Sanghyun Lee

Extracellular HMGB1 Released by NMDA Treatment Confers Neuronal Apoptosis via RAGE-p38 MAPK/ERK Signaling Pathway

High mobility group box 1 (HMGB1) was originally identified as ubiquitously expressed nonhistone DNA-binding protein, but recently, it was found to act as an endogenous danger molecule, which signals danger and traumatic cell death. Previously, the authors showed that HMGB1 is massively released immediately after an ischemic insult and that it subsequently activates microglia and induces inflammation in the postischemic brain. Here, we showed the endogenous danger molecule-like function of HMGB1 in primary cortical cultures. HMGB1 was found to be accumulated in NMDA-treated primary cortical culture media, and media collected from these cultures were able to induce neuronal cell death when added to fresh primary cortical cultures. However, HMGB1-depleted NMDA-conditioned media produced by HMGB1 siRNA transfection or by preincubation with anti-HMGB1 antibody or with HMGB1 A box failed to induce neuronal cell death. Furthermore, siRNA-mediated HMGB1 knockdown substantially suppressed NMDA- or Zn2+-induced cell death. It was interesting to find that extracellular HMGB1-induced neuronal apoptosis, as evidenced by TUNEL staining and caspase 3 assay in combination with double immunofluorescence staining. A series of RAGE and HMGB1 co-immunoprecipitation experiments in the presence of SB203580 and PD98059 (p38 MAPK and ERK inhibitors, respectively) demonstrated that RAGE-p38 MAPK and RAGE-ERK pathway might underlie extracellular HMGB1-mediated neuronal apoptosis. These results together with our previous reports regarding microglial activation by extracellular HMGB1 indicate that HMGB1 functions as a novel danger signal, which aggravates brain damage via autocrine and paracrine manners.

The effect of biodegradable gelatin microspheres on the neuroprotective effects of high mobility group box 1 A box in the postischemic brain.

High mobility group box 1 (HMGB1) is a family of endogenous molecules that is released by necrotic cells and causes neuronal damages by triggering inflammatory processes. In the cerebral ischemic brain, sustained and regulated suppression of HMGB1 has been emerged as a therapeutic means to grant neuroprotection. HMGB1 consists of two HMG boxes (A and B) and an acidic C-terminal tail, and the A box peptide antagonistically competes with HMGB1 for its receptors. In the middle cerebral artery occlusion (MCAO) in rats, a murine model of transient cerebral ischemia, administration of HMGB1 A box intraparenchymally, after encapsulated in biodegradable gelatin microspheres (GMS), which enhances the stability of peptide inside and allows its sustained delivery, at 1 h, 3 h, or 6 h after MCAO, reduced mean infarct volumes by, respectively, 81.3%, 42.6% and 30.7% of the untreated MCAO-brain, along with remarkable improvement of neurological deficits. Furthermore, the administration of HMGB1 A box/GMS suppressed proinflammatory cytokine inductions more strongly than the injection of non-encapsulated HMGB1 A box. Given that insulted brains-like ischemia have enhanced gelatinase activity than the normal brain, our results suggest that GMS-mediated delivery of therapeutic peptides is a promising means to provide efficient neuroprotection in the postischemic brain.

Effect of hypoxia-inducible VEGF gene expression on revascularization and graft function in mouse islet transplantation

For gene transfer strategies to improve islet engraftment, vascular endothelial growth factor (VEGF) expression should be regulated in a way that matches the transient nature of revascularization with simultaneously avoiding undesirable effects of overexpression. The aim of this study was to investigate the effects of hypoxia‐inducible VEGF gene transfer using the RTP801 promoter on islet grafts. We implanted pSV‐hVEGF transfected, pRTP801‐hVEGF transfected or nontransfected mouse islets under the kidney capsule of streptozotocin‐induced diabetic syngeneic mice. Human VEGF immunostaining of day 3 grafts revealed that the pRTP801‐hVEGF transfected group had higher hVEGF expression compared with the pSV‐hVEGF transfected group. BS‐1 staining of day 3 grafts from the pRTP801‐hVEGF transfected group showed the highest vascular density, which was comparable with day 6 grafts from the nontransfected group. In 360 islet equivalent (IEQ)‐transplantation which reverted hyperglycemia in all mice, the area under the curve of glucose levels during intraperitoneal glucose tolerance test 7 weeks post‐transplant was lower in mice transplanted with pRTP801‐hVEGF transfected grafts compared with mice transplanted with nontransfected grafts. In 220 IEQ‐transplantations, diabetic mice transplanted with pRTP801‐hVEGF islets became normoglycemic more rapidly compared with mice transplanted with pSV‐hVEGF or nontransfected islets, and diabetes reversal rate after 50 days was 90%, 68%, and 50%, respectively. In conclusion, our results indicate that regulated overexpression of hVEGF in a hypoxia‐inducible manner enhances islet vascular engraftment and preserves islet function overtime in transplants.

Enhanced protection of Ins-1 β cells from apoptosis under hypoxia by delivery of DNA encoding secretion signal peptide-linked exendin-4

In this study, we developed an expression system of exendin-4, a glucagon-like peptide (GLP-1) analog, using a secretion signal peptide (SP) to facilitate exendin-4 secretion. For delivery of the exendin-4 expression system, high-molecular-weight polyethylenimine (25 kDa, PEI25k), low-molecular-weight polyethylenimine (2 kDa, PEI2k), and polyamidoamine (PAMAM) dendrimers were evaluated as gene carriers to Ins-1 β cells. As a result, PEI25k showed the highest transfection efficiency. For the construction of the exendin-4 expression vector, DNA coding the SP sequence was inserted upstream of the exendin-4 cDNA, resulting in the construction of pβ-SP-Ex-4. Transfection assay showed that the secretion level of exendin-4 increased in the pβ-SP-Ex-4 transfected cells, compared with the pβ-Ex-4 transfected cells. To identify the β-cell protection effect of pβ-SP-Ex-4 delivery, the Ins-1 β cells were transfected with pβ-SP-Ex-4 or pβ-Ex-4 and incubated under normoxia or hypoxia. An MTT assay showed that the pβ-SP-Ex-4 transfected cells had higher β-cell viability than the pβ-Ex-4 transfected cells under hypoxia. In addition, the pβ-SP-Ex-4 transfected cells exhibited lower caspase-3 activity than the pβ-Ex-4 transfected cells. Therefore, PEI25k/pβ-SP-Ex-4 complex may be useful to protect isolated β cells from apoptosis during transplantation.

Antitumor activity of methyl gallate by inhibition of focal adhesion formation and Akt phosphorylation in glioma cells

BACKGROUND Methyl gallate (MG) possesses a wide range of biological properties that include anti-oxidant, anti-inflammatory, and anti-microbial activities. However, its anti-tumor activity has not been extensively examined in cancer cells. Thus, we examined the effect of MG in both glutamate-induced rat C6 and human U373 glioma cell proliferation and migration. METHODS MG was isolated from the stem bark of Acer barbinerve. Cell viability and migration were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and scratch wound-healing assay, respectively. Focal adhesion formation was detected with immunofluorescence. RESULTS Treatment of C6 and U373 glioma cells with MG significantly reduced cell viability, migration, and Akt phosphorylation level. Glutamate stimulation markedly increased the level of ERK1/2 phosphorylation. However, cells treated with MG displayed decreased ERK1/2 phosphorylation. Inhibition of ERK1/2 by MG or MEK1/2 inhibitor significantly inhibited paxillin phosphorylation at Ser(83) and focal adhesion turn-over produced inefficient glioma cell migration. In addition, activation of Akt and ERK1/2 upon glutamate stimulation was independently regulated by Ca(2+) and protein kinase C activity, respectively, via the α-amino-3-hydroxy-5-methy-4-isoxazolepropionate acid glutamate receptor and metabotropic glutamate receptor. GENERAL SIGNIFICANCE Our results clearly indicate that MG has a strong anti-tumor effect through the down-regulation of the Akt and ERK1/2 signaling pathways. Thus, methyl gallate is a potent anti-tumor and novel therapeutic agent for glioma.

Combined delivery of dexamethasone and plasmid DNA in an animal model of LPS-induced acute lung injury

Dexamethasone was conjugated to low molecular weight polyethylenimine (2kDa, PEI2k). Dexamethasone conjugated PEI2k (PEI2k-Dexa) was evaluated as a combined delivery carrier of dexamethasone and plasmid DNA (pDNA) in an animal model of lipopolysaccharide (LPS) induced acute lung injury (ALI). In vitro transfection of L2 lung epithelial cells, PEI2k-Dexa exhibited higher transfection efficiency than PEI2k or a simple mixture of PEI2k and dexamethasone. In addition, the PEI2k-Dexa/pβ-Luc complexes reduced the levels of pro-inflammatory cytokines in LPS activated Raw 264.7 macrophage cells. The anti-inflammatory effect of PEI2k-Dexa was higher than that of controls. The PEI2k-Dexa/pβ-Luc complexes were administered to mice via intratracheal injection. PEI2k-Dexa had higher pDNA delivery efficiency than PEI2k in the lung and decreased TNF-α and IL-6 in the lung homogenates and bronchoalveolar lavage (BAL) fluid compared with the controls. Furthermore, total protein and immunoglobulin M (IgM) concentrations in BAL fluid were reduced by the PEI2k-Dexa/pβ-Luc complexes. The intratracheal injection of the PEI2k-Dexa/pcDNA-EGFP complexes in the ALI model showed higher EGFP expression compared with PEI2k. Hematoxylin and eosin (H&E) staining showed that PEI2k-Dexa reduced inflammatory reaction in the lung. Therefore, PEI2k-Dexa may be useful for combination gene and drug therapy for ALI.

Comparisons of the Pentax-AWS, Glidescope, and Macintosh Laryngoscopes for Intubation Performance during Mechanical Chest Compressions in Left Lateral Tilt: A Randomized Simulation Study of Maternal Cardiopulmonary Resuscitation

Purpose. Rapid advanced airway management is important in maternal cardiopulmonary resuscitation (CPR). This study aimed to compare intubation performances among Pentax-AWS (AWS), Glidescope (GVL), and Macintosh laryngoscope (MCL) during mechanical chest compression in 15° and 30° left lateral tilt. Methods. In 19 emergency physicians, a prospective randomized crossover study was conducted to examine the three laryngoscopes. Primary outcomes were the intubation time and the success rate for intubation. Results. The median intubation time using AWS was shorter than that of GVL and MCL in both tilt degrees. The time to visualize the glottic view in GVL and AWS was significantly lower than that of MCL (all P < 0.05), whereas there was no significant difference between the two video laryngoscopes (in 15° tilt, P = 1; in 30° tilt, P = 0.71). The progression of tracheal tube using AWS was faster than that of MCL and GVL in both degrees (all P < 0.001). Intubations using AWS and GVL showed higher success rate than that of Macintosh laryngoscopes. Conclusions. The AWS could be an appropriate laryngoscope for airway management of pregnant women in tilt CPR considering intubation time and success rate.

Proper target depth of an accelerometer-based feedback device during CPR performed on a hospital bed: a randomized simulation study

PURPOSE Feedback devices are used to improve chest compression (CC) quality related to survival rates in cardiac arrest. However, several studies have shown that feedback devices are not sufficiently reliable to ensure adequate CC depth on soft surfaces. Here, we determined the proper target depth of feedback (TDF) using an accelerometer during cardiopulmonary resuscitation in hospital beds. METHODS In prospective randomized crossover study, 19 emergency physicians performed CCs for 2 minutes continuously on a manikin in 2 different beds with 3 TDFs (5, 6, and 7 cm). We measured CC depth, the proportion of accurate compression depths, CC rate, the proportion of incomplete chest decompressions, the velocity of CC (CC velocity), the proportion of time spent in CC relative to compression plus decompression (duty cycle), and the time spent in CC (CC time). RESULTS Mean (SD) CC depths at TDF 5, 6, and 7 were 45.42 (5.79), 52.68 (4.18), and 58.47 (2.48) on one bed and 46.26 (4.49), 53.58 (3.15), and 58.74 (2.10) mm on the other bed (all P<.001), respectively. The proportions of accurate compression depths and CC velocity at TDF 5, 6, and 7 differed significantly according to TDF on both beds (all P<.001).The CC rate, CC time, and proportion of incomplete chest decompression did not differ on both beds (all P>.05). The duty cycle differed significantly on only B2. CONCLUSIONS The target depth of the real-time feedback device should be at least 6 cm but should not exceed 7 cm for optimal CC on patients on hospital beds.

Comparison of the Pentax Airwayscope, Glidescope Video Laryngoscope, and Macintosh Laryngoscope During Chest Compression According to Bed Height.

Abstract We aimed to investigate whether bed height affects intubation performance in the setting of cardiopulmonary resuscitation and which type of laryngoscope shows the best performance at each bed height. A randomized crossover manikin study was conducted. Twenty-one participants were enrolled, and they were randomly allocated to 2 groups: group A (n = 10) and group B (n = 11). The participants underwent emergency endotracheal intubation (ETI) using the Airwayscope (AWS), Glidescope video laryngoscope, and Macintosh laryngoscope in random order while chest compression was performed. Each ETI was conducted at 2 levels of bed height (minimum bed height: 68.9 cm and maximum bed height: 101.3 cm). The primary outcomes were the time to intubation (TTI) and the success rate of ETI. The P value for statistical significance was set at 0.05 and 0.017 in post-hoc test. The success rate of ETI was always 100% regardless of the type of laryngoscope or the bed height. TTI was not significantly different between the 2 bed heights regardless of the type of laryngoscope (all P > 0.05). The time for AWS was the shortest among the 3 laryngoscopes at both bed heights (13.7 ± 3.6 at the minimum bed height and 13.4 ± 4.7 at the maximum bed height) (all P < 0.017). The TTI of Glidescope video laryngoscope was not significantly shorter than that of Macintosh laryngoscope at the minimum height (17.6 ± 4.0 vs 19.6 ± 4.8; P = 0.02). The bed height, whether adjusted to the minimum or maximum setting, did not affect intubation performance. In addition, regardless of the bed height, the intubation time with the video laryngoscopes, especially AWS, was significantly shorter than that with the direct laryngoscope during chest compression.

The box a domain of high mobility group box-1 protein as an efficient siRNA carrier with anti-inflammatory effects

High mobility group box‐1 (HMGB‐1) is a DNA binding nuclear protein and pro‐inflammatory cytokine. The box A domain of HMGB‐1 (rHMGB‐1A) exerts an anti‐inflammatory effect, inhibiting wild‐type HMGB‐1 (wtHMGB‐1). In this study, HMGB‐1A was evaluated as an siRNA carrier with anti‐inflammatory effects. HMGB‐1A was expressed and purified by consecutive nickel chelate chromatography, cationic exchange chromatography, and polymixin B chromatography. Purified rHMGB‐1A demonstrated an anti‐inflammatory effect, reducing tumor necrosis factor‐α (TNF‐α) in wtHMGB‐1 or lipopolysaccharide (LPS) activated macrophages. In gel retardation assay, rHMGB‐1A formed a stable complex with siRNA at or above a 1:2 weight ratio (siRNA:rHMGB‐1A). A heparin competition assay showed that an siRNA/rHMGB‐1A complex released siRNA more easily than an siRNA/polyethylenimine (PEI, 25 kDa) complex. Luciferase siRNA/rHMGB‐1A reduced firefly luciferase expression at a similar level as luciferase siRNA/PEI complex. Furthermore, TNF‐α siRNA/rHMGB‐1A synergistically reduced TNF‐α expression in LPS activated macrophages. Therefore, rHMGB‐1A may be useful as an siRNA carrier with anti‐inflammatory effects in siRNA therapy for various inflammatory diseases. J. Cell. Biochem. 113: 122–131, 2012.