Eun-Ju Choi

Effect of the protein corona on nanoparticles for modulating cytotoxicity and immunotoxicity

Although the cytotoxicity of nanoparticles (NPs) is greatly influenced by their interactions with blood proteins, toxic effects resulting from blood interactions are often ignored in the development and use of nanostructured biomaterials for in vivo applications. Protein coronas created during the initial reaction with NPs can determine the subsequent immunological cascade, and protein coronas formed on NPs can either stimulate or mitigate the immune response. Along these lines, the understanding of NP-protein corona formation in terms of physiochemical surface properties of the NPs and NP interactions with the immune system components in blood is an essential step for evaluating NP toxicity for in vivo therapeutics. This article reviews the most recent developments in NP-based protein coronas through the modification of NP surface properties and discusses the associated immune responses.

Integrin-Linked Kinase Is Required in Hypoxic Mesenchymal Stem Cells for Strengthening Cell Adhesion to Ischemic Myocardium†‡

Mesenchymal stem cells (MSCs) therapy has limitations due to the poor viability of MSCs after cell transplantation. Integrin‐mediated adhesion is a prerequisite for cell survival. As a novel anti‐death strategy to improve cell survival in the infarcted heart, MSCs were genetically modified to overexpress integrin‐linked kinase (ILK). The survival rate of ILK‐transfected MSCs (ILK‐MSCs) was augmented by about 1.5‐fold and the phosphorylation of ERK1/2 and Akt in ILK‐MSCs were increased by about three and twofold, respectively. ILK‐MSCs demonstrated an increase of twofold in the ratio of Bcl‐2/Bax and inhibited caspase‐3 activation, compared with hypoxic MSCs. The adhesion rate of ILK‐MSCs also had a 32.2% increase on the cardiac fibroblast‐derived three‐dimensional matrix and ILK‐MSCs showed higher retention by about fourfold compared to unmodified MSCs. Six animals per group were used for the in vivo experiments analyzed at 1 week after occlusion of the left coronary artery. ILK‐MSC transplanted rats had a 12.0% ± 3.1% smaller infarct size than MSC‐treated rats after ligation of left anterior descending coronary artery. Transplantation of ILK‐MSCs not only led to a 16.0% ± 0.4% decrease in the fibrotic heart area, but also significantly reduced the apoptotic positive index by two‐thirds when compared with ligation only. The mean microvessel count per field in the infarcted myocardium of ILK‐MSCs group was increased relative to the sham group and MSCs group. In conclusion, the ILK gene transduction of MSCs further assisted cell survival and adhesion, and improved myocardial damage when compared with MSC only after transplantation. STEM CELLS 2009;27:1358–1365

Eupatilin Protects Gastric Epithelial Cells from Oxidative Damage and Down-Regulates Genes Responsible for the Cellular Oxidative Stress

PurposeThe formulated ethanol extract (DA-9601) of Artemisia asiatica has pronounced anti-inflammatory activities and exhibits cytoprotective effects against gastrointestinal damage. Here we investigated whether eupatilin, a major component of DA-9601, has a property of antioxidant activity and protects gastric epithelial cells from H2O2-induced damage.MethodsThe protective effect of eupatilin against H2O2-induced damages was studied in gastric epithelial AGS cells by measuring wound healing, cell proliferation, and cell viability. Global gene expression profiling was obtained by high-density microarray.ResultsHydrogen peroxide significantly delayed epithelial migration in wounded area. In contrast, eupatilin prevented the reduction of epithelial migration induced by H2O2. Eupatilin also ameliorated H2O2-induced actin disruption in AGS cells. Interestingly, treatment with eupatilin dramatically inhibited FeSO4-induced ROS production in a dose-dependent manner. In addition, eupatilin protected cells from FeSO4-induced F-actin disruption. With high-density microarray, we identified dozens of genes whose expressions were up-regulated in H2O2-treated cells. We found that eupatilin reduces the expression of such oxidative-responsible genes as HO-1, PLAUR and TNFRSF10A in H2O2-treated cells.ConclusionThese results suggest that eupatilin acts as a novel antioxidant and may play an important role in DA-9601-mediated effective repair of the gastric mucosa.

Eupatilin inhibits lipopolysaccharide-induced expression of inflammatory mediators in macrophages

AIMSnEupatilin (5,7-dihydroxy-3,4,6-trimethoxyflavone) is a pharmacologically active ingredients in Stillen(TM), a drug for the gastric mucosal ulcers. Eupatilin has been known to possess anti-peptic, anti-cancer, and anti-allergy activity. In this report, we defined the effect of eupatilin on the endotoxin-induced inflammation in lipopolysaccharide (LPS)-stimulated macrophages.nnnMAIN METHODSnMouse J774A.1 cell line and mouse peritoneal macrophages were used. Gene expression and production of inflammatory mediators were determined by real-time PCR and Western blot.nnnKEY FINDINGSnEupatilin dose-dependently suppressed LPS-induced expression of inducible nitric oxide synthase (iNOS) and production of nitric oxide (NO). Eupatilin decreased LPS-induced expression of inflammatory mediators and pro-inflammatory cytokines such as cyclooxygenase-2, monocyte chemoattractant protein-1, tumor necrosis factor-α, interleukin (IL)-1β and IL-6. In addition, this suppression of inflammatory mediators was nuclear factor (NF)-κB dependent.nnnSIGNIFICANCEnOur findings imply that eupatilin suppresses inflammatory responses by the inhibition of NF-κB signaling pathway, and downstream inflammatory mediators in endotoxin-stimulated macrophages.

Suppression of dust mite extract and 2,4-dinitrochlorobenzene-induced atopic dermatitis by the water extract of Lindera obtusiloba.

ETHNOPHARMACOLOGICAL RELEVANCEnThe Lindera obtusiloba has been used in traditional medicine for the treatment of inflammation and dermatitis. In this study, we investigated the effect of topical application of Lindera obtusiloba water extract (LOWE) on the house dust mite extract (Dermatophagoides farinae extract, DFE) and 2,4-dinitrochlorobenzene (DNCB)-induced atopic dermatitis (AD).nnnMATERIALS AND METHODSnWe established AD model in BALB/c mice by repeated local exposure of DFE/DNCB to the ears. After a topical application of LOWE on the skin lesions, the epidermal thickness, mast cell infiltration, and serum immunoglobulin E (IgE) and histamine were measured. In addition, the gene expression of interleukin (IL)-4, IL-13, IL-31, and tumor necrosis factor (TNF)-α in the ears was assayed.nnnRESULTSnLOWE reduced AD symptoms based on ear thickness, histopathological analysis, and serum IgE levels. LOWE inhibited mast cell infiltration into the ear and elevation of serum histamine in AD model. Moreover, LOWE suppressed DFE/DNCB-induced expression of IL-4, IL-13, IL-31, and TNF-α in the ears.nnnCONCLUSIONSnOur results showed that topical application of LOWE exerts beneficial effects in AD symptoms, suggesting that LOWE might be a candidate for the treatment of AD.

DA-9601 suppresses 2, 4-dinitrochlorobenzene and dust mite extract-induced atopic dermatitis-like skin lesions.

DA-9601 (Stillen™) is a novel anti-peptic formulation prepared from the ethanol extracts of Artemisia asiatica possessing anti-oxidative, anti-allergic and anti-inflammatory activities. However, their effect on atopic dermatitis (AD) has not been studied yet. In this study, we report that topical application of DA-9601 suppressed house dust mite extract (Dermatophagoides farinae extract, DFE) and 2, 4-dinitrochlorobenzene (DNCB)-induced AD-like skin lesions in BALB/c mice model. We established atopic dermatitis model in BALB/c mice by repeated local exposure of DFE/DNCB to the ears. Repeated alternative treatment of DFE/DNCB caused AD-like lesions. DA-9601 reduced AD-like skin lesions based on ear thickness and histopathological analysis, and serum IgE levels. DA-9601 inhibited mast cell infiltration into the ear and elevation of serum histamine in AD model. In addition, DA-9601 suppressed DFE/DNCB-induced expression of IL-4, IL-13, IL-31, and TNF-α in the ears. Taken together, our results showed that topical application of DA-9601 exerts beneficial effects in animal model of AD, suggesting that DA-9601 might be a candidate for the treatment of AD.

Rosuvastatin inhibits norepinephrine-induced cardiac hypertrophy via suppression of Gh

Statins have recently been shown to produce anti-cardiac hypertrophic effects via the regulation of small GTPases. However, the effects of statins on G protein-mediated cardiac hypertrophy, which is the main pathway of cardiac hypertrophy, have not yet been studied. We sought to evaluate whether statin treatment directly suppresses cardiac hypertrophy through a large G protein-coupled pathway regardless of the regulation of small GTPases. Using neonatal rat cardiomyocytes, we evaluated norepinephrine-induced cardiac hypertrophy for suppressibility of rosuvastatin and the pathways involved by analyzing total protein/DNA content, cell surface area, immunoblotting and RT-PCR for the signal transduction molecule. In a concentration-dependent manner, rosuvastatin inhibited total protein synthesis and downregulated basal and norepinephrine-induced expressions of myosin light chain2 and the c-fos proto-oncogene in cardiomyocytes. Treatment with norepinephrine induced cardiac hypertrophy accompanied by G(h) expression and membrane translocation. Rosuvastatin inhibited G(h) protein activity in cardiomyocytes by inhibiting basal and norepinephrine-stimulated mRNA transcription, protein expression and membrane translocation; however, norepinephrine-stimulated G(q) protein expression was not inhibited. In addition, the norepinephrine-stimulated protein kinase C (PKC)-mitogen-activated protein kinase (MEK 1,2)-extracellular signal-regulated kinases (ERKs) signaling cascade was inhibited by pretreatment with rosuvastatin. Rosuvastatin treatment also helped maintain expression levels of SERCA2a and intracellular calcium concentration. G(h) protein is a novel target of statins in myocardial hypertrophy, and statin treatment may directly suppress cardiac hypertrophy through a large G(h) protein-coupled pathway regardless of the regulation of small GTPases.

The Inhibition of Insulin-stimulated Proliferation of Vascular Smooth Muscle Cells by Rosiglitazone Is Mediated by the Akt-mTOR-P70S6K Pathway

PURPOSEnThiazolidinediones (TZDs) are known to inhibit the proliferation of vascular smooth muscle cell (VSMC) by increasing the activity of p27Kip1 and retinoblastoma protein (RB). However, the upstream signaling mechanisms associated with this pathway have not been elucidated. The Akt-mTOR-P70S6 kinase pathway is the central regulator of cell growth and proliferation, and increases cell proliferation by inhibiting the activities of p27Kip1 and retinoblastoma protein (RB). Therefore, we hypothesized in this study that rosiglitazone inhibits VSMC proliferation through the inhibition of the Akt-TOR-P70S6K signaling pathway.nnnMATERIALS AND METHODSnRat aortic smooth muscle cells (RAoSMCs) were treated with 10microM of rosiglitazone 24 hours before the addition of insulin as a mitogenic stimulus. Western blot analysis was performed to determine the inhibitory effect of rosiglitazone treatment on the Akt-mTOR-P70S6K signaling pathway. Carotid balloon injury was also performed in Otsuka Long-Evans Tokushima Fatty (OLETF) diabetic rats that were pretreated with 3 mg/kg of rosiglitazone.nnnRESULTSnWestern blot analysis demonstrated significant inhibition of activation of p-Akt, p-m-TOR, and p-p70S6K in cells treated with rosiglitazone. The inhibition of the activation of the p-mTOR-p-p70S6K pathway seemed to be mediated by both the upstream PI3K pathway and MEK-ERK complex.nnnCONCLUSIONnThe inhibitory effect of rosiglitazone on RAoSMC proliferation in vitro and in vivo is mediated by the inhibition of the Akt-mTOR-P70S6K pathway.

Phytocomponent p-Hydroxycinnamic acid inhibits T-cell activation by modulation of protein kinase C-θ-dependent pathway.

The phytocomponent p-hydoxycinnamic acid (HCA) has been shown to have many beneficial effects in terms of antioxidant activity, inhibition of melanogenesis, bone resorption, and platelet activity, and stimulation of mineralization. However, effects of HCA in immune functions have not been investigated. Here, we show that HCA has a profound effect on IL-2 production in Jurkat T cells as well as in human peripheral blood leukocytes. HCA, at a concentration that optimally inhibits IL-2 production, had little effect on apoptotic or necrotic cell death of Jurkat T cells, suggesting that apoptosis is not a mechanism for HCA-induced T-cell suppression. On the contrary, HCA dramatically inhibited PKC-θ accumulation and further phosphorylation at the immunological synapse which formed at the contact site between T cells and superantigen SEE-loaded antigen presenting cells. In addition, HCA significantly inhibited ERK and p38 kinase phosphorylation in both anti-CD3/28- and PMA/A23187-stimulated T cells. Consequently, HCA inhibited both AP-1 and NF-κB promoter activities in Jurkat T cells. Collectively, our results provide evidence for the immunosuppressive effect of HCA on activated T cells, through modulation of PKC-θ pathway.

Gene induction by glycyrol to apoptosis through endonuclease G in tumor cells and prediction of oncogene function by microarray analysis.

Glycyrrhiza uralensis (Leguminosae) has long been known as an antiinflammatory agent for gastric ulcers, arthritis, and rheumatism. The flavonoid glycyrol (GC) (10u2009μg/ml) isolated from G. uralensis dramatically inhibits phorbol ester (phorbol 12-myristate 13-acetate)-induced nuclear factor (NF)-κB-dependent transcriptional activity, as determined by luciferase reporter activity in human kidney epithelial 293T cells. To investigate global gene expression profiling in cells by GC, we performed high-density oligonucleotide microarrays. Our microarray analyses showed that GC inhibited phorbol ester-induced NF-κB-dependent transcriptional activity in inflammatory-related gene expression. RT-PCR analysis, based on microarray data, showed that NF-κB-dependent genes (such as CCL2, CCL7, CD44, and HSPB8 in addition to NF-κB itself) were significantly downregulated by GC. Treatment with GC (10u2009μg/ml) inhibited I-κB degradation induced by phorbol 12-myristate 13-acetate. The microarray data also suggested that GC induces gene expression to p53-dependent apoptosis through endonuclease G, instead of CAD/DFF and AIF/PDCD8, as a downstream-apoptosis factor in human kidney epithelial 293T tumor cells, and induces oncogenes with a suppressor role as an added function.