Direct Interaction of Daxx and Androgen Receptor Is Required for Their Regulatory Activity in Cholesterol Biosynthesis.

INTRODUCTION: Homeostasis of cholesterol is crucial for cellular function, and dysregulated cholesterol biosynthesis is a metabolic event that can lead to hepatic and cardiovascular abnormalities. OBJECTIVE: The aim of this study was to investigate the effects and mechanisms of domain-associated protein (Daxx) and androgen receptor (AR) on intracellular cholesterol synthesis. METHODS: HepG2 cells were transfected with pCDNA3.1(+)/Daxx plasmid or treated with testosterone propionate to observe the effects of Daxx and AR on intracellular cholesterol levels. Co-immunoprecipitation experiments were performed to identify the interaction between Daxx and AR and to explore the regulatory effects of this interaction on cholesterol synthesis. RESULTS: Our experiments showed that AR promoted cholesterol synthesis and accumulation by activating sterol-regulatory element-binding protein isoform 2. AR-induced cholesterol synthesis was inhibited by Daxx; however, the expression of AR was not affected. Further studies demonstrated the existence of direct binding between Daxx and AR and this interaction was required to suppress AR activity. CONCLUSIONS: The Daxx-mediated antagonism of AR depicts a more complete picture as to how Daxx regulates intracellular cholesterol level and provides a new target for treatment of atherosclerosis.

The crosstalk: exosomes and lipid metabolism.

Exosomes have been considered as novel and potent vehicles of intercellular communication, instead of "cell dust". Exosomes are consistent with anucleate cells, and organelles with lipid bilayer consisting of the proteins and abundant lipid, enhancing their "rigidity" and "flexibility". Neighboring cells or distant cells are capable of exchanging genetic or metabolic information via exosomes binding to recipient cell and releasing bioactive molecules, such as lipids, proteins, and nucleic acids. Of note, exosomes exert the remarkable effects on lipid metabolism, including the synthesis, transportation and degradation of the lipid. The disorder of lipid metabolism mediated by exosomes leads to the occurrence and progression of diseases, such as atherosclerosis, cancer, non-alcoholic fatty liver disease (NAFLD), obesity and Alzheimer's diseases and so on. More importantly, lipid metabolism can also affect the production and secretion of exosomes, as well as interactions with the recipient cells. Therefore, exosomes may be applied as effective targets for diagnosis and treatment of diseases. Video abstract.

Ablation of endothelial prolyl hydroxylase domain protein-2 promotes renal vascular remodelling and fibrosis in mice.

Accumulating evidence demonstrates that hypoxia-inducible factor (HIF-α) hydroxylase system has a critical role in vascular remodelling. Using an endothelial-specific prolyl hydroxylase domain protein-2 (PHD2) knockout (PHD2EC KO) mouse model, this study investigates the regulatory role of endothelial HIF-α hydroxylase system in the development of renal fibrosis. Knockout of PHD2 in EC up-regulated the expression of HIF-1α and HIF-2α, resulting in a significant decline of renal function as evidenced by elevated levels of serum creatinine. Deletion of PHD2 increased the expression of Notch3 and transforming growth factor (TGF-ß1) in EC, thus further causing glomerular arteriolar remodelling with an increased pericyte and pericyte coverage. This was accompanied by a significant elevation of renal resistive index (RI). Moreover, knockout of PHD2 in EC up-regulated the expression of fibroblast-specific protein-1 (FSP-1) and increased interstitial fibrosis in the kidney. These alterations were strongly associated with up-regulation of Notch3 and TGF-ß1. We concluded that the expression of PHD2 in endothelial cells plays a critical role in renal fibrosis and vascular remodelling in adult mice. Furthermore, these changes were strongly associated with up-regulation of Notch3/TGF-ß1 signalling and excessive pericyte coverage.

Inhibition of smooth muscle cell proliferation by ezetimibe via the cyclin D1-MAPK pathway.

Proliferation of vascular smooth muscle cells (VSMCs) contributes to the development of atherosclerosis. Ezetimibe is a new lipid lowering agent that inhibits cholesterol absorption. In the present study we attempted to investigate whether ezetimibe has any effect on VSMC proliferation and the potential mechanisms involved. Our data showed ezetimibe abrogated the proliferation and migration of primary rat VSMCs induced by Chol:MßCD. Mechanically, we found that ezetimibe was capable of abolishing cyclin D1, CDK2, phospho-Rb (p-Rb), and E2F protein expressions that were upregulated by Chol:MßCD treatment. In addition, Ezetimibe was able to reverse cell cycle progression induced by Chol:MßCD, which was further supported by its down-regulation of cyclin D1 promoter activity in the presence of Chol:MßCD. Furthermore, ezetimibe abrogated the increment of phospho-ERK1/2 (p-ERK1/2) and nuclear accumulation of ERK1/2 in VSMCs induced by Chol:MßCD. Inhibition of the MAPK pathway by using ERK1/2 inhibitor PD98059 attenuated the reduction effect of ezetimibe on the expressions of phosphor-MEK1 (p-MEK1), p-ERK1/2, and cyclin D1. Taken together our data suggest that ezetimibe inhibits Chol:MßCD-induced VSMCs proliferation and leads to cell cycle arrest at the G0/G1 phase by suppressing cyclin D1 expression via the MAPK signaling pathway. These novel findings support the potential pleiotropic effect of ezetimibe in cardiovascular disease.

Ezetimibe-mediated protection of vascular smooth muscle cells from cholesterol accumulation through the regulation of lipid metabolism-related gene expression.

BACKGROUND: Ezetimibe is a potent inhibitor of Niemann-Pick type C1-Like 1 and has been approved for the treatment of hypercholesterolemia. Our preliminary study showed that ezetimibe promotes cholesterol efflux from vascular smooth muscle cells (VSMCs). Our aim was to investigate the cellular mechanisms underlying the ezetimibe actions. METHODS AND RESULTS: Rat VSMCs were converted to foam cells by incubation with cholesterol:methyl-ß-cyclodextrin. The intracellular free cholesterol, total cholesterol, and the ratio of cholesteryl ester to total cholesterol were decreased after the incubation of VSMCs with different concentrations of ezetimibe (3, 10, 30, and 30 µmol/l) or treated with 30 µmol/l of ezetimibe for different time periods (6, 12, 24, and 48 h). Our results also showed that the expression of caveolin-1, liver X receptor α, and ATP-binding cassette transporter ABCA1 was enhanced, but the expression of nSREBP-1c was decreased in a concentration- and time-dependent manner. RNA interference was used to determine the roles of caveolin-1 and SREBP-1 in the lipid-lowering effect of ezetimibe. The results showed that caveolin-1 was involved in the regulation of intracellular cholesterol content, and the expression of caveolin-1 was repressed by SREBP-1. CONCLUSION: The present study indicates that ezetimibe protects VSMCs from cholesterol accumulation by regulating the expression of lipid metabolism-related genes.

Four New Species of Russula Subsection Sardoninae from China.

Four new species of Russula subsection Sardoninae from northern and southwestern China under coniferous and deciduous trees are proposed as R. begonia, R. photinia, R. rhodochroa, and R. rufa. Illustrations and descriptions of R. gracillima, R. leucomarginata, R. roseola, and the above four new species are provided based on evidence of morphological characters and phylogenetic analyses of the internal transcribed spacer (ITS), as well as the multi-locus of mtSSU, nLSU, rpb1, rpb2 and tef1-α. The relationships between these new species and allied taxa are discussed.

[Effect of left ventricular ejection fraction on clinical long-term follow-up outcomes in patients with acute ST-segment elevation myocardial infarction undergoing primary percutaneous coronary intervention].

OBJECTIVE: To evaluate the effect of left ventricular ejection fraction (LVEF) on clinical outcomes in patients with acute ST-segment elevation myocardial infarction undergoing primary percutaneous coronary intervention. METHODS: A total of 158 patients with acute ST-segment elevation myocardial infarction undergoing primary percutaneous coronary intervention between January 2005 to December 2007 were enrolled. They were divided into three groups: LVEF≤40% (n=14), LVEF 41%-55% (n=46) and LVEF>55% group (n=98). The clinical follow-up end-point was major adverse cardiac event (MACE) including death, acute myocardial infarction, stent thrombosis and stent restenosis. The clinical follow-up duration was 43.1±15.2 months. MACE occurred in 15 patients. RESULTS: The rates of infarction site, infarction relative artery, 1-vessel disease, 2-vessel disease, hypertension, diabetes, hyperlipidemia, smoking, obesity and aspirin use were not different in three groups (P>0.05). Average CTnI, CK, CK-MB and duration of clopidogrel use were not different in three groups (P>0.05). The rate of 3-vessel disease was significantly higher in the LVEF≤40% group than that in the LVEF 41%-55% and LVEF>55% groups (P=0.0036). The rates of TIMI flow grades (Grade III) and complete revascularization were significantly higher in the LVEF 41%-55% and LVEF>55% groups than that in the LVEF≤40% group (P=0.0099, P=0.0010). The rates of Killip classification (classes II, III, IV) and average symptom-onset-to balloon-time (SOTB) were significantly lower in the LVEF 41%-55% and LVEF>55% groups than that in the LVEF≤40% group (P=0.0100, P=0.0087). The rate of drug-eluting stents was significantly lower in the LVEF≤40% group and LVEF 41%-55% group than that in LVEF>55% group (P=0.0242). Logistic regression analysis showed that LVEF was independent predictor for MACE in the follow-up period (P=0.0029). With LVEF decrease, incidence of MACE in the follow-up period significantly increased in LVEF>55% group, LVEF 41%-55% group and LVEF≤40% group (6.12% vs 8.7% vs 35.71%, P=0.0019). Incidence of total death and cardiac death in the follow-up period significantly increased in LVEF>55% group, LVEF 41%-55% group and LVEF≤40% group (1.02% vs 4.35% vs 21.43%, P=0.0090; 1.02% vs 2.17 vs 14.29%, P=0.0060). CONCLUSION: In patients with acute ST-segment elevation myocardial infarction undergoing primary percutaneous coronary intervention, LVEF was independent predictor for MACE in the follow-up period. With LVEF decrease, incidence of MACE in the follow-up period significantly increased.

Acid-Sensing Ion Channel 1/Calpain1 Activation Impedes Macrophage ATP-Binding Cassette Protein A1-Mediated Cholesterol Efflux Induced by Extracellular Acidification.

BACKGROUND: Extracellular acidification is a common feature of atherosclerotic lesions, and such an acidic microenvironment impedes ATP-binding cassette transporter A1 (ABCA1)-mediated cholesterol efflux and promotes atherogenesis. However, the underlying mechanism is still unclear. Acid-sensing ion channel 1 (ASIC1) is a critical H+ receptor, which is responsible for the perception and transduction of extracellular acidification signals. AIM: In this study, we explored whether or how ASIC1 influences extracellular acidification-induced ABCA1-mediated cholesterol efflux from macrophage-derived foam cells. METHODS: RAW 264.7 macrophages were cultured in an acidic medium (pH 6.5) to generate foam cells. Then the intracellular lipid deposition, cholesterol efflux, and ASIC1/calpain1/ABCA1 expressions were evaluated. RESULTS: We showed that extracellular acidification enhanced ASIC1 expression and translocation, promoted calpain1 expression and lipid accumulation, and decreased ABCA1 protein expression as well as ABCA1-mediated cholesterol efflux. Of note, inhibiting ASIC1 activation with amiloride or Psalmotoxin 1 (PcTx-1) not only lowered calpain1 protein level and lipid accumulation but also enhanced ABCA1 protein levels and ABCA1-mediated cholesterol efflux of macrophages under extracellular acidification conditions. Furthermore, similar results were observed in macrophages treated with calpain1 inhibitor PD150606. CONCLUSION: Extracellular acidification declines cholesterol efflux via activating ASIC1 to promote calpain1-mediated ABCA1 degradation. Thus, ASIC1 may be a novel therapeutic target for atherosclerosis.

Three new Russula species in sect. Ingratae (Russulales, Basidiomycota) from southern China.

Three new species of Russulasection Ingratae, found in Guizhou and Jiangsu Provinces, southern China, are proposed: R.straminella, R.subpectinatoides and R.succinea. Photographs, line drawings and detailed morphological descriptions for these species are provided with comparisons against closely-related taxa. Phylogenetic analysis of the internal transcribed spacer (ITS) region supported the recognition of these specimens as new species. Additionally, R.indocatillus is reported for the first time from China and morphological and phylogenetic data are provided for the Chinese specimens.

Autophagy in chronic stress induced atherosclerosis.

Atherosclerosis, a complex multifactorial disease, is the leading cause of acute cardiovascular events. Substantial evidence confirms that chronic stress plays a pivot role in the occurrence and development of atherosclerosis, but the specific mechanism remains unclear. Autophagy serves as a safeguard mechanism for sustaining cellular homeostasis via eliminating unnecessary or/and harmful components, and damaged organelles in response to various stress. An increasing number of studies indicate that autophagy plays vital roles in the development of atherosclerosis. Therefore, understanding the role of chronic stress in the regulation of autophagy may provide new insight into prevention and treatment atherosclerotic disease, especially with respect to emerging targeted therapy. In present review, we focus on changes in autophagic function under chronic stress and its relationship to atherosclerosis.

Acid-sensing ion channels: Linking extracellular acidification with atherosclerosis.

Extracellular acidification in atherosclerosis-prone regions of arterial walls is considered pro-atherosclerotic by exerting detrimental effect on macrophages, endothelial cells (ECs) and vascular smooth muscle cells (VSMCs). Acid-sensing ion channels (ASICs), a family of extracellular H+ (proton)-gated cation channels, are present extensively in the nervous system and other tissues, implying physiologic as well as pathophysiologic importance. Aberrant activation of ASICs is thought to be associated in EC dysfunction, macrophage phenotypic switch, and VSMC migration and proliferation. Although in vitro evidence acknowledges the contribution of ASIC activation in atherosclerosis, no direct evidence confirms their pro-atherosclerotic roles in vivo. In this review, the effect of extracellular acidity on three major contributors, ECs, macrophages, and VSMCs, is discussed focusing on the potential roles of ASICs in atherosclerotic development and underlying pathology. A more comprehensive understanding of ASICs in these processes may provide promising new therapeutic targets for treatment and prevention of atherosclerotic diseases.

Autophagy: a killer or guardian of vascular smooth muscle cells.

Vascular smooth muscle cells (VSMCs) is one of the main intracellular components of the blood vessel wall. The abnormalities of VSMCs participate in the development of cardiovascular diseases such as atherosclerosis, hypertension, and restenosis, especially the formation and stability of atherosclerotic plaques. Autophagy is involved in the regulation of proliferation, migration and phenotype switching of VSMCs, which in turn affects the pathological process of atherosclerosis. However, the autophagy of VSMCs has a dual effect on cells survival. Autophagy is induced in VSMCs by various stimuli such as 7-ketocholesterol (7-KC), unsaturated lipid peroxidation-derived aldehyde and excess free cholesterol, thereby promoting VSMCs survival and stabilising atherosclerotic plaque. Conversely, autophagy caused by factors such as osteopontin (OPN), angiotensin II (Ang II) and nicotine can accelerate the death of VSMCs, further accelerating atherosclerotic lesions. In addition, mitophagy and lipophagy as selective autophagy are also involved in the outcome of VSMCs as well as progression of atherosclerotic lesion. Currently, there are only a few drugs available to induce VSMCs autophagy, such as atorvastatin, telmisartan and so on. Due to the important role of VSMCs autophagy in the progression of atherosclerosis plaques, drugs that directly target autophagy of VSMCs are urgently needed to be developed.

Astragaloside VI Promotes Neural Stem Cell Proliferation and Enhances Neurological Function Recovery in Transient Cerebral Ischemic Injury via Activating EGFR/MAPK Signaling Cascades.

Radix Astragali (AR) is a commonly used medicinal herb for post-stroke disability in Traditional Chinese Medicine but its active compounds for promoting neurogenic effects are largely unknown. In the present study, we tested the hypothesis that Astragaloside VI could be a promising active compound from AR for adult neurogenesis and brain repair via targeting epidermal growth factor (EGF)-mediated MAPK signaling pathway in post-stroke treatment. By using cultured neural stem cells (NSCs) and experimental stroke rat model, we investigated the effects of Astragaloside VI on inducing NSCs proliferation and self-renewal in vitro, and enhancing neurogenesis for the recovery of the neurological functions in post-ischemic brains in vivo. For animal experiments, rats were undergone 1.5 h middle cerebral artery occlusion (MCAO) plus 7 days reperfusion. Astragaloside VI (2 µg/kg) was daily administrated by intravenous injection (i.v.) for 7 days. Astragaloside VI treatment promoted neurogenesis and astrogenic formation in dentate gyrus zone, subventricular zone, and cortex of the transient ischemic rat brains in vivo. Astragaloside VI treatment enhanced NSCs self-renewal and proliferation in the cultured NSCs in vitro without affecting NSCs differentiation. Western blot analysis showed that Astragaloside VI up-regulated the expression of nestin, p-EGFR and p-MAPK, and increased neurosphere sizes, whose effects were abolished by the co-treatment of EGF receptor inhibitor gefitinib and ERK inhibitor PD98059. Behavior tests revealed that Astragaloside VI promoted the spatial learning and memory and improved the impaired motor function in transient cerebral ischemic rats. Taken together, Astragaloside VI could effectively activate EGFR/MAPK signaling cascades, promote NSCs proliferation and neurogenesis in transient cerebral ischemic brains, and improve the repair of neurological functions in post-ischemic stroke rats. Astragaloside VI could be a new therapeutic drug candidate for post-stroke treatment.

Notch3 deficiency impairs coronary microvascular maturation and reduces cardiac recovery after myocardial ischemia.

RATIONALE: Vascular maturation plays an important role in wound repair post-myocardial infarction (MI). The Notch3 is critical for pericyte recruitment and vascular maturation during embryonic development. OBJECTIVE: This study is to test whether Notch3 deficiency impairs vascular maturation and blunts cardiac functional recovery post-MI. APPROACH AND RESULTS: Wild type (WT) and Notch3 knockout (Notch3KO) mice were subjected to MI by the ligation of left anterior descending coronary artery (LAD). Cardiac function and coronary blood flow reserve (CFR) were measured by echocardiography. The expression of angiogenic growth factor, pericyte/capillary coverage and arteriolar formation were analyzed. Loss of Notch3 in mice resulted in a significant reduction of pericytes and small arterioles. Notch3 KO mice had impaired pericyte/capillary coverage and CFR compared to WT mice. Notch3 KO mice were more prone to ischemic injury with larger infarcted size and higher rates of mortality. The expression of CXCR-4 and VEGF/Ang-1 was significantly decreased in Notch3 KO mice. Notch3 KO mice also had few NG2+/Sca1+ and NG2+/c-kit+ progenitor cells in the ischemic area and exhibited worse cardiac function recovery at 2weeks after MI. These were accompanied by a significant reduction of pericyte/capillary coverage and arteriolar maturation. Furthermore, Notch3 KO mice subjected to MI had increased intracellular adhesion molecule-2 (ICAM-2) expression and CD11b+ macrophage infiltration into ischemic areas compared to that of WT mice. CONCLUSION: Notch3 mutation impairs recovery of cardiac function post-MI by the mechanisms involving the pre-existing coronary microvascular dysfunction conditions, and impairment of pericyte/progenitor cell recruitment and microvascular maturation.

Oxidized low-density lipoprotein induced mouse hippocampal HT-22 cell damage via promoting the shift from autophagy to apoptosis.

AIMS: Although oxidized low-density lipoprotein (ox-LDL) in the brain induces neuronal death, the mechanism underlying the damage effects remains largely unknown. Given that the ultimate outcome of a cell is depended on the balance between autophagy and apoptosis, this study was performed to explore whether ox-LDL induced HT-22 neuronal cell damage via autophagy impairment and apoptosis enhancement. METHODS: Flow cytometry and transmission electron microscopy (TEM) were used to evaluate changes in cell apoptosis and autophagy, respectively. The protein expression of LC3-II, p62, Bcl-2, and Bax in HT-22 cells was measured by Western bolt analysis. RESULTS: Our study confirmed that 100 µg/mL of ox-LDL not only promoted TH-22 cell apoptosis, characterized by elevated cell apoptosis rate and Bax protein expression, decreased Bcl-2 protein expression, and damaged cellular ultrastructures, but also impaired autophagy as indicated by the decreased LC3-II levels and the increased p62 levels. Importantly, all of these effects of ox-LDL were significantly aggravated by cotreatment with chloroquine (an inhibitor of autophagy flux). In contrast, cotreatment with rapamycin (an inducer of autophagy) remarkably reversed these effects of ox-LDL. CONCLUSIONS: Taken together, our results indicated that ox-LDL-induced shift from autophagy to apoptosis contributes to HT-22 cell damage.

Loss of prolyl hydroxylase domain protein 2 in vascular endothelium increases pericyte coverage and promotes pulmonary arterial remodeling.

Pulmonary arterial hypertension (PAH) is a leading cause of heart failure. Although pulmonary endothelial dysfunction plays a crucial role in the progression of the PAH, the underlying mechanisms are poorly understood. The HIF-α hydroxylase system is a key player in the regulation of vascular remodeling. Knockout of HIF-2α has been reported to cause pulmonary hypertension. The present study examined the role of endothelial cell specific prolyl hydroxylase-2 (PHD2) in the development of PAH and pulmonary vascular remodeling. The PHD2f/f mouse was crossbred with VE-Cadherin-Cre promoter mouse to generate an endothelial specific PHD2 knockout (Cdh5-Cre-PHD2ECKO) mouse. Pulmonary arterial pressure and the size of the right ventricle was significantly elevated in the PHD2ECKO mice relative to the PHD2f/f controls. Knockout of PHD2 in EC was associated with vascular remodeling, as evidenced by an increase in pulmonary arterial media to lumen ratio and number of muscularized arterioles. The pericyte coverage and vascular smooth muscle cells were also significantly increased in the PA. The increase in vascular pericytes was associated with elevated expression of fibroblast specific protein-1 (FSP-1). Moreover, perivascular interstitial fibrosis of pulmonary arteries was significantly increased in the PHD2ECKO mice. Mechanistically, knockout of PHD2 in EC increased the expression of Notch3 and transforming growth factor (TGF-ß) in the lung tissue. We conclude that the expression of PHD2 in endothelial cells plays a critical role in preventing pulmonary arterial remodeling in mice. Increased Notch3/TGF-ß signaling and excessive pericyte coverage may be contributing to the development of PAH following deletion of endothelial PHD2.