Sheng-Qing Wang

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.

A simple and effective coumarin-based fluorescent probe for cysteine

Acrylic acid 3-acetyl-2-oxo-2 H-chromen-7-yl ester (ACA) was rationally designed and synthesized as a simple and effective fluorescent probe for sensing cysteine with high selectivity and naked-eye detection. The probe can detect cysteine by fluorescence spectrometry with a detection limit of 0.657 μM and can be used with calf serum and in live cell imaging. The conjugate addition/cyclization sequence mechanism of the reaction between ACA and cysteine was confirmed by ESI-MS and fluorescence spectra.

Novel pyrazoline-based fluorescent probe for detecting glutathione and its application in cells

A novel compound, 2-(1,5-diphenyl-4,5-dihydro-1H-pyrazol-3-yl)phenyl acrylate (probe L), was designed and synthesized as a highly sensitive and selective fluorescent probe for recognizing and detecting glutathione among cysteine, homocysteine and other amino acids. The structures of related compounds were characterized using IR, NMR and HRMS spectroscopy analysis. The probe is a non-fluorescent compound. On being mixed with glutathione in buffered EtOH:PBS=3:7 solution at pH 7.4, the probe exhibited the blue emission of the pyrazoline at 474 nm and a 83-fold enhancement in fluorescence intensity. This probe is very sensitive and displayed a linear fluorescence off-on response to glutathione with fluorometric detection limit of 8.2 × 10(-8)M. The emission of the probe is pH independent in the physiological pH range. Live-cell imaging of HeLa cells confirmed the cell permeability of the probe and its ability to selectively discriminate GSH from Cys and Hcy in cells. The toxicity of the probe was low in cultured HeLa cells.

A novel pyrazoline-based selective fluorescent probe for detecting reduced glutathione and its application in living cells and serum

A new fluorescent probe, N-(4-(1,5-diphenyl-4,5-dihydro-1H-pyrazol-3-yl)phenyl)-2,4-dinitrobenzenesulfonamide (probe 3), was designed and synthesized as a highly sensitive and selective fluorescent probe for recognizing and detecting glutathione among biological thiols in aqueous media. Probe 3 is a nonfluorescent compound. On being mixed with biothiols under neutral aqueous conditions, the 2,4-dinitrobenzenesulfoyl moiety can be cleaved off by glutathione, and the blue emission of the pyrazoline at 464 nm is switched on, with a fluorescence enhancement of 488-fold for glutathione. Furthermore, probe 3 was highly selective for glutathione without interference from some biologically relevant analytes. The detection limit of glutathione was 4.11 × 10(-7) M. The emission of the probe is pH independent in the physiological pH range. Moreover, the probe can be used for fluorescent imaging of cellular glutathione and can be used for detecting glutathione in calf serum.

A highly sensitive fluorescent probe based on simple pyrazoline for Zn2+ in living neuron cells

We develop a pyrazoline-based fluorescent sensor for biological Zn(2+) detection. The sensor shows good binding selectivity for Zn(2+) over competing metal with 40-fold fluorescence enhancement in response to Zn(2+). The new probe is cell-permeable and can be used to detect intracellular zinc ions in living neuron cells.

Novel pyrazoline-based selective fluorescent probe for the detection of hydrazine

A novel pyrazoline-based fluorescent probe, 2-[4-(3,5-diphenyl-4,5-dihydro-pyrazol-1-yl)-benzylidene]-malononitrile, with a simple structure and low detection limit (6.16×10(-6)M) for the detection of hydrazine is designed and synthesized. The probe responds selectively to hydrazine over other molecules with marked fluorescence enhancement. The probe can detect hydrazine effectively at pH 5.0-9.0 with a special emission wavelength at 520nm. Moreover, the probe can be used to detect hydrazine from variety of natural source water.

Novel pyrazoline-based fluorescent probe for detecting thiols and its application in cells

A new compound, N-(4-(1,5-diphenyl-4,5-dihydro-1H-pyrazol-3-yl)phenyl)-acrylamide (probe L), was designed and synthesized as a highly sensitive and selective fluorescent probe for recognizing and detecting thiol from other amino acids. On being mixed with thiol in buffered DMSO:HEPES=1:1 solution at pH 7.4, the probe exhibited the blue emission at 474 nm. This probe is very sensitive and displayed a linear fluorescence off-on response to thiol. The fluorescence emission of the probe is pH independent in the physiological pH range. Living cell imaging of HeLa cells confirmed its cell permeability and its ability to selectively detect thiol in cells. The structure of the probe was characterized by IR, NMR and HRMS spectroscopy analysis.

Identification of a small molecule targeting annexin A7.

Autophagy involves multiple membrane trafficking and fusion events. Annexin A7 (ANXA7) is postulated to play a role in membrane fusion during exocytosis, while the contribution of ANXA7 to autophagy is poorly understood. Our recent studies demonstrated that ABO could promote autophagy via elevation of ANXA7 and triggering ANXA7 subcellular redistribution. However, little is known about the molecular mechanisms how ANXA7 regulates autophagy. As molecular disruption of ANXA7 in mice results in several unwished phenotypes, small molecule modulators may be efficacious in defining the mechanisms of ANXA7 action. However, so far no compounds that selectively target ANXA7 have been identified. So, we hypothesize that ABO might be a potent modulator of ANXA7. We also have detected the colocalization of ANXA7 and microtubule-associated protein 1 light chain 3 (LC3), and ANXA7 was essential for LC3 accumulation in VEC autophagy. As a GTPase, whether ANXA7 affects the phosphorylation of LC3 or other proteins needs further investigation. In this study, we performed site-directed mutagenesis and found that ABO directly bound to Thr(286) of ANXA7 and inhibited its phosphorylation. By yeast two-hybrid screening, we found that ANXA7 could interact with grancalcin (GCA). ABO promoted the interaction and inhibited GCA phosphorylation, leading to the decrease of intracellular Ca(2+) concentration. At the same time, ABO inhibited the phosphorylation of LC3. Hence, by identifying ABO as an unprecedented modulator of ANXA7 as well as GCA and LC3 as interacting proteins of ANXA7, we demonstrated the possible mechanisms how ANXA7 regulates autophagy for the first time.

A Chemical Small Molecule Induces Mouse Embryonic Stem Cell Differentiation into Functional Vascular Endothelial Cells via Hmbox1

Embryonic stem cells (ESCs) can differentiate to endothelial progenitor cells and vascular endothelial cells (VECs), but the mechanism is largely unknown. In this study, we synthesized 2 chiral compounds (R-ABO and S-ABO) and identified R-ABO as an effective inducer of ESC differentiation into VECs. Furthermore, we found that R-ABO induced ESC differentiation into VECs via homeobox containing 1 (Hmbox1) that acted upstream of fibroblast growth factor 2 (FGF-2). The data suggest that R-ABO is a novel tool for ESC differentiation into VECs, and Hmbox1 is a key regulator in this differentiation process. These findings provide information on a novel target and a new platform for further investigating the gene control of ESC differentiation to VECs.

Relationship between annexin A7 and integrin β4 in autophagy.

We recently found that both annexin A7 and integrin β4 were involved in autophagy of vascular endothelial cells. But, their relation is not clear. In this study, we addressed this question by using a small molecule ABO that promoted autophagy by targeting annexin A7. The results showed that knockdown of integrin β4 partly inhibited ABO-induced autophagy in vascular endothelial cells. Furthermore, in HEK293 cells that express integrin β4 too low to detect by western blot, ABO could not induce autophagy. If integrin β4 was overexpressed in HEK293 cells, ABO could evoke autophagy. On the other hand, knockdown of annexin A7 also blocked ABO-induced autophagy although the level of integrin β4 was elevated. Moreover, by co-immunoprecipitation, we identified the interaction of integrin β4 and annexin A7, and found that ABO could modulate the interaction, at the same time, the phosphorylation of Y-1494 in integrin β4 cytoplasmic domain was inhibited significantly in vitro and in vivo. Hence, by identifying the interaction between integrin β4 and annexin A7, we demonstrated that both annexin A7 and integrin β4 were essential for small molecule ABO-induced autophagy and targeting annexin A7 by ABO could modulate integrin β4 phosphorylation, while Y-1494 phosphorylation of integrin β4 may negatively regulate autophagy.