ZNF667 Suppressed LPS-induced Macrophages Inflammation through mTOR-dependent Aerobic Glycolysis Regulation.

BACKGROUND: Macrophages participate in all stages of the inflammatory response, and the excessive release of inflammatory mediators and other cytokines synthesized and secreted by macrophages is fundamentally linked to an uncontrolled inflammatory response. The zinc finger 667 (ZNF667) protein, a novel DNAbinding protein, has been shown to play a vital role in oxidative stress. However, none of the target genes in macrophages or the potential roles of ZNF667 have been elucidated to date. > Objectives: The present study was designed to investigate the effects of ZNF667 on LPS-induced inflammation in macrophages. > Methods: The RAW264.7 macrophage cell line was selected as a model system. Inflammatory response-related gene expression levels and phosphorylation levels of PI3K, AKT, and mTOR were detected in LPS-treated macrophages via RT-PCR and western blotting, respectively. > Results: We found that LPS resulted in the up-regulation of ZNF667 in macrophages and a peak response in ZNF667 protein expression levels when used at a concentration of 100 ng/mL. ZNF667 overexpression significantly inhibited the LPS-induced up-regulation of iNOS, and IL-1ß mRNA and protein expression levels, together with the secretion of IL-1ß, IL-6, and TNF-α. ZNF667 overexpression also inhibited PI3K, AKT, and mTOR hyperphosphorylation and had no effect on the phosphorylation of NF-κB p65, ERK1/2, MAPK p38, and the transcriptional activity of NF-κB in macrophages. The up-regulation of ZNF667 inhibited the levels of expression of HK2 and PFKFB3, glucose consumption, and lactate production in LPS-stimulated macrophages. The up-regulation of mRNA levels of LPS-induced glycolytic genes HK2 and PFKFB3 and the increased mRNA expression of pro-inflammatory cytokines (IL-1ß and iNOS) were abolished by hexokinase inhibitor 2-DG in ZNF667-deficient macrophages. Meanwhile, glucose consumption and lactate production were abrogated in macrophages when cells were treated with the specific mTOR inhibitor RPM. > Conclusion: Our results demonstrate that ZNF667 suppressed LPS-stimulated RAW264.7 macrophage inflammation by regulating mTOR-dependent aerobic glycolysis.>.

Hydrogen Sulfide Ameliorates Angiotensin II-Induced Atrial Fibrosis Progression to Atrial Fibrillation Through Inhibition of the Warburg Effect and Endoplasmic Reticulum Stress.

Atrial fibrosis is the basis for the occurrence and development of atrial fibrillation (AF) and is closely related to the Warburg effect, endoplasmic reticulum stress (ERS) and mitochondrion dysfunctions-induced cardiomyocyte apoptosis. Hydrogen sulfide (H2S) is a gaseous signalling molecule with cardioprotective, anti-myocardial fibrosis and improved energy metabolism effects. Nevertheless, the specific mechanism by which H2S improves the progression of atrial fibrosis to AF remains unclear. A case-control study of patients with and without AF was designed to assess changes in H2S, the Warburg effect, and ERS in AF. The results showed that AF can significantly reduce cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate thiotransferase (3-MST) expression and the H2S level, induce cystathionine-ß-synthase (CBS) expression; increase the Warburg effect, ERS and atrial fibrosis; and promote left atrial dysfunction. In addition, AngII-treated SD rats had an increased Warburg effect and ERS levels and enhanced atrial fibrosis progression to AF compared to wild-type SD rats, and these conditions were reversed by sodium hydrosulfide (NaHS), dichloroacetic acid (DCA) or 4-phenylbutyric acid (4-PBA) supplementation. Finally, low CSE levels in AngII-induced HL-1 cells were concentration- and time-dependent and associated with mitochondrial dysfunction, apoptosis, the Warburg effect and ERS, and these effects were reversed by NaHS, DCA or 4-PBA supplementation. Our research indicates that H2S can regulate the AngII-induced Warburg effect and ERS and might be a potential therapeutic drug to inhibit atrial fibrosis progression to AF.

Hydrogen sulfide improves ox­LDL­induced expression levels of Lp­PLA2 in THP­1 monocytes via the p38MAPK pathway.

Hydrogen sulfide (H2S) exerts an anti­atherosclerotic effect and decreases foam cell formation. Lipoprotein­associated phospholipase A2 (Lp­PLA2) is a key factor involved in foam cell formation. However, the association between H2S and Lp­PLA2 expression levels with respect to foam cell formation has not yet been elucidated. The present study investigated whether H2S can affect foam cell formation and potential signalling pathways via regulation of the expression and activity of Lp­PLA2. Using human monocytic THP­1 cells as a model system, it was observed that oxidized low­density lipoprotein (ox­LDL) not only upregulates the expression level and activity of Lp­PLA2, it also downregulates the expression level and activity of Cystathionine γ lyase. Exogenous supplementation of H2S decreased the expression and activity of Lp­PLA2 induced by ox­LDL. Moreover, ox­LDL induced the expression level and activity of Lp­PLA2 via activation of the p38MAPK signalling pathway. H2S blocked the expression levels and activity of Lp­PLA2 induced by ox­LDL via inhibition of the p38MAPK signalling pathway. Furthermore, H2S inhibited Lp­PLA2 activity by blocking the p38MAPK signaling pathway and significantly decreased lipid accumulation in ox­LDL­induced macrophages, as detected by Oil Red O staining. The results of the present study indicated that H2S inhibited ox­LDL­induced Lp­PLA2 expression levels and activity by blocking the p38MAPK signalling pathway, thereby improving foam cell formation. These findings may provide novel insights into the role of H2S intervention in the progression of atherosclerosis.

Macrophage polarization in atherosclerosis.

Atherosclerosis is a chronic inflammatory response that increases the risk of cardiovascular diseases. An in-depth study of the pathogenesis of atherosclerosis is critical for the treatment of atherosclerotic cardiovascular disease. The development of atherosclerosis involves many cells, such as endothelial cells, vascular smooth muscle cells, macrophages, and others. The considerable effects of macrophages in atherosclerosis are inextricably linked to macrophage polarization and the resulting phenotype. Moreover, the significant impact of macrophages on atherosclerosis depend not only on the function of the different macrophage phenotypes but also on the relative ratio of different phenotypes in the plaque. Research on atherosclerosis therapy indicates that the reduced plaque size and enhanced stability are partly due to modulating macrophage polarization. Therefore, regulating macrophage polarization and changing the proportion of macrophage phenotypes in plaques is a new therapeutic approach for atherosclerosis. This review provides a new perspective for atherosclerosis therapy by summarizing the relationship between macrophage polarization and atherosclerosis, as well as treatment targeting macrophage polarization.

Regulating the Warburg effect on metabolic stress and myocardial fibrosis remodeling and atrial intracardiac waveform activity induced by atrial fibrillation.

Atrial fibrillation (AF) is associated with metabolic stress and induces myocardial fibrosis reconstruction by increasing glycolysis. One goal in the treatment of paroxysmal AF (p-AF) is to improve myocardial fibrosis reconstruction and myocardial metabolic stress caused by the Warburg effect. Adopted male canine that rapid right atrial pacing (RAP) for 6 days to establish a p-AF model. The canines were pre-treated with phenylephrine (PE) or dichloroacetic acid (DCA) before exposure to p-AF or non-p-AF. P-wave duration (Pmax), minimum P-wave duration (Pmin), P wave dispersion (PWD), atrial effective refractory period (AERP) and AERP dispersion (AERPd) were measured in canine atrial cardiomyocytes. Pyruvate dehydrogenase kinase-1 (PDK-1), PDK-4, lactate dehydrogenase A (LDHA), pyruvate dehydrogenase (PDH), citrate synthase (CS), isocitrate dehydrogenase (IDH), and matrix metalloproteinase 9 (MMP-9) were evaluated by western blotting and reverse transcription polymerase chain reaction (RT-PCR), content of adenosine monophosphate (AMP), adenosine triphosphate (ATP), lactic acid and glycogen, and activity of LDHA, PDK-1 and PDK-4 were evaluated by enzyme-linked immunosorbent assay (ELISA), myocardial tissue glycogen content was evaluated by PAS, myocardial fibrosis remodeling was evaluated by hematoxylin and eosin (H&E) and Masson staining. Our findings demonstrated that p-AF increases the Warburg effect-related metabolic stress and myocardial fibrosis remodeling by increasing the expression and activity of PDK-1, PDK-4, and LDHA, content of AMP and lactic acid, and the ratio of AMP/ATP and decreasing the expression of PDH, CS, and IDH, and glycogen content. In addition, p-AF can induce cardiomyocyte fibrosis remodeling and increase MMP-9 expression, and p-AF also increases atrial intracardiac waveform activity by prolonging Pmax, Pmin, PWD, and AERPd and shortening AERP. PDK isoforms agonists (PE) produce a similar p-AF pathological effect and can produce synergistic effects with p-AF, further increasing Warburg effect-related metabolic stress, myocardial fibrosis remodeling, and atrial intracardiac waveform activity. In contrast, the use of PDK-specific inhibitors (DCA) completely reverses these pathophysiological changes induced by p-AF. We demonstrate that p-AF can induce the Warburg effect in canine atrial cardiomyocytes and significantly improve p-AF-induced metabolic stress, myocardial fibrosis remodeling, and atrial intracardiac waveform activity by inhibiting the Warburg effect.

Fibroblast growth factor-21 alleviates hypoxia/reoxygenation injury in H9c2 cardiomyocytes by promoting autophagic flux.

Fibroblast growth factor (FGF)­21, a member of the family of FGFs, exhibits protective effects against myocardial ischemia and ischemia/reperfusion injury; it is also an enhancer of autophagy. However, the mechanisms underlying the protective role of FGF­21 against cardiomyocyte hypoxia/reoxygenation (H/R) injury remain unclear. The present study aimed to investigate the effect of FGF­21 on H9c2 cardiomyocyte injury induced by H/R and the mechanism associated with changes in autophagy. Cultured H9c2 cardiomyocytes subjected to hypoxia were treated with a vehicle or FGF­21 during reoxygenation. The viability of H9c2 rat cardiomyocytes was measured using Cell Counting Kit­8 and trypan blue exclusion assays. The contents of creatine kinase (CK) and creatine kinase isoenzymes (CK­MB), cardiac troponin I (cTnT), cardiac troponin T (cTnI) and lactate dehydrogenase (LDH) in culture medium were detected with a CK, CK­MB, cTnT, cTnI and LDH assay kits. The protein levels were examined by western blot analysis. Autophagic flux was detected by Ad­mCherry­GFP­LC3B autophagy fluorescent adenovirus reagent. The results indicated that FGF­21 alleviated H/R­induced H9c2 myocardial cell injury and enhanced autophagic flux during H/R, and that this effect was antagonized by co­treatment with 3­methyladenine, an autophagy inhibitor. Furthermore, FGF­21 increased the expression levels of Beclin­1 and Vps34 proteins, but not of mechanistic target of rapamycin. These data indicate that FGF­21 treatment limited H/R injury in H9c2 cardiomyocytes by promoting autophagic flux through upregulation of the expression levels of Beclin­1 and Vps34 proteins.

Endothelial to mesenchymal transition in atherosclerotic vascular remodeling.

Endothelial cells are the main components of the heart, blood vessels, and lymphatic vessels, which play an important role in regulating the physiological functions of the cardiovascular system. Endothelial dysfunction is involved in a variety of acute and chronic cardiovascular diseases. As a special type of epithelial-mesenchymal transition (EMT), endothelium to mesenchymal transition (EndMT) regulates the transformation of endothelial cells into mesenchymal cells accompanied by changes in the expression of various transcription factors and cytokines, which is closely related to vascular endothelial injury, vascular remodeling, myocardial fibrosis and valvar disease. Endothelial cells undergoing EndMT lose their endothelial characteristics and undergo a transition toward a more mesenchymal-like phenotype. However, the molecular mechanism of EndMT remains unclear. EndMT, as a type of endothelial dysfunction, can cause vascular remodeling which is a major determinant of atherosclerotic luminal area. Therefore, exploring the important signaling pathways in the process of EndMT may provide novel therapeutic strategies for treating atherosclerotic diseases.

Role of autophagy in advanced atherosclerosis (Review).

Atherosclerosis (AS) remains the leading cause for global cardiovascular disease morbidity and mortality, and a major cause of cardiopathy, myocardial infarction and peripheral vascular diseases. Macrophages serve a critical role in atherosclerotic plaque stabilization and rupture, and the selective removal of macrophages may be beneficial in improving plaque stability. Autophagy is a process of self­feeding, during which cytoplasmic proteins or organelles are packaged into vesicles and fused with the lysosome to form an autophagosome. The newly formed autophagosome can degrade internalized proteins, and this process may be used to serve the metabolic and self­renewal requirements of the cell. Autophagy serves an important role in maintaining cell homeostasis and promoting cell survival, and therefore an imbalance in autophagy is closely associated with multiple diseases.

PI3K/Akt/FoxO3a signaling mediates cardioprotection of FGF-2 against hydrogen peroxide-induced apoptosis in H9c2 cells.

Cardiovascular disease is a growing major global public health problem. Oxidative stress is regarded as one of the key regulators of pathological physiology, which eventually leads to cardiovascular disease. However, mechanisms by which FGF-2 rescues cells from oxidative stress damage in cardiovascular disease is not fully elucidated. Herein this study was designed to investigate the protective effects of FGF-2 in H2O2-induced apoptosis of H9c2 cardiomyocytes, as well as the possible signaling pathway involved. Apoptosis of H9c2 cardiomyocytes was induced by H2O2 and assessed using methyl thiazolyl tetrazolium assay, Hoechst, and TUNEL staining. Cells were pretreated with PI3K/Akt inhibitor LY294002 to investigate the possible PI3K/Akt pathways involved in the protection of FGF-2. The levels of p-Akt, p-FoxO3a, and Bim were detected by immunoblotting. Stimulation with H2O2 decreased the phosphorylation of Akt and FoxO3a, and induced nuclear localization of FoxO3a and apoptosis of H9c2 cells. These effects of H2O2 were abrogated by pretreatment with FGF-2. Furthermore, the protective effects of FGF-2 were abolished by PI3K/Akt inhibitor LY294002. In conclusion, our data suggest that FGF-2 protects against H2O2-induced apoptosis of H9c2 cardiomyocytes via activation of the PI3K/Akt/FoxO3a pathway.

Janus-like role of fibroblast growth factor 2 in arteriosclerotic coronary artery disease: atherogenesis and angiogenesis.

Angiogenic stimulation is a promising new strategy for treating patients with arteriosclerotic coronary artery disease. This strategy aims to ameliorate cardiac function by improving myocardial perfusion and lowering the risk of myocardial infarction. However, angiogenesis may contribute to the growth of atherosclerotic lesions. Atherogenesis is also a potential side effect of angiogenic therapy. Early clinical trials were performed using fibroblast growth factor 2 (FGF2) protein, which enhances the formation of new collateral vessels to reduce ischaemic symptoms. Conversely, angiogenic stimulation by FGF2 is a dilemma because it could cause negative angiogenic effects, such as atherosclerosis. Thus far, clinical trials in patients with recombinant FGF2 protein therapy have not yet yielded undisputable beneficial effects. Future trials should determine whether an improvement can be obtained in patients with coronary artery disease using a combination of FGF2 and other growth factors or a combination of the FGF2 gene and stem cell therapy. This review summarises the multiple roles of FGF2 in the progression of atherosclerosis, its effect on pro-angiogenesis and improvement of cardiac function in coronary artery disease, and the potentially unfavourable effect of angiogenesis on the prevention and treatment of atherogenesis.

Hydrogen sulfide, the next potent preventive and therapeutic agent in aging and age-associated diseases.

Hydrogen sulfide (H(2)S) is the third endogenous signaling gasotransmitter, following nitric oxide and carbon monoxide. It is physiologically generated by cystathionine-γ-lyase, cystathionine-ß-synthase, and 3-mercaptopyruvate sulfurtransferase. H(2)S has been gaining increasing attention as an important endogenous signaling molecule because of its significant effects on the cardiovascular and nervous systems. Substantial evidence shows that H(2)S is involved in aging by inhibiting free-radical reactions, activating SIRT1, and probably interacting with the age-related gene Klotho. Moreover, H(2)S has been shown to have therapeutic potential in age-associated diseases. This article provides an overview of the physiological functions and effects of H(2)S in aging and age-associated diseases, and proposes the potential health and therapeutic benefits of H(2)S.