Wenjun Ding

PM2.5-induced oxidative stress triggers autophagy in human lung epithelial A549 cells.

Exposure to higher levels of air pollution particulate matter (PM) with an aerodynamic diameter of less than 2.5 μm (PM2.5) links with an increased risk of cardiovascular and respiratory deaths and hospital admission as well as lung cancer. Although the mechanism underlying the correlation between PM2.5 exposure and adverse effects has not fully elucidated, PM2.5-induced oxidative stress has been considered as an important molecular mechanism of PM2.5-mediated toxicity. In this work, human lung epithelial A549 cells were used to further investigate the biological effects of PM2.5 on autophagy. The cell viability showed both time- and concentration-dependent decrease when exposure to PM2.5, which can be attributed to increase of the levels of extracellular lactate dehydrogenase (LDH) release and intracellular reactive oxygen species (ROS) generation in A549 cells. Moreover, PM2.5-induced oxidative damage in A549 cells was observed through the alteration of superoxide dismutase (SOD) and catalase (CAT) activities compared to the unexposed control cells. PM2.5-induced autophagy was indicated by an increase in microtubule-associated protein light chain-3 (LC3) puncta, and accumulation of LC3 in both time- and concentration-dependent manner. PM2.5-induced mRNA expression of autophagy-related protein Atg5 and Beclin1 was also observed compared with those of the unexposed control cells. These results suggest the possibility that PM2.5-induced oxidative stress probably plays a key role in autophagy in A549 cells, which may contribute to PM2.5-induced impairment of pulmonary function.

The imbalance of Th17/Th1/Tregs in patients with type 2 diabetes: relationship with metabolic factors and complications

Immune disorders are linked to the development of type 2 diabetes (T2D) and its complications. The relationship of CD4+CD25hi T regulatory cells (Treg) and pro-inflammatory Th17 and Th1 subsets in T2D patients with metabolic disorders and complications need to be determined. The ratios of CD4+CD25hi Treg/Th17 cells and CD4+CD25hi Treg/Th1 cells, but not Th17/Th1 cells, were significantly decreased in T2D patients. The thymic output CD4+Foxp3+Helios+ Tregs were normal but peripheral induced CD4+Foxp3+Helios− Tregs were decreased in T2D patients. The Bcl-2/Bax ratio decreased in CD4+CD25hi Tregs in T2D patients, supporting the increased sensitivity to cell death of these cells in T2D. CD4+CD25hiCD127− Tregs in T2D patients with microvascular complications were significantly less than T2D patients with macrovascular complications. Importantly, CD4+CD25hiCD127− Tregs were positively correlated with plasma IL-6, whereas IL-17+CD4+cells were negatively related to high-density lipoprotein (HDL). Our data offered evidence for the skewed balance of anti- and pro-inflammatory T cell subsets in T2D patients and identified that HDL closely modulate T cell polarization. These results opened an alternative explanation for the substantial activation of immune cells as well as the development of T2D and complications, which may have significant impacts on the prevention and treatment of T2D patients.

ZnO nanoparticle-induced oxidative stress triggers apoptosis by activating JNK signaling pathway in cultured primary astrocytes

It has been documented in in vitro studies that zinc oxide nanoparticles (ZnO NPs) are capable of inducing oxidative stress, which plays a crucial role in ZnO NP-mediated apoptosis. However, the underlying molecular mechanism of apoptosis in neurocytes induced by ZnO NP exposure was not fully elucidated. In this study, we investigated the potential mechanisms of apoptosis provoked by ZnO NPs in cultured primary astrocytes by exploring the molecular signaling pathways triggered after ZnO NP exposure. ZnO NP exposure was found to reduce cell viability in MTT assays, increase lactate dehydrogenase (LDH) release, stimulate intracellular reactive oxygen species (ROS) generation, and elicit caspase-3 activation in a dose- and time-dependent manner. Apoptosis occurred after ZnO NP exposure as evidenced by nuclear condensation and poly(ADP-ribose) polymerase-1 (PARP) cleavage. A decrease in mitochondrial membrane potential (MMP) with a concomitant increase in the expression of Bax/Bcl-2 ratio suggested that the mitochondria also mediated the pathway involved in ZnO NP-induced apoptosis. In addition, exposure of the cultured cells to ZnO NPs led to phosphorylation of c-Jun N-terminal kinase (JNK), extracellular signal-related kinase (ERK), and p38 mitogen-activated protein kinase (p38 MAPK). Moreover, JNK inhibitor (SP600125) significantly reduced ZnO NP-induced cleaved PARP and cleaved caspase-3 expression, but not ERK inhibitor (U0126) or p38 MAPK inhibitor (SB203580), indicating that JNK signaling pathway is involved in ZnO NP-induced apoptosis in primary astrocytes.

Inhibition of protein tyrosine phosphatase 1B and alkaline phosphatase by bis(maltolato)oxovanadium (IV)

Vanadate has been recognized as a specific and potent phosphatase inhibitor since its structure is similar to that of phosphate. In this study, we measured the inhibition of glutathione S-transferase-tagged protein tyrosine phosphatase 1B (GST-PTP1B) and alkaline phosphatase (ALP) by the insulin enhancing compounds, bis(maltolato)oxovanadium(IV) (BMOV). The results showed that the activity of GST-PTP1B was reversibly inhibited by solutions of BMOV with an IC(50) value of 0.86+/-0.02 microM. Steady state kinetic studies showed that inhibition of GST-PTP1B by BMOV was of a mixed competitive and noncompetitive type. In addition, incubation of GST-PTP1B with BMOV showed a time-dependent biphasic inactivation of the protein. On the other hand, the inhibitory behavior of BMOV on ALP activity was reversible and competitive with an IC(50) value of 32.1+/-0.6 microM. Incubation with BMOV did not show biphasic inactivation of ALP. The reversible inhibition of GST-PTP1B by BMOV is more potent than that of ALP, but solutions of BMOV inhibited both enzymes. This data support the suggestion that mechanisms for the inhibitory effects of BMOV on GST-PTP1B and ALP are very different.

Chloro-substituted dipicolinate vanadium complexes: synthesis, solution, solid-state, and insulin-enhancing properties.

Three vanadium complexes of chlorodipicolinic acid (4-chloro-2,6-dipicolinic acid) in oxidation states III, IV, and V were prepared and their properties characterized across the oxidation states. In addition, the series of hydroxylamido, methylhydroxylamido, dimethylhydroxylamido, and diethylhydroxylamido complexes were prepared from the chlorodipicolinato dioxovanadium(V) complex. The vanadium(V) compounds were characterized in solution by (51)V and (1)H NMR and in the solid-state by X-ray diffraction and (51)V NMR. Density Functional Theory (DFT) calculations were performed to evaluate the experimental parameters and further describes the electronic structure of the complex. The small structural changes that do occur in bond lengths and angles and partial charges on different atoms are minor compared to the charge features that are responsible for the majority of the electric field gradient tensor. The EPR parameters of the vanadium(IV) complex were characterized and compared to the corresponding dipicolinate complex. The chemical properties of the chlorodipicolinate compounds are discussed and correlated with their insulin-enhancing activity in streptozoticin (STZ) induced diabetic Wistar rats. The effect of the chloro-substitution on lowering diabetic hyperglycemia was evaluated and differences were found depending on the compounds oxidation state similar as was observed for the vanadium III, IV and V dipicolinate complexes (P. Buglyo, D.C. Crans, E.M. Nagy, R.L. Lindo, L. Yang, J.J. Smee, W. Jin, L.-H. Chi, M.E. Godzala III, G.R. Willsky, Inorg. Chem. 44 (2005) 5416-5427). However, a linear correlation of oxidation states with efficacy was not observed, which suggests that the differences in mode of action are not simply an issue of redox equivalents. Importantly, our results contrast the previous observation with the vanadium-picolinate complexes, where the halogen substituents increased the insulin-enhancing properties of the complex (T. Takino, H. Yasui, A. Yoshitake, Y. Hamajima, R. Matsushita, J. Takada, H. Sakurai, J. Biol. Inorg. Chem. 6 (2001) 133-142).

Cellular Metabolism and Macrophage Functional Polarization

Macrophages are a functionally heterogeneous cell population that is mainly shaped by a variety of microenvironmental stimuli. Interferon γ (IFN-γ), interleukin-1β (IL-1β), and lipopolysaccharide (LPS) induce a classical activation of macrophages (M1), whereas IL-4 and IL-13 induce an alternative activation program in macrophages (M2). Reprogramming of intracellular metabolisms is required for the proper polarization and functions of activated macrophages. Similar to the Warburg effect observed in tumor cells, M1 macrophages increase glucose consumption and lactate release and decreased oxygen consumption rate. In comparison, M2 macrophages mainly employ oxidative glucose metabolism pathways. In addition, fatty acids, vitamins, and iron metabolisms are also related to macrophage polarization. However, detailed metabolic pathways involved in macrophages have remained elusive. Understanding the bidirectional interactions between cellular metabolism and macrophage functions in physiological and pathological situations and the regulatory pathways involved may offer novel therapies for macrophage-associated diseases.

CXCL12 Enhances Human Neural Progenitor Cell Survival Through a CXCR7‐ and CXCR4‐Mediated Endocytotic Signaling Pathway

Chemokine CXCL12 is widely expressed in the central nervous system and essential for the proper functioning of human neural progenitor cells (hNPCs). Although CXCL12 is known to function through its receptor CXCR4, recent data have suggested that CXCL12 binds to chemokine receptor CXCR7 with higher affinity than to CXCR4. However, little is known about the function of CXCR7 in hNPCs. Using a primary hNPC culture system, we demonstrated that CXCL12 promotes hNPC survival in the events of camptothecin‐induced apoptosis or growth factor deprivation, and that this effect requires both CXCR7 and CXCR4. Through fluorescence‐activated cell sorting analysis and immunocytochemistry, we determined that CXCR7 is mainly localized in the early endosome, while CXCR4 is more broadly expressed at the cell surface and on both early and recycling endosomes. Furthermore, we found that endocytosis is required for the prosurvival function of CXCL12. Using dual‐color total internal reflection fluorescence microscopy and immunoprecipitation, we demonstrated that CXCR7 quickly trafficks to plasma membrane in mediating CXCL12 endocytosis and colocalizes with CXCR4 after CXCL12 treatment. Investigating the molecular mechanisms, we found that ERK1/2 endocytotic signaling pathway is essential for hNPC survival upon apoptotic challenges. Consistent with these findings, a significantly higher number of apoptotic NPCs were found in the developing brain of CXCR7 knockout mice. In conclusion, CXCL12 protects hNPCs from apoptotic challenges through CXCR7‐ and CXCR4‐mediated endocytotic signaling. Since survival of hNPCs is important for neurogenesis, CXCR7 may become a new therapeutic target to properly regulate critical processes of brain development. STEM CELLS2012;30:2571–2583

The phenotype and functional alterations of macrophages in mice with hyperglycemia for long term

Abnormal immunity and its related complications are the major causes of mortality and morbidity in diabetes patients. Macrophages, as one of the important innate cells, play pivotal roles in controlling immune homeostasis, immunity, and tolerance. The effects of hyperglycemia on the function of macrophages in hosts remain to be determined. Here we used mice with streptozotocin (STZ)‐induced diabetes for long term to study the changes of macrophages. We found that F4/80+ peritoneal exudate macrophages (PEMs) from mice with diabetes for 4 months displayed significantly reduced CD86 and CD54 expression and tumor necrosis factor (TNF)‐α and IL‐6 production but enhanced nitric oxide (NO) secretion compared with control mice when treated with interferon (IFN)‐γ and lipopolysaccharide (LPS), while the activity of arginase in PEMs from diabetic mice was significantly higher than control mice when stimulating with IL‐4. These dysfunctions of macrophages could be efficiently reversed by insulin treatment. Importantly, in vitro bone marrow‐derived macrophages showed similar functional changes, indicating the epigenetic alteration of macrophage precursors in these mice. In an in vitro culture system, high glucose and insulin significantly altered TNF‐α, IL‐6, and NO production and arginase activity of macrophages, which was reversed by the treatment with AKT and ERK inhibitors. Therefore, hyperglycemia and insulin deficiency can modify macrophage function through AKT‐mTOR and ERK pathways and through epigenetic effects on macrophage precursors. To further identify different components of diabetes on the dysfunction of macrophages is important for efficient prevention of diabetic complications. J. Cell. Physiol. 227: 1670–1679, 2012.

PM2.5‐induced oxidative stress increases adhesion molecules expression in human endothelial cells through the ERK/AKT/NF‐κB‐dependent pathway

The aim of this study was to explore the intracellular mechanisms underlying the cardiovascular toxicity of air particulate matter (PM) with an aerodynamic diameter of less than 2.5 µm (PM2.5) in a human umbilical vein cell line, EA.hy926. We found that PM2.5 exposure triggered reactive oxygen species (ROS) generation, resulting in a significant decrease in cell viability. Data from Western blots showed that PM2.5 induced phosphorylation of Jun N‐terminal kinase (JNK), extracellular signal regulatory kinase (ERK), p38 mitogen‐activated protein kinase (MAPK) and protein kinase B (AKT), and activation of nuclear factor kappa B (NF‐κB). We further observed a significant increase in expressions of intercellular adhesion molecule‐1 (ICAM‐1) and vascular adhesion molecule‐1 (VCAM‐1) in a time‐ and dose‐dependent manner. Moreover, the adhesion of monocytic THP‐1 cells to EA.hy926 cells was greatly enhanced in the presence of PM2.5. However, N‐acetylcysteine (NAC), a scavenger of ROS, prevented the increase of ROS generation, attenuated the phosphorylation of the above kinases, and decreased the NF‐κB activation as well as the expression of ICAM‐1 and VCAM‐1. Furthermore, ERK inhibitor (U0126), AKT inhibitor (LY294002) and NF‐κB inhibitor (BAY11‐7082) significantly down‐regulated PM2.5‐induced ICAM‐1 and VCAM‐1 expression as well as adhesion of THP‐1 cells, but not JNK inhibitor (SP600125) and p38 MAPK inhibitor (SB203580), indicating that ERK/AKT/NF‐κB is involved in the signaling pathway that leads to PM2.5‐induced ICAM‐1 and VCAM‐1 expression. These findings suggest PM2.5‐induced ROS may function as signaling molecules triggering ICAM‐1 and VCAM‐1 expressions through activating the ERK/AKT/NF‐κB‐dependent pathway, and further promoting monocyte adhesion to endothelial cells. Copyright

Biodegradable polymeric microcarriers with controllable porous structure for tissue engineering.

Porous microspheres fabricated by biodegradable polymers show great potential as microcarriers for cell cultivation in tissue engineering. Herein biodegradable poly(DL-lactide) (PLA) was used to fabricate porous microspheres through a modified double emulsion solvent evaporation method. The influence of fabrication parameters, such as the stirring speed of the primary and secondary emulsion, the polymer concentration of the oil phase, and solvent type, as well as the post-hydrolysis treatment of the porous structure of the PLA microspheres are discussed. Good attachment and an active spread of MG-63 cells on the microspheres is observed, which indicates that the PLA microspheres with controllable porous structure are of great potential as cell delivery carriers for tissue engineering.