Chan Wha Kim

Dexamethasone coordinately regulates angiopoietin-1 and VEGF : A mechanism of glucocorticoid-induced stabilization of blood-brain barrier

Glucocorticoids stabilize the blood-brain barrier (BBB), leading to attenuation of vasogenic brain edema. However, the action mechanism of glucocorticoids has been poorly elucidated. To elucidate the mechanism, we investigated whether dexamethasone (Dex), a synthetic glucocorticoid hormone, regulates the levels of key permeability regulating factors such as angiopoietin-1, angiopoietin-2, and vascular endothelial growth factor (VEGF) in the three types of cells comprising BBB. Dex increased the level of angiopoietin-1 mRNA and protein and decreased VEGF mRNA and protein in brain astrocytes and pericytes, but not in endothelial cells. The mRNA and protein of angiopoietin-2 were detected only in endothelial cells and not regulated by Dex. The Dex-induced regulation of angiopoietin-1 and VEGF was inhibited by RU486, suggestive of glucocorticoid receptor mediation. The mRNA stability of angiopoietin-1 and VEGF was not changed by Dex treatment, implying that Dex increases angiopoietin-1 and decreases VEGF through transcriptional regulation. This is the first study showing the coordinate regulation of angiopoietin-1 and VEGF by glucocorticoids, suggesting a novel mechanism underlying glucocorticoids-induced stabilization of BBB.

Electromagnetic fields induce neural differentiation of human bone marrow derived mesenchymal stem cells via ROS mediated EGFR activation

Even though the inducing effect of electromagnetic fields (EMF) on the neural differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs) is a distinctive, the underlying mechanism of differentiation remains unclear. To find out the signaling pathways involved in the neural differentiation of BM-MSCs by EMF, we examined the CREB phosphorylation and Akt or ERK activation as an upstream of CREB. In hBM-MSCs treated with ELF-EMF (50 Hz, 1 mT), the expression of neural markers such as NF-L, MAP2, and NeuroD1 increased at 6 days and phosphorylation of Akt and CREB but not ERK increased at 90 min in BM-MSCs. Moreover, EMF increased phosphorylation of epidermal growth factor receptor (EGFR) as an upstream receptor tyrosine kinase of PI3K/Akt at 90 min. It has been well documented that ELF-MF exposure may alter cellular processes by increasing intracellular reactive oxygen species (ROS) concentrations. Thus, we examined EMF-induced ROS production in BM-MSCs. Moreover, pretreatment with a ROS scavenger, N-acetylcystein, and an EGFR inhibitor, AG-1478, prevented the phosphorylation of EGFR and downstream molecules. These results suggest that EMF induce neural differentiation through activation of EGFR signaling and mild generation of ROS.

Proteomic and metabolomic analysis of H2O2-induced premature senescent human mesenchymal stem cells

Stress induced premature senescence (SIPS) occurs after exposure to many different sublethal stresses including H(2)O(2), hyperoxia, or tert-butylhydroperoxide. Human mesenchymal stem cells (hMSCs) exhibit limited proliferative potential in vitro, the so-called Hayflick limit. According to the free-radical theory, reactive oxygen species (ROS) might be the candidates responsible for senescence and age-related diseases. H(2)O(2) may be responsible for the production of high levels of ROS, in which the redox balance is disturbed and the cells shift into a state of oxidative stress, which subsequently leads to premature senescence with shortening telomeres. H(2)O(2) has been the most commonly used inducer of SIPS, which shares features of replicative senescence (RS) including a similar morphology, senescence-associated β-galactosidase activity, cell cycle regulation, etc. Therefore, in this study, the senescence of hMSC during SIPS was confirmed using a range of different analytical methods. In addition, we determined five differentially expressed spots in the 2-DE map, which were identified as Annexin A2 (ANXA2), myosin light chain 2 (MLC2), peroxisomal enoyl-CoA hydratase 1 (ECH1), prosomal protein P30-33K (PSMA1) and mutant β-actin by ESI-Q-TOF MS/MS. Also, proton ((1)H) nuclear magnetic resonance spectroscopy (NMR) was used to elucidate the difference between metabolites in the control and hMSCs treated with H(2)O(2). Among these metabolites, choline and leucine were identified by (1)H-NMR as up-regulated metabolites and glycine and proline were identified as down-regulated metabolites.

Transgelin Promotes Migration and Invasion of Cancer Stem Cells

Recent studies have suggested the existence of a small subset of cancer cells called cancer stem cells (CSCs), which possess the ability to initiate malignancies, promote tumor formation, drive metastasis, and evade conventional chemotherapies. Elucidation of the specific signaling pathway and mechanism underlying the action of CSCs might improve the efficacy of cancer treatments. In this study, we analyzed differentially expressed proteins between tumerigenic and nontumorigenic cells isolated from the human hepatocellular carcinoma (HCC) cell line, Huh7, via proteomic analysis to identify proteins correlated with specific features of CSCs. The expression level of Transgelin was 25-fold higher in tumorigenic cells than nontumorigenic cells. Similar results were also observed in tumorigenic cells derived from colorectal adenocarcinoma and prostate carcinoma. More importantly, the elevated levels of Transgelin significantly increased the invasiveness of tumorigenic cells, whereas reduced levels decreased the invasive potential. Moreover, in tumors derived from Huh7-induced xenografts, Transgelin was also co-expressed with CXCR4, which is responsible for tumor invasion. Taken together, these results indicate that the metastatic potential of CSCs arises from highly expressed Transgelin.

Streptozotocin-induced diabetes can be reversed by hepatic oval cell activation through hepatic transdifferentiation and pancreatic islet regeneration

Hepatic oval cells have shown the potential to transdifferentiate into insulin-producing cells when cultured with high glucose concentrations. However, it remains unknown whether the oval cells can contribute to insulin production in diabetic mice. In this study, our aim was to investigate the response of activated hepatic oval cells to hyperglycemic conditions. C57BL/6 mice were fed a diet containing 0.1% 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) for 4 weeks to activate the hepatic oval cell population before inducing hyperglycemia with streptozotocin (STZ). Despite the initial hyperglycemia (341±15 mg/dl), the blood glucose levels of DDC-STZ-treated mice were significantly improved within 6 weeks (185±12 mg/dl). During the initial hyperglycemic stage, DDC-STZ-treated livers expressed pancreatic developmental, endocrine and exocrine genes. Hepatic insulin production was confirmed by immunohistochemistry and ELISA. These results suggested that transdifferentiated hepatic oval cell population contributed to the amelioration of hyperglycemia. We additionally determined that DDC-STZ-treated pancreata played a critical role in complete reversal of hyperglycemia as evidenced by extensive β-cell regeneration and increase of pancreatic insulin content after STZ treatment, which is rarely observed in other adult STZ models. Reversal of hyperglycemia in this model seems to be accomplished by biphasic insulin augmentation, first by hepatic transdifferentiation, and followed by endogenous β-cell regeneration in the pancreas. The DDC-STZ treatment provides a novel injury model for better understanding of the functional behavior of hepatic and pancreatic stem/progenitor cell population under hyperglycemic condition, which may yield critical information for developing β-cell-based therapies to treat diabetes.

Comparison of tear proteins between healthy and early diabetic retinopathy patients

OBJECTIVES To identify potential prognostic or diagnostic marker tear proteins for early diabetic retinopathy (DR) and to investigate the pathogenesis of this disease using proteomics techniques. DESIGN AND METHODS The tear proteins expressed in patients suffering from diabetes mellitus without the retinopathy symptoms, nonproliferative diabetic retinopathy and healthy volunteers were analyzed by 2-DE. The differentially expressed proteins in patients were identified by ESI-Q-TOF and confirmed by Western blotting. RESULTS Proteins which were differentially expressed with statistical significance (P<0.05) in two diabetic groups as compared to those in healthy group were selected and identified by ESI-Q-TOF MS/MS. Among these proteins, three proteins (LCN-1, HSP27 and B2M) were found to exhibit a progressive reduction in two disease groups. The expression levels of which might be useful as diagnostic biomarkers of DR were verified by Western blotting CONCLUSIONS Proteomic analysis using tear is a novel approach that can provide insight into possible biomarker and the pathogenesis of early DR.

Extremely low-frequency electromagnetic fields induce neural differentiation in bone marrow derived mesenchymal stem cells.

Extremely low-frequency electromagnetic fields (ELF-EMF) affect numerous biological functions such as gene expression, cell fate determination and even cell differentiation. To investigate the correlation between ELF-EMF exposure and differentiation, bone marrow derived mesenchymal stem cells (BM-MSCs) were subjected to a 50-Hz electromagnetic field during in vitro expansion. The influence of ELF-EMF on BM-MSCs was analysed by a range of different analytical methods to understand its role in the enhancement of neural differentiation. ELF-EMF exposure significantly decreased the rate of proliferation, which in turn caused an increase in neuronal differentiation. The ELF-EMF-treated cells showed increased levels of neuronal differentiation marker (MAP2), while early neuronal marker (Nestin) was down-regulated. In addition, eight differentially expressed proteins were detected in two-dimensional electrophoresis maps, and were identified using ESI-Q-TOF LC/MS/MS. Among them, ferritin light chain, thioredoxin-dependent peroxide reductase, and tubulin β-6 chain were up-regulated in the ELF-EMF-stimulated group. Ferritin and thioredoxin-dependent peroxide reductase are involved in a wide variety of functions, including Ca2+ regulation, which is a critical component of neurodegeneration. We also observed that the intracellular Ca2+ content was significantly elevated after ELF-EMF exposure, which strengthens the modulatory role of ferritin and thioredoxin-dependent peroxide reductase, during differentiation. Notably, western blot analysis indicated significantly increased expression of the ferritin light chain in the ELF-EMF-stimulated group (0.60 vs. 1.08; P < 0.01). These proteins may help understand the effect of ELF-EMF stimulation on BM-MSCs during neural differentiation and its potential use as a clinically therapeutic option for treating neurodegenerative diseases.

Human β-defensin 2 is induced by interleukin-1β in the corneal epithelial cells

Mammalian epithelia produce the various antimicrobial peptides against the bacterial or viral infection, thereby acting as the active immune modulators in the innate immunity. In this study, we examined the effects of the various proinflammatory cytokines or LPS on cell viability and antimicrobial β-defensin gene expressions in human corneal epithelial cells. Results showed that the cytokines or LPS did not exert severe cytotoxic effects on the cells, and that β-defensin 1 was constitutively expressed, while β-defensin 2 was specifically induced by IL-1β, supporting the idea that these cytokines or LPS involve the defense mechanism in the cornea. Furthermore, the reporter and gel shift assay to define the induction mechanism of β-defensin 2 by IL-1β demonstrated that the most proximal NF-kB site on the promoter region of β-defensin 2 was not critical for the process. Data obtained from the normal or patients with the varying ocular diseases showed that our in vitro results were relevant in the clinical settings. Our results clearly demonstrated that β-defensin 1 and 2 are important antimicrobial peptides in the corneal tissues, and that the mechanistic induction process of β-defensin 2 by IL-1β is not solely dependent on proximal NF-kB site activation, thus suggesting that the long distal portion of the promoter is needed for the full responsiveness toward IL-1β.

Differentially Expressed Proteins of Mesenchymal Stem Cells Derived from Human Cord Blood (hUCB) during Osteogenic Differentiation

Multipotent mesenchymal stem cells (MSCs) derived from human umbilical cord blood (hUCB) represent promising candidates for the development of future cellular therapy strategies. MSCs have been found to be able to differentiate into various tissues. One of the primary limitations in our understanding of the biology of human MSCs is the absence of prospective markers required for the monitoring of lineage-specific differentiation. hUCB-derived MSCs have been found to have significantly greater osteogenic potential. In this study, we focused on proteins that were differentially expressed during osteogenic differentiation of hUCB-MSCs. And we analyzed the protein expression inherent to osteogenic differentiation by two-dimensional gel electrophoresis, ESI-Q-TOF, and Western blotting. Eleven differentially expressed spots were observed between the two groups (before and after differentiation) on the 2-DE map. These might also be proved as useful cytosolic biomarker proteins for osteogenesis, and might be employed in quality control of osteoblasts in cell-therapy applications.

Proteomic analysis of cancer stem cells in human prostate cancer cells

Results from recent studies support the hypothesis that cancer stem cells (CSCs) are responsible for tumor initiation and formation. Here, we applied a proteome profiling approach to investigate the mechanisms of CSCs and to identify potential biomarkers in the prostate cancer cell line DU145. Using MACS, the DU145 prostate cancer cell line was isolated into CD44+ or CD44- cells. In sphere culture, CD44+ cells possessed stem cell characteristics and highly expressed genes known to be important in stem cell maintenance. In addition, they showed strong tumorigenic potential in the clonogenic assay and soft agar colony formation assay. We then analyzed and identified proteins that were differentially expressed between CD44+ and CD44- using two-dimensional gel electrophoresis and LC-MS/MS. Cofilin and Annexin A5, which are associated with proliferation or metastasis in cancer, were found to be positively correlated with CD44 expression. These results provide information that will be important to the development of new cancer diagnostic tools and understanding the mechanisms of CSCs although a more detailed study is necessary to investigate the roles of Cofilin and Annexin A5 in CSCs.