Yanxia Ning

Overexpression of mitochondrial cholesterol delivery protein, StAR, decreases intracellular lipids and inflammatory factors secretion in macrophages

Hyperlipidemia is one of the most important risk factors for atherosclerosis. This can be amplified by a localized inflammatory response mediated by macrophages. Macrophages are capable of taking up excess cholesterol, and it is well known that delivery of cholesterol to the mitochondria by steroidogenic acute regulatory (StAR) protein is the rate-limiting step for cholesterol degradation in the liver. It has also been shown that overexpression of StAR in hepatocytes dramatically increases the amount of regulatory oxysterols in the nucleus, which play an important role in the maintenance of intracellular lipid homeostasis. The goal of the present study was to determine whether StAR plays a similar role in macrophages. We have found that overexpression of StAR in human THP-1 monocyte-derived macrophages decreases intracellular lipid levels, activates liver X receptor alpha (LXRalpha) and proliferation peroxysome activator receptor gamma (PPARgamma), and increases ABCG1 and CYP27A1 expression. Furthermore, it reduces the secretion of inflammatory factors, and prevents apoptosis. These results suggest that StAR delivers cholesterol to mitochondria where regulatory oxysterols are generated. Regulatory oxysterols can in turn activate nuclear receptors, which increase expression of cholesterol efflux transporters, and decrease secretion of inflammatory factors. These effects can prevent macrophage apoptosis. These results imply a potential role of StAR in the prevention of atherosclerosis.

The effects of inflammatory cytokines on steroidogenic acute regulatory protein expression in macrophages

Abstract.Objective:To investigate the expression of steroidogenic acute regulatory protein (StAR) in macrophages and the effects of inflammatory cytokines on StAR expression.Methods:The macrophages isolated from ApoE knockout mice and C57BL/6J mice and RAW264.7 cells (a cell line from mouse macrophage. ATCC Number: TIB-71TM) were cultured in DMEM containing 10% fetal bovine serum. RAW264.7 cells were treated with different inflammatory cytokines (TNF-α, IFN-γ and TGF-β1) and 8-Br-cAMP, a cAMP analog. RT-PCR and Western blot analysis were applied to evaluate the effects of inflammatory cytokines on StAR expression.Results:RT-PCR and Western blot analysis demonstrated the expression of StAR in the macrophages isolated from ApoE knockout mice, C57BL/6J mice and RAW264.7 cells. Proinflammatory cytokines TNF-α and IFN-γ significantly decreased StAR mRNA and protein levels in RAW264.7 cells. The inhibition was dose- and time-dependent. In contrast, anti-inflammatory cytokine TGF-β1 increased StAR mRNA and protein levels. At 1:15 molecular ratio, TGF-β1 blocked the down-regulation of StAR expression mediated by TNF-α. cAMP also induced StAR expression in RAW264.7 cells. When the cells were co-treated with 8-Br-cAMP and TNF-α, 8-Br-cAMP failed to induce StAR expression.Conclusion:Our results provide interesting evidence that inflammatory cytokines regulate StAR expression in macrophages.

Tryptase promotes atherosclerotic plaque haemorrhage in ApoE-/- mice.

Tryptase, the most abundant mast cell (MC) granule protein, plays an important role in atherosclerosis plaque development. To test the hypothesis that tryptase participates directly in atherosclerosis plaque haemorrhage, the gene sequence and siRNA for tryptase were cloned into a lentivirus carrier and atherosclerosis plaque haemorrhage models in ApoE-/- mice were constructed. After a cuffing-cervical artery operation, the mice were randomly divided into 6 groups. Hematoxylin and eosin(HE) staining showed that the cervical artery plaque area was much larger in the tryptase overexpression group compared to the other groups, and there was greater artery stenosis. The artery stenosis from the cuff-side in all groups was more than 90%, except the siRNA group. Tryptase promotes plaque haemorrhage distinctively because 50% of the mice in the tryptase overexpression group had plaque haemorrhage, while only 10% in the siRNA group did. The immunohistochemistry of the cervical artery plaque showed that plasminogen activator inhibitor-1 (PAI-1) expression was the lowest while tissue plasminogen activator (tPA), CD31, CD34 and VEGF was the highest in the tryptase overexpression groups. This observation was completely contrary to what was observed in the siRNA group. Tryptase promoted bEnd.3 cell growth, migration and capillary-like tube formation, which suggests that tryptase can promote microvessel angiogenesis. PAI-1 expression was inhibited, while tPA expression was increased by tryptase in bEnd.3 cells. Our in vivo and in vitro studies suggest that trypase can promote atherosclerotic plaque haemorrhage by promoting angiogenesis and regulating the balance of PAI-1 and tPA. Thus, regulating tryptase expression in MCs may provide a potential target for atherosclerosis treatment.

Overexpression of steroidogenic acute regulatory protein in rat aortic endothelial cells attenuates palmitic acid-induced inflammation and reduction in nitric oxide bioavailability

BackgroundEndothelial dysfunction is a well documented evidence for the onset of atherosclerosis and other cardiovascular diseases. Lipids disorder is among the main risk factors for endothelial dysfunction in these diseases. Steroidogenic acute regulatory protein (StAR), one of the cholesterol transporters, plays an important role in the maintenance of intracellular lipid homeostasis. However, the effect of StAR on endothelial dysfunction is not well understood. Palmitic acid (PA) has been shown to decrease eNOS activity and induce inflammation, both are the causes of endothelial dysfunction, in an endothelial cell culture model.MethodsStAR gene was introduced into primary rat aortic endothelial cells by adenovirus infection. Real-time PCR and Western blotting were performed to determine the relative genes and proteins expression level to elucidate the underlying mechanism. The free fatty acid and cholesterol quantification kits were used to detect total cellular free fatty acid and cholesterol. The levels of inflammatory factors and nitric oxide were determined by ELISA and classic Griess reagent methods respectively.ResultsWe successfully overexpressed StAR in primary rat aortic endothelial cells. Following StAR overexpression, mRNA levels of IL-1β, TNFα, IL6 and VCAM-1 and protein levels of IL-1β, , TNFα and IL-6 in culture supernatant were significantly decreased, which duing to blocke NFκB nuclear translocation and activation. Moreover, StAR overexpression attenuated the PA-induced reduction of nitric oxide bioavailability by protecting the bioactivity of pAkt/peNOS/NO pathway. Furthermore, the key genes involved in lipid metabolism were greatly reduced following StAR overexpression. In order to investigate the underlying mechanism, cerulenin and lovastatin, the inhibitor of fatty acid and cholesterol synthase, were added prior to PA treatment. The results showed that both cerulenin and lovastatin had a similar effect as StAR overexpression. On the other hand, the role of StAR was inhibited when siRNA was introduced to reduce StAR expression.ConclusionsOur results showed that StAR attenuated lipid synthesis and uptake as well as PA-induced inflammation and reduction in NO bioavailability in aortic endothelial cells. StAR can ameliorate endothelial dysfunction induced by PA via reducing the intracellular lipid levels.

Mitochondrial Cholesterol Transporter, StAR, Inhibits Human THP-1 Monocyte-Derived Macrophage Apoptosis

Steroidogenic acute regulatory protein (StAR) plays an important role in the maintenance of intracellular lipid homeostasis. Macrophages are the key cellular player in the pathophysiology of atherosclerosis. Imbalance of macrophage lipid homeostasis causes cellular apoptosis, which is the key process in the initiation of atherosclerosis. The present study has investigated the effects of StAR in the apoptotic process of human THP-1 derived macrophages induced by serum withdrawal or Ox-LDL. Overexpression of StAR significantly decreased the number of apoptotic macrophages by decreasing the expression of pro-apoptotic genes Caspase-3 and Bax mRNA and protein levels, as well as through increasing expression of anti-apoptotic gene Bcl-2 mRNA and protein levels in the absence and presence of Ox-LDL. The results indicate that StAR plays an important role in macrophage and foam cell apoptotic processing, which may provide a potential method for preventing atherosclerosis.

Overexpression of the steroidogenic acute regulatory protein increases the expression of ATP-binding cassette transporters in microvascular endothelial cells (bEnd.3)

ObjectiveTo determine the effect of steroidogenic acute regulatory protein (StAR) overexpression on the levels of adenosine triphosphate (ATP)-binding cassette transporter A1 (ABCA1) and ATP-binding cassette transporter G1 (ABCG1) in an endothelial cell line (bEnd.3).MethodsThe StAR gene was induced in bEnd.3 cells with adenovirus infection. The infection efficiency was detected by fluorescence activated cell sorter (FACS) and fluorescence microscopy. The expressions of StAR gene and protein levels were detected by real-time polymerase chain reaction (PCR) and Western blot. The gene and protein levels of ABCA1 and ABCG1 were detected by real-time PCR and Western blot after StAR overexpression.ResultsThe result shows that StAR was successfully overexpressed in bEnd.3 cells by adenovirus infection. The mRNA and protein expressions of ABCA1 and ABCG1 were greatly increased by StAR overexpression in bEnd.3 cells.ConclusionOverexpression of StAR increases ABCA1 and ABCG1 expressions in endothelial cells.

Effects of RNA interference-induced tryptase down-regulation in P815 cells on IL-6 and TNF-α release of endothelial cells

ObjectiveTo explore the effects of down-regulated tryptase expression in mast cells on the synthesis and release of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) of vascular endothelial cells.MethodsTryptase-siRNA (small-interfering RNA) vector was constructed to inhibit tryptase expression in P815 cells. The medium of P815 cells treated by the tryptase-siRNA (RNAi-P815 group) or pure vector (P815 group) was collected and used to culture bEnd.3 cells. The messenger RNAs (mRNAs) of IL-6 and TNF-α in bEnd.3 cells and their protein levels in the medium were measured by reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively.ResultsIL-6 and TNF-α mRNAs in bEnd.3 cells cultured in RNAi-P815-conditioned medium decreased significantly compared to those in P815-conditioned medium. Consistently, IL-6 and TNF-α protein levels in the medium of bEnd.3 of RNAi-P815 group were lower than those of P815 group.ConclusionReduced tryptase expression significantly inhibited the synthesis and release of IL-6 and TNF-α in vascular endothelial cells. RNA interference targeting tryptase expression may be a new anti-inflammatory strategy for vascular diseases.

Steroidogenic acute regulatory protein (StAR) overexpression attenuates HFD-induced hepatic steatosis and insulin resistance

Non-alcoholic fatty liver disease (NAFLD) covers a wide spectrum of liver pathology. Intracellular lipid accumulation is the first step in the development and progression of NAFLD. Steroidogenic acute regulatory protein (StAR) plays an important role in the synthesis of bile acid and intracellular lipid homeostasis and cholesterol metabolism. We hypothesize that StAR is involved in non-alcoholic fatty liver disease (NAFLD) pathogenesis. The hypothesis was identified using free fatty acid (FFA)-overloaded NAFLD in vitro model and high-fat diet (HFD)-induced NAFLD mouse model transfected by recombinant adenovirus encoding StAR (StAR). StAR expression was also examined in pathology samples of patients with fatty liver by immunohistochemical staining. We found that the expression level of StAR was reduced in the livers obtained from fatty liver patients and NAFLD mice. Additionally, StAR overexpression decreased the levels of hepatic lipids and maintained the hepatic glucose homeostasis due to the activation of farnesoid x receptor (FXR). StAR overexpression attenuated the impairment of insulin signaling in fatty liver. This protective role of StAR was owing to a reduction of intracellular diacylglycerol levels and the phosphorylation of PKCε. Furthermore, FXR inactivation reversed the observed beneficial effects of StAR. The present study revealed that StAR overexpression can reduce hepatic lipid accumulation, regulate glucose metabolism and attenuate insulin resistance through a mechanism involving the activation of FXR. Our study suggests that StAR may be a potential therapeutic target for NAFLD.