Yuqi Fan

The anti-malarial artemisinin inhibits pro-inflammatory cytokines via the NF-κB canonical signaling pathway in PMA-induced THP-1 monocytes

Several kinds of sesquiterpene lactones have been proven to inhibit NF-κB and to retard atherosclerosis by reducing lesion size and changing plaque composition. The anti-malarial artemisinin (Art) is a pure sesquiterpene lactone extracted from the Chinese herb Artemisia annua (qinghao, sweet wormwood). In the present study, we demonstrate that artemisinin inhibits the secretion and the mRNA levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6 in a dose-dependent manner in phorbol 12-myristate 13-acetate (PMA)-induced THP-1 human monocytes. We also found that the NF-κB specific inhibitor, Bay 11-7082, inhibited the expression of these pro-inflammatory cytokines, suggesting that the NF-κB pathway may be involved in the decreased cytokine release. At all time-points (1-6 h), artemisinin impeded the phosphorylation of IKKα/ß, the phosphorylation and degradation of IκBα and the nuclear translocation of the NF-κB p65 subunit. Additionally, artemisinin inhibited the translocation of the NF-κB p65 subunit as demonstrated by confocal laser scanning microscopic analysis and by NF-κB binding assays. Our data indicate that artemisinin exerts an anti-inflammatory effect on PMA-induced THP-1 monocytes, suggesting the potential role of artemisinin in preventing the inflammatory progression of atherosclerosis.

Iron deficiency activates pro-inflammatory signaling in macrophages and foam cells via the p38 MAPK-NF-κB pathway

BACKGROUND/OBJECTIVES Major bleeding in patients with acute coronary syndrome (ACS) increases the risk of recurrent ACS and mortality. However, the mechanism involved is poorly understood. Bleeding induces iron deficiency. Iron deficiency enhances inflammation in other diseases. Thus, in this paper, the particular effect of iron deficiency on atherosclerotic plaque destabilization, especially the pro-inflammatory role of iron deficiency in atheroma and the mechanism involved were investigated. METHODS Extracellular matrix metalloproteinase inducer (EMMPRIN) and matrix metalloproteinase-9 (MMP-9) mRNA levels were investigated by RT-PCR. EMMPRIN and MMP-9 protein levels, nuclear factor (NF)-κB-p65 protein levels, peroxisome proliferator-activated receptor γ (PPARγ) protein levels, and mitogen-activated protein kinase (MAPK) phosphorylation were determined by western blotting. MMP-9 enzymatic activity was assayed by gelatin zymography. RESULTS Iron deficiency enhanced EMMPRIN, MMP-9 production, and MMP-9 enzymatic activity in THP-1 derived macrophages and foam cells. Iron deficiency elicited the activation of NF-κB and p38 MAPK. By using the p38 inhibitor and NF-κB inhibitor, the study established that EMMPRIN and MMP-9 inductions by iron deficiency required the consecutive upstream activation of p38 MAPK and NF-κB. This pro-inflammatory action was not prevented by PPARγ agonist. Meanwhile, iron deficiency did not modulate PPARγ expression. Retinoid X receptor agonist suppressed the effects of iron deficiency on EMMPRIN , MMP-9, and NF-κB, but not on MAPK activation. CONCLUSIONS Iron deficiency enhances atheroma inflammation through p38 MAPK-NF-κB-EMMPRIN/MMP-9 pathway. Our findings provide a potential mechanism for the association of major bleeding with recurrent ACS and mortality in patients with ACS.

MicroRNA-9 Inhibits NLRP3 Inflammasome Activation in Human Atherosclerosis Inflammation Cell Models through the JAK1/STAT Signaling Pathway

Background/Aims: MicroRNA-9 (miR-9) is involved in inflammatory reaction in atherosclerosis; however, its function and regulatory mechanisms remain unclear. We aimed to uncover the exact roles of miR-9 and downstream signaling pathways using in vitro human atherosclerosis models. Methods: We used oxidized low-density lipoprotein (oxLDL)-stimulated human THP-1 derived macrophages, oxLDL-stimulated human primary peripheral blood monocytes and lipopolysaccharides (LPS) or Alum-stimulated human THP-1 derived macrophages as in vitro atherosclerosis inflammation models. Transient transfection of over-expression vectors, small interference RNAs (siRNAs) or antisense oligonucleotides was used to regulate intracellular protein or miR-9 levels. Cell responses and signal transduction were detected by multiple assays including Western blotting, enzyme-linked immunosorbent assay (ELISA) and luciferase reporter assay. Results: MiR-9 inhibited while anti-miR-9 antisense oligonucleotides induced interleukin-1 beta (IL-1β) and NLRP3 inflammasome activation in all in vitro models. Janus kinase 1 (JAK1) and matrix metalloproteinase 13 (MMP-13) were identified as the target genes of miR-9. In oxLDL-stimulated human THP-1 derived macrophages, knockdown of JAK1 by siRNA blocked the phosphorylation of signal transducer and activator of transcription 1 (STAT1) and mimicked the effects of miR-9. In the same model, JAK1 knockdown blocked the phosphorylation of NF-κB p65 in the nuclei and the phosphorylation of NF-κB IκBα in the cytoplasm. Conclusions: Our study demonstrated that miR-9 could inhibit activation of the NLRP3 inflammasome and attenuate atherosclerosis-related inflammation, likely through the JAK1/STAT1 signaling pathway. Therefore, miR-9 may serve as a potential therapeutic target for atherosclerosis.

Long-term prescription of beta-blocker delays the progression of heart failure with preserved ejection fraction in patients with hypertension: A retrospective observational cohort study:

Background Hypertension complicated with left ventricular hypertrophy (LVH) and diastolic dysfunction is one of the most common risks for heart failure with preserved ejection fraction (HFpEF). This study was designed to evaluate the influences of long-term beta-blocker prescription in these patients. Methods This retrospective analysis included eligible patients diagnosed with hypertension, LVH (left ventricular (LV) mass index >125 g/m2 for men and >110 g/m2 for women) and suspected diastolic dysfunction (E/E’ ratio between 8 and 15) and without clinical signs or symptoms of heart failure in our hospital medical record database (January 2005–December 2009). A total of eligible 1498 patients were enrolled, of whom 803 received beta-blocker prescription and 695 accepted non-beta-blocker therapy. Results With a median follow-up of 7.2 years, the new-onset symptomatic HFpEF occurred in 48 of 803 patients in the beta-blocker group (6.0%) and 92 of 695 patients in the non-beta-blocker group (13.2%, p < 0.001). Beta-blockers also generated more prominent improvement in diastolic function and LVH. And Cox proportional hazards model revealed that beta-blocker (hazard ratio (HR) 0.327, 95% confidence interval (CI): 0.121–0.540, p = 0.009) or angiotensin-converting enzyme inhibitor/angiotensin II receptor blocker (ACEI/ARB) exposure (HR 0.422, 95% CI: 0.210–0.699, p = 0.015) was associated with a reduced risk of new onset of symptomatic HFpEF, and the elevation of LVMI (HR 1.210, 95% CI: 1.069–1.362, p = 0.040) or E/E’ (HR 1.398, 95% CI: 1.306–1.541, p = 0.032) was associated with a high risk of new onset of symptomatic HFpEF. Conclusions Long-term beta-blocker exposure was associated with protective effects in terms of the incidence of new-onset symptomatic HFpEF, LV diastolic dysfunction and LVH, which might be beneficial for the delay of HFpEF progression.

Artemisinin inhibits monocyte adhesion to HUVECs through the NF-κB and MAPK pathways in vitro

The adhesion of monocytes to human umbilical vein endothelial cells (HUVECs) plays a crucial role in the initiation of atherosclerosis. Intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) are two important molecules involved in the adhesion of monocytes to HUVECs. Previous studies have suggested that artemisinin, apart from an anti-malarial agent, also has other effects. In the present study, we found that artemisinin significantly decreased the adhesion of monocytes to tumor necrosis factor-α (TNF-α)-stimulated HUVECs in a dose-dependent manner and suppressed the mRNA and protein level of ICAM-1 and VCAM-1 in the TNF-α-stimulated HUVECs. In addition, the nuclear factor-κB (NF-κB) inhibitor, Bay 11-7082, and mitogen-activated protein kinase (MAPK) inhibitors (SB203580 and U0126) respectively reduced the adhesion of monocytes to TNF-α-stimulated HUVECs, and suppressed ICAM-1 and VCAM-1 expression in TNF-α stimulated HUVECs. Moreover, artemisinin impeded the activation of the NF-κB and MAPK signaling pathways. Furthermore, Bay 11-7082 significantly decreased the phosphorylation of levels extracellular signal-regulated protein kinase (ERK)1/2, p38 and c-Jun N-terminal kinase (JNK). Taken together, the findings of our study indicated that artemisinin blocked monocyte adhesion to TNF-α-stimulated to HUVECs by downregulating ICAM-1 and VCAM-1 expression in the TNF-α-stimulated HUVECs. Artemisinin may thus have potential for use in the protection against the early development of atherosclerotic lesions.

Optimizing methods for the study of intravascular lipid metabolism in zebrafish

The zebrafish (Danio rerio) is a useful vertebrate model for use in cardiovascular drug discovery. The present study aimed to construct optimized methods for the study of intravascular lipid metabolism of zebrafish. The lipophilic dye, Oil Red O, was used to stain fasting zebrafish one to eight days post-fertilization (dpf) and to stain 7-dpf zebrafish incubated in a breeding system containing 0.1% egg yolk as a high-fat diet (HFD) for 48 h. Three-dpf zebrafish were kept in CholEsteryl boron-dipyrromethene (BODIPY) 542/563 C11 water for 24 h which indicated the efficiency of CholEsteryl BODIPY 542/563 C11 intravascular cholesterol staining. Subsequently, 7-dpf zebrafish were incubated in water containing the fluorescent probe CholEsteryl BODIPY 542/563 C11 and fed a high-cholesterol diet (HCD) for 10 d. Two groups of 7-dpf zebrafish were incubated in regular breeding water and fed with a regular or HCD containing CholEsteryl BODIPY 542/563 C11 for 10 d. Finally, blood lipids of adult zebrafish fed with regular or HFD for seven weeks were measured. Oil Red O was not detected in the blood vessels of 7-8-dpf zebrafish. Increased intravascular lipid levels were detected in 7-dpf zebrafish incubated in 0.1% egg yolk, indicated by Oil Red O staining. Intravascular cholesterol was efficiently stained in 3-dpf zebrafish incubated in breeding water containing CholEsteryl BODIPY 542/563 C11; however, this method was inappropriate for the calculation of intravascular fluorescence intensity in zebrafish >7‑dpf. In spite of this, intra-aortic fluorescence intensity of zebrafish fed a HCD containing CholEsteryl BODIPY 542/563 C11 was significantly higher (P<0.05) than that of those fed a regular diet containing CholEsteryl BODIPY 542/563 C11. The serum total cholesterol and triglyceride levels of adult zebrafish fed a HFD were markedly increased compared to those of the control group (P<0.05). In conclusion, the use of Oil Red O staining and CholEsteryl BODIPY 542/563 C11 may have applications in zebrafish intravascular lipid metabolism research and screens for novel lipid-regulating drugs.

Non-Invasive Assessment of Early Atherosclerosis Based on New Arterial Stiffness Indices Measured with an Upper-Arm Oscillometric Device

The clinical significance of detecting early atherosclerosis is now widely recognized. Measurement methods available at present are usually not suitable for use in primary care where rapid screening for a large population is needed. The Arterial Velocity-pulse Index (AVI) and Arterial Pressure-volume Index (API) are new noninvasive arterial stiffness indices that can be rapidly measured using an oscillometric device. The purpose of this study was to determine whether high AVI and API values are predictive of early atherosclerosis prior to the onset of obstructive coronary artery disease (CAD). A total of 183 patients were enrolled and allocated to the CAD group (n = 109), early atherosclerosis (AS) group (n = 34) or an apparently healthy (non-AS) group (n = 40) based on the results of angiographic examinations. Measurements for arterial blood pressure, AVI, API and brachial-ankle pulse wave velocity (baPWV) were collected. Statistical analyses revealed that AVIs were significantly lower in the non-AS group than in the AS group and the CAD group. The inter-group differences in API were not statistically significant among the 3 patient groups. As a reference, baPWV was found to be statistically higher in the CAD group than in the non-AS group, whereas there was no significant difference between the CAD group and the AS group, or between the AS group and the non-AS group. The AVI and API were both significantly correlated with baPWV. This study demonstrated that AVI was more sensitive than baPWV and API in indicating early atherosclerosis, although elevated AVI and baPWV were both predictive of CAD.

Galectin-3-induced oxidized low-density lipoprotein promotes the phenotypic transformation of vascular smooth muscle cells

Oxidized low-density lipoprotein (oxLDL) is involved in the pathological phenotypic transformation of vascular smooth muscle cells in atherosclerosis. Galectin-3 also has an important role in atherosclerosis. However, little is currently known regarding the effects of galectin-3 on the oxLDL-induced phenotypic transformation of vascular smooth muscle cells. In the present study, primary culture human umbilical vascular smooth muscle cells were treated with various oxLDL concentrations (0-50 μg/ml) for 72 h, and phenotypic changes were subsequently recorded. The results of the present study suggested that oxLDL increases the expression levels of galectin-3, and induces the phenotypic transformation of vascular smooth muscle cells. The oxLDL-induced cells exhibited increased expression levels of osteopontin, a smooth muscle synthetic protein, and calponin and α-actin, smooth muscle contractile proteins. The oxLDL-induced changes in cellular phenotype were associated with increased migration, proliferation, and phagocytosis. Concordant with these results, oxLDL-treated smooth muscle cells exhibited activation of canonical Wnt signaling, as determined by an increase in the protein expression levels of β-catenin. Silencing of galectin-3 by small interfering RNA reversed the phenotypic transformation and functional changes observed in the oxLDL-treated cells, suggesting these changes were dependent on the activation of galectin-3. In addition, galectin-3 knockdown decreased the protein expression levels of β-catenin in both the cytoplasm and nucleus; however, the mRNA expression levels of β-catenin remained unchanged. These results suggest that galectin-3 is responsible for the phenotypic transformation of human umbilical vascular smooth muscle cells, and the canonical Wnt/β-catenin signaling pathway may be involved in this process.

Serum uric acid is associated with incidence of heart failure with preserved ejection fraction and cardiovascular events in patients with arterial hypertension

The objective of the present study was to evaluate clinical implications of serum uric acid (UA) on the progression of heart failure with preserved ejection fraction (HFpEF) in hypertensive patients. A total of 1009 adult patients with left ventricular hypertrophy and suspected left ventricular diastolic dysfunction were enrolled at our hospital from January 2008 to December 2011. With a median follow‐up of 7.2 years, 136 (13.2%) patients developed new‐onset HFpEF and 151 (15.0%) had major adverse cardiovascular events (MACEs). Compared with the lowest UA tertile of UA (<302 μmol L−1), subjects in the highest tertile (>367 μmol L−1) had a higher risk of developing new‐onset HFpEF (HR: 1.761, 95% CI: 1.119‐2.772, P = .015) as well as MACEs (HR: 1.664, 95% CI: 1.086‐2.547, P = .019). Our findings indicate that hyperuricemia is associated with detrimental effects in terms of the incidence of new‐onset HFpEF as well as MACEs in hypertensive patient.

Resveratrol suppresses doxorubicin-induced cardiotoxicity by disrupting E2F1 mediated autophagy inhibition and apoptosis promotion

Graphical abstract Figure. No Caption available. Abstract The clinical use of doxorubicin (DOX) is limited by cardiotoxicity, involving the dysregulation of autophagy and apoptosis in the myocardium, which were partly reversed by resveratrol (RSV) supplement. However, a definitive mechanisms accounting for DOX’s cardiotoxicity and the protective role of RSV remain poorly defined. The aim of the present study was to clarify the specific role of E2F transcription factor 1 (E2F1) in regulating autophagy and apoptosis in DOX‐induced cardiotoxicity as well as the protective effects of RSV. Autophagy and apoptosis were successfully induced by the serum deprivation strategy in H9c2 cardiomyocytes. DOX inhibited autophagy through activating E2F1/mammalian target of rapamycin complex 1 (mTORC1) pathway and further induced apoptosis by activating E2F1/AMP‐activated protein kinase &agr;2 (AMPK&agr;2) pathway in starved H9C2 cells. And RSV supplement showed increased autophagy and decreased apoptosis, accompanied with inhibitory effect on E2F1/mTORC1 as well as E2F1/AMPK&agr;2 pathway. Moreover, the favorable effect of RSV on autophagy and apoptosis was dependent on E2F1. The same result was observed in the mouse model of DOX‐induced cardiotoxicity in both non‐myocardial ischemia and myocardial ischemia condition. Injection with DOX and RSV in combination, resulted in a reduced apoptotic ratio and activated autophagy process compared with the DOX treatment alone. In conclusions, RSV, which has been suggested to attenuate DOX‐induced cytotoxicity, significantly blocks induction of E2F1/mTORC1 and E2F1/AMPK&agr;2 pathway by DOX, leading to acceleratory autophagy and inhibitory apoptosis. And E2F1 plays a key role for the protective effect of RSV.