Le Su

An activator of mTOR inhibits oxLDL-induced autophagy and apoptosis in vascular endothelial cells and restricts atherosclerosis in apolipoprotein E -/- mice

Oxidized low-density lipoprotein (oxLDL) inhibits mammalian target of rapamycin (mTOR) and induces autophagy and apoptosis in vascular endothelial cells (VECs) that play very critical roles for the cardiovascular homostasis. We recently defined 3-benzyl-5-((2-nitrophenoxy) methyl)-dihydrofuran-2(3H)-one (3BDO) as a new activator of mTOR. Therefore, we hypothesized that 3BDO had a protective role in VECs and thus stabilized atherosclerotic lesions in apolipoprotein E-/- (apoE-/-) mice. Our results showed that oxLDL inhibited the activity of mTOR and increased the protein level of autophagy-related 13 (ATG13) and its dephosphorylation, thus inducing autophagy in human umbilical vein endothelial cells (HUVECs). All of these effects were strongly inhibited by 3BDO. In vivo experiments confirmed that 3BDO activated mTOR and decreased the protein level of ATG13 in the plaque endothelium of apoE-/- mice. Importantly, 3BDO did not affect the activity of mTOR and autophagy in macrophage cell line RAW246.7 and vascular smooth muscle cells of apoE-/- mice, but suppressed plaque endothelial cell death and restricted atherosclerosis development in the mice. 3BDO protected VECs by activating mTOR and thus stabilized atherosclerotic lesions in apoE-/- mice.

Identification of a novel MTOR activator and discovery of a competing endogenous RNA regulating autophagy in vascular endothelial cells

MTOR, a central regulator of autophagy, is involved in cancer and cardiovascular and neurological diseases. Modulating the MTOR signaling balance could be of great significance for numerous diseases. No chemical activators of MTOR have been found, and the urgent challenge is to find novel MTOR downstream components. In previous studies, we found a chemical small molecule, 3-benzyl-5-((2-nitrophenoxy) methyl)–dihydrofuran-2(3H)-one (3BDO), that inhibited autophagy in human umbilical vein endothelial cells (HUVECs) and neuronal cells. Here, we found that 3BDO activated MTOR by targeting FKBP1A (FK506-binding protein 1A, 12 kDa). We next used 3BDO to detect novel factors downstream of the MTOR signaling pathway. Activation of MTOR by 3BDO increased the phosphorylation of TIA1 (TIA1 cytotoxic granule-associated RNA binding protein/T-cell-restricted intracellular antigen-1). Finally, we used gene microarray, RNA interference, RNA-ChIP assay, bioinformatics, luciferase reporter assay, and other assays and found that 3BDO greatly decreased the level of a long noncoding RNA (lncRNA) derived from the 3′ untranslated region (3′UTR) of TGFB2, known as FLJ11812. TIA1 was responsible for processing FLJ11812. Further experiments results showed that FLJ11812 could bind with MIR4459 targeting ATG13 (autophagy-related 13), and ATG13 protein level was decreased along with 3BDO-decreased FLJ11812 level. Here, we provide a new activator of MTOR, and our findings highlight the role of the lncRNA in autophagy.

A new microRNA signal pathway regulated by long noncoding RNA TGFB2-OT1 in autophagy and inflammation of vascular endothelial cells

TGFB2-OT1 (TGFB2 overlapping transcript 1) is a newly discovered long noncoding RNA (lncRNA) derived from the 3′UTR of TGFB2. It can regulate autophagy in vascular endothelial cells (VECs). However, the mechanisms of TGFB2-OT1 action are unclear, and whether it is involved in VECs dysfunction needs investigation. Here, the level of TGFB2-OT1 was markedly increased by lipopolysaccharide and oxidized low-density lipoprotein, 2 VECs inflammation triggers. A chemical small molecule, 3-benzyl-5-((2-nitrophenoxy) methyl)-dihydrofuran-2(3H)-one (3BDO) significantly decreased TGFB2-OT1 levels and inhibited the effect of LPS and oxLDL. The NUPR1 level was upregulated by the 2 inflammation inducers and modulated the TGFB2-OT1 level by promoting the expression of TIA1, responsible for TGFB2-OT1 processing. We focused on how TGFB2-OT1 regulated autophagy and inflammation. Use of miRNA chip assay, TGFB2-OT1 overexpression technology and 3BDO revealed that TGFB2-OT1 regulated the levels of 3 microRNAs, MIR3960, MIR4488 and MIR4459. Further studies confirmed that TGFB2-OT1 acted as a competing endogenous RNA, bound to MIR3960, MIR4488 and MIR4459, then regulated the expression of the miRNA targets CERS1 (ceramide synthase 1), NAT8L (N-acetyltransferase 8-like [GCN5-related, putative]), and LARP1 (La ribonucleoprotein domain family, member 1). CERS1 and NAT8L participate in autophagy by affecting mitochondrial function. TGFB2-OT1 increased the LARP1 level, which promoted SQSTM1 (sequestosome 1) expression, NFKB RELA and CASP1 activation, and then production of IL6, IL8 and IL1B in VECs. Thus, NUPR1 and TIA1 may control the level of TGFB2-OT1, and TGFB2-OT1 bound to MIR3960, MIR4488 and MIR4459, which targeted CERS1, NAT8L, and LARP1, respectively, the key proteins involved in autophagy and inflammation.

Lipopolysaccharide activated phosphatidylcholine-specific phospholipase C and induced IL-8 and MCP-1 production in vascular endothelial cells†

Secretion of proinflammatory cytokines by lipopolysaccharide (LPS) activated vascular endothelial cells (VECs) contributes substantially to the pathogenesis of several inflammatory diseases such as atherosclerosis and septic shock. However, the mechanisms involved in this process are not well understood. Here, we investigated the role of phosphatidylcholine‐specific phospholipase C (PC‐PLC) in LPS‐induced IL‐8 and MCP‐1 production in VECs. The results showed that LPS elevated the level of PC‐PLC and the production of IL‐8 and MCP‐1 in Human umbilical vein vascular endothelial cells (HUVECs). Blocking the function of PC‐PLC by exploiting the neutralization antibody of PC‐PLC or tricyclodecan‐9‐yl‐xanthogenate (D609), an inhibitor of PC‐PLC, significantly inhibited LPS‐induced production of IL‐8 and MCP‐1 in HUVECs. Furthermore, the in vivo experimental results showed that the levels of PC‐PLC, IL‐8, and MCP‐1 in the aortic endothelium and serum were increased in mice injected with LPS. The increased levels of these molecules were also inhibited by the treatment with D609. The data suggested that blocking PC‐PLC function significantly inhibited LPS‐induced IL‐8 and MCP‐1 production in cultured HUVECs and in vivo. PC‐PLC might be a potential target for therapy in inflammation associated‐diseases such as atherosclerosis. J. Cell. Physiol. 226: 1694–1701, 2011.

D609 Inhibits Progression of Preexisting Atheroma and Promotes Lesion Stability in Apolipoprotein E−/− Mice A Role of Phosphatidylcholine-Specific Phospholipase in Atherosclerosis

Objective—Atherosclerosis is considered to be a chronic inflammatory disease. Previous research has demonstrated that phosphatidylcholine-specific phospholipase C (PC-PLC) plays critical roles in various inflammatory responses. However, the association between PC-PLC and atherosclerosis is undetermined. Therefore, we sought to investigate whether PC-PLC was implicated in atherosclerosis. Methods and Results—Immunofluorescence analysis revealed an upregulation of PC-PLC in the aortic endothelium from apolipoprotein E-deficient (apoE−/−) mice. PC-PLC level and activity were also increased in human umbilical vein endothelial cells in response to oxidized low-density lipoprotein treatment. Pharmacological blockade of PC-PLC by D609 inhibited the progression of preexisting atherosclerotic lesions in apoE−/− mice and changed the lesion composition into a more stable phenotype. Using a combination of pharmacological inhibition, polyclonal antibodies, confocal laser scanning microscopy and Western blotting, we demonstrated that PC-PLC was required for endothelial expression of lectin-like oxidized low-density lipoprotein receptor-1. In addition, D609 treatment significantly decreased the aortic endothelial expression of the vascular cell adhesion molecule-1 and the intercellular adhesion molecule-1. Furthermore, inhibition of PC-PLC in human umbilical vein endothelial cells reduced the oxidized low-density lipoprotein induced expression of vascular cell adhesion molecule-1, intercellular adhesion molecule-1, and monocyte chemotactic protein-1. Conclusion—Our data suggest that PC-PLC contributes to the progression of atherosclerosis.

A ratiometric fluorescence sensor for HOCl based on a FRET platform and application in living cells

Studies showed that the intravascular pH of neutrophils fell to 4.5–5.0 with stimulation for a few minutes. Under this condition, myeloperoxidase was activated to catalyze H2O2 and Cl− to form hypochlorous acid (HOCl). Therefore, it is of significance to develop fluorescence probes for sensing HOCl in acid conditions. In this work, we reported a ratiometric probe CRSH based on a fluorescence resonance energy transfer (FRET) platform for detecting HOCl under acid conditions. Probe CRSH exhibited excellent sensitivity, high selectivity and a rapid response toward HOCl and is suitable for imaging endogenous HOCl in living cells.

Regulation of apoptosis and autophagy by sphingosylphosphorylcholine in vascular endothelial cells

Sphingosylphosphorylcholine (SPC), an important cardiovascular mediator derived from sphingomyelin that has atheroprotective effects via actions on vascular endothelial cells (VECs) at normal levels in vivo. However, the underlying mechanism is not well known. To clarify this question, we first investigated the effect of SPC on VEC apoptosis and autophagy induced by deprivation of serum and fibroblast growth factor 2 (FGF‐2). SPC at 5–20 µM inhibited apoptosis and induced autophagy in vitro. To understand the underlying mechanism, we investigated the role of integrin β4 in SPC‐induced autophagy in VECs. SPC significantly decreased the level of integrin β4, whereas overexpression of integrin β4 inhibited SPC‐induced autophagy. Moreover, knockdown of integrin β4 promoted VEC autophagy. To understand the downstream factors of integrin β4 in this process, we observed the effects of SPC on phosphatidylcholine‐specific phospholipase C (PC‐PLC) activity and level of p53. PC‐PLC activity and p53 level in cytoplasm was decreased during autophagy induced by SPC, and knockdown of integrin β4 inhibited the activity of PC‐PLC and the cytoplasmic level of p53. SPC may promote autophagy via integrin β4. Moreover, PC‐PLC and p53 may be the downstream factors of integrin β4 in autophagy of VECs deprived of serum and FGF‐2. J. Cell. Physiol. 226: 2827–2833, 2011.

Lipopolysaccharide induces autophagy through BIRC2 in human umbilical vein endothelial cells.

Lipopolysaccharide (LPS), as an important proinflammatory agent, targets the endothelium. However, almost all in vitro experiments of the effect of LPS on vascular endothelial cells (VECs) were performed under an artificially decreased concentration of serum that was not enough to maintain the cell growth for a long time. The mechanism underlying LPS action on VECs cultured in a nutrient‐rich condition is not clear. To address this question and mimic the in vivo condition, we investigated the effect of LPS on VEC autophagy, which is involved in numerous physiological processes. The effect of LPS on microtubule‐associated protein 1 light chain 3 (LC3) distribution, LC3‐II accumulation and p62 degradation showed that LPS effectively induced autophagy in VECs cultured in the presence of 20% serum. To understand the mechanism by which LPS triggers the cell autophagy, we first investigated the effects of LPS on the expression of BIRC2 (cIAP1), a well‐known apoptosis inhibitor, and on the kinase activity of mammalian target of rapamycin (mTOR) and nuclear translocation of p53. LPS increased BIRC2 expression in a dose‐ and time‐dependent manner and elevated the intranuclear level of p53 but had no effect on the mTOR pathway when it triggered VEC autophagy. Furthermore, knockdown of BIRC2 by RNA interference inhibited the autophagy and the translocation of p53 to nuclei induced by LPS. These data suggest a novel role for BIRC2 in LPS‐induced autophagy in VECs. J. Cell. Physiol. 225: 174–179, 2010.

Long Noncoding RNA LOC100129973 Suppresses Apoptosis by Targeting miR-4707-5p and miR-4767 in Vascular Endothelial Cells

Accumulating evidence has demonstrated that long non-coding RNAs (lncRNAs) are key regulators of multiple biological processes by altering gene expression at various levels. Apoptosis in vascular endothelial cells (VECs) is closely linked to numerous cardiovascular diseases, such as arteriosclerosis, thrombus formation and plaque erosion. However, studies on lncRNAs in the cardiovascular system are just beginning. And thus far, no anti-apoptosis lncRNAs have been identified in VECs. Here, we focused on the anti-apoptosis roles of lncRNAs in the serum and FGF-2 starvation-induced apoptosis of VECs. Using microarray analysis, we found a novel lncRNA LOC100129973 which acted as an apoptosis inhibitor in VECs. Through sponging miR-4707-5p and miR-4767, lncRNA LOC100129973 upregulated the expression of two apoptosis repressors gene, Apoptosis Inhibitor 5 (API5) and BCL2 like 12 (BCL2L12), and thus alleviated the serum and FGF-2 starvation-induced apoptosis in VECs. This evidence suggests that lncRNA LOC100129973 is an attractive target to improve endothelial function and for therapy of apoptosis related cardiovascular diseases.

A high sensitive fluorescence turn-on probe for imaging Zn2+ in aqueous solution and living cells

A new pyrazoline-based fluorescent probe was designed and synthesized. The probe can induce a 60-fold fluorescence enhancement after 5 equiv. of Zn2+ was added, which is superior to a previous report (13-fold), and exhibits high sensitivity and selectivity for response to Zn2+ in aqueous solution. The detection limit and association constant were calculated as 1.23 × 10−7 M and 3.89 × 103 M−1 by a fluorescence titration experiment. The 1 : 1 binding stoichiometry of L–Zn2+ complex was analysed by Jobs plot. The probe showed good reversibility upon addition of EDTA or TPEN. Furthermore, the probe L showed good membrane permeability and can be applied to monitor Zn2+ ions in living HeLa cells.