGF protein production after 12 hours treatment in EA.Hy926 cells. The expression of transforming growth factor-β1 (TGF-β1) mRNA was almost unaffected in cells treated with visfatin, whereas the expression of hypoxia inducible factor-1α (HIF-1α) was increased significantly. Moreover, knockdown of HIF-1α by its specific shRNA inhibits the effect of visfatin on CTGF expression. In conclusion, the up-regulation of CTGF expression by visfatin might be mediated via HIF-1α -dependent pathway, but not the TGF-β1 pathway in EA.Hy926 cells.

Role of microRNA-124 in cardiomyocyte hypertrophy inducedby angiotensin II.

Cardiac hypertrophy is a crucial predictor of heart failure and is regulated by microRNAs. MicroRNA-124 (miR-124) is regarded as a prognostic indicator for outcomes after cardiac arrest. However, whether miR-124 participates in cardiac hypertrophy remains unclear. Therefore, our study aimed to determine the role of miR-124 in angiotensin II(AngII)-induced myocardial hypertrophy and the possible mechanism. Primary cultured rat neonatal cardiomyocytes(NCMs) were transfected with miR-124 mimics or inhibitor, followed by AngII stimulation. Quantitative RT-PCR, western blot analysis and determination of cell surface area of NCMs were used to detect the hypertrophic phenotypes. We observed that miR-124 was elevated in AngII-induced hypertrophic cardiomyocytes. Cell surface area of NCMs and mRNA expression of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and β-myosin heavy chain (β-MHC), indicators of myocardial hypertrophy, were higher in NCMs transfected with miR-124 mimics in the presence of AngII. On the contrary, knockdown of miR-124 by its specific inhibitor could restore these courses. Furthermore, downregulation of miR-124 alleviated the increased protein level of endoplasmic reticulum (ER) stress markers 78-kDa glucose-regulated protein (Grp78) and calreticulin(CRT) in AngII-induced NCMs. In conclusion, our study shows that inhibition of miR-124 effectively suppresses AngII-induced myocardial hypertrophy, which is associated with attenuation of ER stress.

Silencing calreticulin gene might protect cardiomyocytes from angiotensin II-induced apoptosis

Aims Calreticulin (CRT), as a chaperone, contributes to protein folding and quality control cycle. CRT is an important factor regulating Ca2+ that participates in cell apoptosis. However, the function of CRT in the heart is still controversial. Therefore, we aimed to investigate the potential role of CRT in angiotensin II‐induced cardiomyocytes apoptosis. Main methods Primary cultured neonatal cardiomyocytes were stimulated with angiotensin II to induce the apoptosis. Expression of CRT and endoplasmic reticulum (ER) stress associated protein was detected by western blotting after angiotensin II stimulation for 24 h. The reactive oxygen species (ROS) production and mitochondrial membrane potential (MMP) were also detected. Additionally, the function of CRT on cardiomyocytes apoptosis and ER stress/unfolded protein response signaling pathway was investigated by transfecting specific CRT‐targeting siRNA. Key findings Cardiomyocytes apoptosis was induced by angiotensin II. The protein level of CRT was elevated after angiotensin ‐II stimulation for 24 h. Additionally, the protein levels of GRP78, ATF4, C‐ATF6, CHOP and the ROS production were elevated, but the Bcl‐2 expression and the level of MMP were down‐regulated. After silencing CRT gene in the process of angiotensin II‐induced cardiomyocytes apoptosis, cardiomyocytes apoptosis rate decreased, meanwhile the protein expression of CRT, GRP78, ATF4, C‐ATF6 and CHOP were down‐regulated. However, the Bcl‐2 expression was up‐regulated, and the increase of ROS and the loss of MMP were alleviated. Significance Our study demonstrated that CRT might protect cardiomyocytes from apoptosis induced by angiotensin II, in which ER stress and mitochondria function were identified as possible underlying molecular bases.

Inhibition of Nogo-B promotes cardiac hypertrophy via endoplasmic reticulum stress

AIMS Nogo-B is a key endoplasmic reticulum (ER) protein that regulates ER stress signaling. However, its role in cardiac hypertrophy remains poorly understood. ER stress is interrelated with autophagy in the process of cardiac hypertrophy. Therefore, we aimed to test the hypothesis that both ER stress and autophagy signaling mediate the function of Nogo-B in cardiac hypertrophy. MAIN METHODS Rat models of transverse aortic constriction (TAC), neonatal rat cardiomyocytes (NRCMs) stimulated with norepinephrine (Ne) and primary cardiac fibroblasts treated with transforming growth factor β1 (TGF-β1) were used in this study. The expression of Nogo-B and markers of ER stress were determined by quantitative RT-PCR, western blotting and immunofluorescence. Autophagy was measured by monitoring autophagic flux. Specific small interfering RNA (siRNA) of Nogo-B was transfected to investigate the role of Nogo-B in regulating cardiac hypertrophy. KEY FINDINGS In TAC-induced hypertrophic heart tissues, Ne-treated hypertrophic cardiomyocytes and TGF-β1-stimulated cardiac fibroblasts, the expression of Nogo-B, and markers of ER stress were significantly elevated. Impairment of autophagic flux was observed in the activated cardiac fibroblasts. Down-regulation of Nogo-B by siRNA further exacerbated Ne-induced cardiomyocyte hypertrophy and TGF-β1-induced cardiac fibroblast activation. Gene silencing of Nogo-B promoted the activation of the ER stress pathway and the impairment of autophagic flux. Moreover, inhibition of Nogo-B activated the protein kinase RNA-like ER kinase (PERK)/activating transcriptional factor 4 (ATF4) and activating transcriptional factor 6 (ATF6) branches of ER stress pathways. SIGNIFICANCE These findings suggest that inhibition of Nogo-B promotes cardiomyocyte hypertrophy and cardiac fibroblast activation by activating the PERK/ATF4 signaling pathway and defects of autophagic flux.

Involvement of TRPC1 in Nampt-induced cardiomyocyte hypertrophy through the activation of ER stress

Nicotinamide phosphoribosyltransferase (Nampt) is involved in the development of cardiac hypertrophy. Transient receptor potential canonical channel 1 (TRPC1) and endoplasmic reticulum stress (ER stress) are regarded as critical pathways in cardiac hypertrophy. Therefore, we hypothesizedthat TRPC1 might be associated with ER stress in Nampt-induced cardiac hypertrophy. CulturedH9c2cardiomyocyteswereexposed to Namptfor different timesand the expression of markers of cardiomyocyte hypertrophy and ER stress, as well as TRPC1 were detected. Moreover, specific TRPC1-shRNA (short hairpin RNA) expressing plasmid was transfected to knockdown TRPC1 expression before Nampt stimulation. Thapsigargin was used as an agonist and pravastatin was employed as an inhibitor of ER stress. The results demonstrated that exposure of H9c2 cells to 100 ng/mL Nampt for 24h, 48h or 72h significantly increased the expression of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), markers of ER stress and TRPC1. The Nampt-induced expression of TRPC1 was attenuated by pre-treatment with pravastatin, whereas promoted by pre-treatment with thapsigargin. Furthermore, transfection of TRPC1-shRNA for 48h partially inhibited Nampt-induced expression of ER stress markers and BNP in H9c2 cells. Our data suggest that TRPC1 might play an important role in cardiomyocyte hypertrophy induced by Namptinan ER stress-dependent way.