SIRT3 suppression resulting from the enhanced ß-catenin signaling drives glycolysis and promotes hypoxia-induced cell growth in hepatocellular carcinoma cells.

The precise mechanisms underlying the inhibitory effects of SIRT3, a mitochondrial sirtuin protein, on hepatocellular carcinoma (HCC) development, as well as its impact on mitochondrial respiration, remain poorly understood. We assessed sirtuins 3 (SIRT3) levels in HCC tissues and Huh7 cells cultured under hypoxic condition. We investigated the effects of SIRT3 on cell proliferation, glycolytic metabolism, mitochondrial respiration, mitophagy, and mitochondrial biogenesis in Huh7 cells. Besides, we explored the potential mechanisms regulating SIRT3 expression in hypoxically cultured Huh7 cells. Gradual reduction in SIRT3 expressions were observed in both adjacent tumor tissues and tumor tissues. Similarly, SIRT3 expressions were diminished in Huh7 cells cultured under hypoxic condition. Forced expression of SIRT3 attenuated the growth of hypoxically cultured Huh7 cells. SIRT3 overexpression led to a decrease in extracellular acidification rate while increasing oxygen consumption rate. SIRT3 downregulated the levels of hexokinase 2 and pyruvate kinase M2. Moreover, SIRT3 enhanced mitophagy signaling, as indicated by mtKeima, and upregulated key proteins involved in various mitophagic pathways while reducing intracellular reactive oxygen species levels. Furthermore, SIRT3 increased proxisome proliferator-activated receptor-gamma coactivator 1α levels and the amount of mitochondrial DNA in Huh7 cells. Notably, ß-catenin expressions were elevated in Huh7 cells cultured under hypoxic condition. Antagonists and agonists of ß-catenin respectively upregulated and downregulated SIRT3 expressions in hypoxically cultured Huh7 cells. The modulationsof glycolysis and mitochondrial respiration represent the primary mechanism through which SIRT3, suppressed by ß-catenin, inhibits HCC cell proliferation.

IL-33 promotes IL-10 production in macrophages: a role for IL-33 in macrophage foam cell formation.

We evaluated the role of IL-10- in IL-33-mediated cholesterol reduction in macrophage-derived foam cells (MFCs) and the mechanism by which IL-33 upregulates IL-10. Serum IL-33 and IL-10 levels in coronary artery disease patients were measured. The effects of IL-33 on intra-MFC cholesterol level, IL-10, ABCA1 and CD36 expression, ERK 1/2, Sp1, STAT3 and STAT4 activation, and IL-10 promoter activity were determined. Core sequences were identified using bioinformatic analysis and site-specific mutagenesis. The serum IL-33 levels positively correlated with those of IL-10. IL-33 decreased cellular cholesterol level and upregulated IL-10 and ABCA1 but had no effect on CD36 expression. siRNA-IL-10 partially abolished cellular cholesterol reduction and ABCA1 elevation by IL-33 but did not reverse the decreased CD36 levels. IL-33 increased IL-10 mRNA production but had little effect on its stability. IL-33 induced ERK 1/2 phosphorylation and increased the luciferase expression driven by the IL-10 promoter, with the highest extent within the -2000 to -1752 bp segment of the 5'-flank of the transcription start site; these effects were counteracted by U0126. IL-33 activated Sp1, STAT3 and STAT4, but only the STAT3 binding site was predicted in the above segment. Site-directed mutagenesis of the predicted STAT3-binding sites (CTGCTTCCTGGCAGCAGAA→︀CTGCCTGGCAGCAGAA) reduced luciferase activity, and a STAT3 inhibitor blocked the regulatory effects of IL-33 on IL-10 expression. Chromatin immunoprecipitation (CHIP) confirmed the STAT3-binding sequences within the -1997 to -1700 and -1091 to -811 bp locus regions. IL-33 increased IL-10 expression in MFCs via activating ERK 1/2 and STAT3, which subsequently promoted IL-10 transcription and thus contributed to the beneficial effects of IL-33 on MFCs.

Macrophage-derived foam cells impair endothelial barrier function by inducing endothelial-mesenchymal transition via CCL-4.

Recently, endothelial-mesenchymal transition (EndMT) has been demonstrated to play an important role in the development of atherosclerosis, the molecular mechanisms of which remain unclear. In the present study, scanning electron microscopy directly revealed a widened endothelial space and immunohistofluorescence demonstrated that EndMT was increased in human aorta atherosclerotic plaques. M1 macrophage-derived foam cell (M1-FC) supernatants, but not M2 macrophage-derived foam cell (M2-FC) supernatants, induced EndMT. A protein array and enzyme-linked immunosorbent assay identified that the levels of several cytokines, including C-C motif chemokine ligand 4 (CCL-4) were increased in M1-FC supernatants, in which EndMT was promoted, accompanied by increased endothelial permeability and monocyte adhesion. Furthermore, anti-CCL-4 antibody abolished the effects of M1-FC supernatants on EndMT. At the same time, CCL-4 activated its receptor, C-C motif chemokine receptor-5 (CCR-5), and upregulated transforming growth factor-ß (TGF-ß) expression. Further experiments revealed that EndMT induced by CCL-4 was reversed by treatment with CCR-5 antagonist and the RNA-mediated knockdown of TGF-ß. On the whole, the data of the present study suggest that M1-FCs induce EndMT by upregulating CCL-4, and increase endothelial permeability and monocyte adhesion. These data may help to elucidate the important role of EndMT in the development of atherosclerosis.

Atorvastatin Attenuates TNF-alpha Production via Heme Oxygenase-1 Pathway in LPS-stimulated RAW264.7 Macrophages.

OBJECTIVE: To assess the effect of atorvastatin on lipopolysaccharide (LPS)-induced TNF-α production in RAW264.7 macrophages. METHODS: RAW264.7 macrophages were treated in different LPS concentrations or at different time points with or without atorvastatin. TNF-α level in supernatant was measured. Expressions of TNF-α mRNA and protein and heme oxygenase-1 (HO-1) were detected by ELISA, PCR, and Western blot, respectively. HO activity was assayed. RESULTS: LPS significantly increased the TNF-α expression and secretion in a dose- and time-dependent manner. The HO-1 activity and HO-1 expression level were significantly higher after atorvastatin treatment than before atorvastatin treatment and attenuated by SB203580 and PD98059 but not by SP600125, suggesting that the ERK and p38 mitogen-activated protein kinase (MAPK) pathways participate in regulating the above-mentioned effects of atorvastatin. Moreover, the HO-1 activity suppressed by SnPP or the HO-1 expression inhibited by siRNA significantly attenuated the effect of atorvastatin on TNF-α expression and production in LPS-stimulated macrophages. CONCLUSION: Atorvastatin can attenuate LPS-induced TNF-α expression and production by activating HO-1 via the ERK and p38 MAPK pathways, suggesting that atorvastatin can be used in treatment of inflammatory diseases such as sepsis, especially in those with atherosclerotic diseases.

Angiotensin II-derived reactive oxygen species promote angiogenesis in human late endothelial progenitor cells through heme oxygenase-1 via ERK1/2 and AKT/PI3K pathways.

Angiotensin II (Ang II), the main component of renin-angiotensin system, could mediate pathogenic angiogenesis in cardiovascular disorders. Late endothelial progenitor cells (EPCs) possess potent self-renewal and angiogenic potency superior to early EPCs, but few study focused on the cross-talk between Ang II and late EPCs. We observed that Ang II could increase reactive oxygen species (ROS) and promote capillary formation in late EPCs. Ang II-derived ROS could also upregulate heme oxygenase-1 (HO-1) expression, and treating late EPCs with HO-1 small interfering RNA or heme oxygenase inhibitor (HO inhibitor) could inhibit Ang II-induced tube formation and increase ROS level and apoptosis rate. In addition, PD98059 and LY294002 pretreatment attenuated Ang II-induced HO-1 expression. Accordingly, Ang II-derived ROS could promote angiogenesis in late EPCs by inducing HO-1 expression via ERK1/2 and AKT/PI3K pathways, and we believe HO-1 might be a promising intervention target in EPCs due to its potent proangiogenic, antioxidant, and antiapoptosis potentials.

Poor coronary collateral vessel development in patients with mild to moderate renal insufficiency.

BACKGROUND: The development of coronary collaterals is crucial to survival through acute ischemia. Mild to moderate loss of renal function has been suggested to play a role in this event, but evidential data are scarce. The aim of this study was to investigate the relationship between mild to moderate renal insufficiency and coronary collateral development in patients with chronic total coronary artery occlusion. METHODS AND RESULTS: A total of 83 patients with mild to moderate loss of renal function (30 mL/min/1.73 m(2) ≤ eGFR < 90 mL/min/1.73 m(2)) with chronic total coronary artery occlusion were included in our study. The collateral circulation was graded according to Rentrop classification and the function of collateral circulation was graded according to Werner collateral connection (CC) grades. Compared to patients with good collateral circulation (Rentrop = 2,3), eGFR was found to be lower in those patients with poor coronary collateral circulation (Rentrop = 0,1) (63.30 ± 10.51 vs. 54.13 ± 10.56, P = 0.02). eGFR was also found to be lower in poorly functioning coronary collateral circulation (CC = 0,1) than in efficiently functioning coronary collateral circulation (CC = 2) (55.22 ± 9.98 vs. 66.28 ± 9.16, P = 0.03). Multiple logistic regression analysis showed that low eGFR was independently associated with poor coronary collateral circulation (Rentrop = 0,1, 95% CI, 0.09-1.09, P = 0.044) and poor function of coronary collateral circulation (CC = 0,1, 95% CI, 0.02-0.17, P = 0.02). CONCLUSIONS: Lower eGFR is associated with poorer coronary collateral vessel development in patients experiencing mild to moderate renal insufficiency. Moreover, eGFR represents an independent factor affecting coronary collateral vessel development.