Eunsil Hahm

Platelets generated from human embryonic stem cells are functional in vitro and in the microcirculation of living mice

Platelets play an essential role in hemostasis and atherothrombosis. Owing to their short storage time, there is constant demand for this life-saving blood component. In this study, we report that it is feasible to generate functional megakaryocytes and platelets from human embryonic stem cells (hESCs) on a large scale. Differential-interference contrast and electron microscopy analyses showed that ultrastructural and morphological features of hESC-derived platelets were indistinguishable from those of normal blood platelets. In functional assays, hESC-derived platelets responded to thrombin stimulation, formed microaggregates, and facilitated clot formation/retraction in vitro. Live cell microscopy demonstrated that hESC-platelets formed lamellipodia and filopodia in response to thrombin activation, and tethered to each other as observed in normal blood. Using real-time intravital imaging with high-speed video microscopy, we have also shown that hESC-derived platelets contribute to developing thrombi at sites of laser-induced vascular injury in mice, providing the first evidence for in vivo functionality of hESC-derived platelets. These results represent an important step toward generating an unlimited supply of platelets for transfusion. Since platelets contain no genetic material, they are ideal candidates for early clinical translation involving human pluripotent stem cells.

Glucagon-Like Peptide-1 Gene Therapy in Obese Diabetic Mice Results in Long-Term Cure of Diabetes by Improving Insulin Sensitivity and Reducing Hepatic Gluconeogenesis

Long-term treatment with glucagon-like peptide (GLP)-1 or its analog can improve insulin sensitivity. However, continuous administration is required due to its short half-life. We hypothesized that continuous production of therapeutic levels of GLP-1 in vivo by a gene therapy strategy may remit hyperglycemia and maintain prolonged normoglycemia. We produced a recombinant adenovirus expressing GLP-1 (rAd-GLP-1) under the cytomegalovirus promoter, intravenously injected it into diabetic ob/ob mice, and investigated the effect of this treatment on remission of diabetes, as well as the mechanisms involved. rAd-GLP-1–treated diabetic ob/ob mice became normoglycemic 4 days after treatment, remained normoglycemic over 60 days, and had reduced body weight gain. Glucose tolerance tests found that exogenous glucose was cleared normally. rAd-GLP-1–treated diabetic ob/ob mice showed improved β-cell function, evidenced by glucose-responsive insulin release, and increased insulin sensitivity, evidenced by improved insulin tolerance and increased insulin-stimulated glucose uptake in adipocytes. rAd-GLP-1 treatment increased basal levels of insulin receptor substrate (IRS)-1 in the liver and activation of IRS-1 and protein kinase C by insulin in liver and muscle; increased Akt activation was only observed in muscle. rAd-GLP-1 treatment reduced hepatic glucose production and hepatic expression of phosphoenolpyruvate carboxykinase, glucose-6-phosphatase, and fatty acid synthase in ob/ob mice. Taken together, these results show that a single administration of rAd-GLP-1 results in the long-term remission of diabetes in ob/ob mice by improving insulin sensitivity through restoration of insulin signaling and reducing hepatic gluconeogenesis.

Platelet protein disulfide isomerase is required for thrombus formation but not for hemostasis in mice.

Protein disulfide isomerase (PDI) derived from intravascular cells is required for thrombus formation. However, it remains unclear whether platelet PDI contributes to the process. Using platelet-specific PDI-deficient mice, we demonstrate that PDI-null platelets have defects in aggregation and adenosine triphosphate secretion induced by thrombin, collagen, and adenosine diphosphate. Such defects were rescued by wild-type but not mutant PDI, indicating that the isomerase activity of platelet surface PDI is critical for the regulatory effect. PDI-deficient platelets expressed increased levels of intracellular ER protein 57 (ERp57) and ERp72. Platelet PDI regulated αIIbβ3 integrin activation but not P-selectin exposure, Ca(2+) mobilization, β3-talin1 interaction, or platelet spreading on immobilized fibrinogen. Inhibition of ERp57 further diminished αIIbβ3 integrin activation and aggregation of activated PDI-deficient platelets, suggesting distinct roles of PDI and ERp57 in platelet functions. We found that platelet PDI is important for thrombus formation on collagen-coated surfaces under shear. Intravital microscopy demonstrates that platelet PDI is important for platelet accumulation but not initial adhesion and fibrin generation following laser-induced arteriolar injury. Tail bleeding time in platelet-specific PDI-deficient mice were not significantly increased. Our results provide important evidence that platelet PDI is essential for thrombus formation but not for hemostasis in mice.

Sodium Ascorbate (Vitamin C) Induces Apoptosis in Melanoma Cells via the Down-Regulation of Transferrin Receptor Dependent Iron Uptake

Sodium ascorbate (vitamin C) has a reputation for inconsistent effects upon malignant tumor cells, which vary from growth stimulation to apoptosis induction. Melanoma cells were found to be more susceptible to vitamin C toxicity than any other tumor cells. The present study has shown that sodium ascorbate decreases cellular iron uptake by melanoma cells in a dose‐ and time‐dependent fashion, indicating that intracellular iron levels may be a critical factor in sodium ascorbate‐induced apoptosis. Indeed, sodium ascorbate‐induced apoptosis is enhanced by the iron chelator, desferrioxamine (DFO) while it is inhibited by the iron donor, ferric ammonium citrate (FAC). Moreover, the inhibitory effects of sodium ascorbate on intracellular iron levels are blocked by addition of transferrin, suggesting that transferrin receptor (TfR) dependent pathway of iron uptake may be regulated by sodium ascorbate. Cells exposed to sodium ascorbate demonstrated down‐regulation of TfR expression and this precedes sodium ascorbate‐induced apoptosis. Taken together, sodium ascorbate‐mediated apoptosis appears to be initiated by a reduction of TfR expression, resulting in a down‐regulation of iron uptake followed by an induction of apoptosis. This study demonstrates the specific mechanism of sodium ascorbate‐induced apoptosis and these findings support future clinical trial of sodium ascorbate in the prevention of human melanoma relapse.

L-ascorbic acid (vitamin C) induces the apoptosis of B16 murine melanoma cells via a caspase-8-independent pathway.

Abstractl-Ascorbic acid (vitamin C) has been reported to play a role in the treatment and prevention of cancer. However, its specific mechanistic pathways remain obscure. This study was carried out to identify the sodium ascorbate–induced apoptotic pathway in B16F10 murine melanoma cells. Sodium ascorbate was found to induce the apoptosis of B16F10 murine melanoma in a time- and dose-dependent manner, and this was prevented by pretreatment with N-acetyl-l-cysteine (NAC), a well-known antioxidant. In fact, sodium ascorbate–treated B16F10 melanoma cells showed increased intracellular reactive oxygen species generation (ROS) levels. These results indicate that sodium ascorbate induced apoptosis in B16F10 murine melanoma cells by acting as a prooxidant. We examined the involvement of caspase-8 using a specific caspase-8 inhibitor (z-IETD-fmk) on the sodium ascorbate–induced apoptotic pathway. Cell death was found not to be inhibited by z-IETD-fmk treatment, indicating that sodium ascorbate–induced apoptosis is not mediated by caspase-8. In addition, we detected a reduction in the mitochondrial membrane potential during apoptosis and confirmed cytochrome-c release from mitochondria by immunoblotting. Taken together, it appears that the induction of a prooxidant state by sodium ascorbate and a subsequent reduction in mitochondrial membrane potential are involved in the apoptotic pathway of B16F10 murine melanoma cells, and that this occurs in a caspase-8–independent manner.

Extracellular protein disulfide isomerase regulates ligand-binding activity of αMβ2 integrin and neutrophil recruitment during vascular inflammation

β2 integrins play a crucial role during neutrophil recruitment into the site of vascular inflammation. However, it remains unknown how ligand-binding activity of the integrin is regulated. Using fluorescence intravital microscopy in mice generated by crossing protein disulfide isomerase (PDI) floxed mice with lysozyme-Cre transgenic mice, we demonstrate that neutrophil PDI is required for neutrophil adhesion and crawling during tumor necrosis factor-α-induced vascular inflammation in vivo. Rescue experiments show that the isomerase activity of extracellular PDI is critical for its regulatory effect on neutrophil recruitment. Studies with blocking anti-PDI antibodies and αLβ2 or αMβ2 null mice suggest that extracellular PDI regulates αMβ2 integrin-mediated adhesive function of neutrophils during vascular inflammation. Consistently, we show that neutrophil surface PDI is important for αMβ2 integrin-mediated adhesion of human neutrophils under shear and static conditions and for binding of soluble fibrinogen to activated αMβ2 integrin. Confocal microscopy and biochemical studies reveal that neutrophil surface PDI interacts with αMβ2 integrin in lipid rafts of stimulated neutrophils and regulates αMβ2 integrin clustering, presumably by changing the redox state of the integrin. Thus, our results provide the first evidence that extracellular PDI could be a novel therapeutic target for preventing and treating inappropriate neutrophil sequestration.

Neutrophil AKT2 regulates heterotypic cell-cell interactions during vascular inflammation

Interactions between platelets, leukocytes, and activated endothelial cells are important during microvascular occlusion; however, the regulatory mechanisms of these heterotypic cell-cell interactions remain unclear. Here, using intravital microscopy to evaluate mice lacking specific isoforms of the serine/threonine kinase AKT and bone marrow chimeras, we found that hematopoietic cell-associated AKT2 is important for neutrophil adhesion and crawling and neutrophil-platelet interactions on activated endothelial cells during TNF-α-induced venular inflammation. Studies with an AKT2-specific inhibitor and cells isolated from WT and Akt KO mice revealed that platelet- and neutrophil-associated AKT2 regulates heterotypic neutrophil-platelet aggregation under shear conditions. In particular, neutrophil AKT2 was critical for membrane translocation of αMβ2 integrin, β2-talin1 interaction, and intracellular Ca2+ mobilization. We found that the basal phosphorylation levels of AKT isoforms were markedly increased in neutrophils and platelets isolated from patients with sickle cell disease (SCD), an inherited hematological disorder associated with vascular inflammation and occlusion. AKT2 inhibition reduced heterotypic aggregation of neutrophils and platelets isolated from SCD patients and diminished neutrophil adhesion and neutrophil-platelet aggregation in SCD mice, thereby improving blood flow rates. Our results provide evidence that neutrophil AKT2 regulates αMβ2 integrin function and suggest that AKT2 is important for neutrophil recruitment and neutrophil-platelet interactions under thromboinflammatory conditions such as SCD.

Vitamin C suppresses proliferation of the human melanoma cell SK-MEL-2 through the inhibition of cyclooxygenase-2 (COX-2) expression and the modulation of insulin-like growth factor II (IGF-II) production†

Vitamin C plays a crucial role in the suppression of proliferation of several types of cancer. Over‐expression of cyclooxygenase (COX)‐2 and type I insulin‐like growth factor (IGF) receptor are important for proliferation and protection from apoptosis in malignancies. However, its specific mechanisms, especially the interaction between COX‐2 expression and IGF‐I axis mediated by vitamin C, remain yet to be clarified. Therefore, we investigated the effects of vitamin C on the proliferation of melanoma cells via the modulation of COX‐2 expression and IGF‐I axis. As a result, we found that 1.0 mM vitamin C inhibits the proliferation of SK‐MEL‐2 without induction of apoptosis. At that moment, IGF‐II production was decreased, followed by the inhibition of COX‐2 activity. IGF‐IR expression was also down‐regulated by vitamin C treatment. It coincided with the result from the inhibition of COX‐2 by NS‐398 and COX‐2 siRNA. In addition, the decreased IGF‐IR expression by vitamin C was restored by the treatment of recombinant prostaglandin E2. Finally, we determined whether the signal pathway would be involved in vitamin C‐induced IGF‐II and IGF‐IR down‐regulation. When the cells were exposed to SB203580, a specific inhibitor of p38 MAPK, COX‐2 expression was dramatically recovered. In addition, phosphorylated p38 MAPK was increased after vitamin C treatment. Taken together, vitamin C suppresses proliferation of the human melanoma cell line SK‐MEL2 via the down‐regulation of IGF‐II production and IGF‐IR expression, which is followed by the activation of p38 MAPK and the inhibition of COX‐2 expression. J. Cell. Physiol. 216: 180–188, 2008.

The enhanced IL-18 production by UVB irradiation requires ROI and AP-1 signaling in human keratinocyte cell line (HaCaT)

Based on our recent observation that enhanced IL-18 expression positively correlates with malignant skin tumors, such as SCC and melanoma, we examined the possible role of UVB, known to be associated with skin cancer development, in the enhancement of IL-18 production using primary human epidermal keratinocytes and human keratinocyte cell line HaCaT. After cells were exposed to UVB irradiation in vitro, IL-18 production was examined by Northern blot analysis and ELISA, and it was found that IL-18 production is enhanced by UVB irradiation in a dose- and time-dependent manner. In addition, we confirmed that it is functionally active form of IL-18 using the inhibitor of caspase-1. The effect of UVB irradiation was blocked by antioxidant, N-acetyl-L-cysteine (NAC), which suggested the involvement of reactive oxygen intermediates (ROI) in the signal transduction of UVB irradiation-enhanced IL-18 synthesis. We also found that UVB irradiation increased AP-1 binding activity by using EMSA with AP-1-specific oligonucleotide. Furthermore, inhibitors of UVB-induced AP-1 activity, such as PD98059, blocked enhanced IL-18 production, indicating that AP-1 activation is required for UVB-induced IL-18 production. Taken together, our results suggest that UVB irradiation-enhanced IL-18 production is selectively mediated through the generation of ROI and the activation of AP-1.

The Molecular Mechanisms of Vitamin C on Cell Cycle Regulation in B16F10 Murine Melanoma

Vitamin C has inconsistent effects on malignant tumor cells, which vary from growth stimulation to apoptosis induction. It is well known that melanoma cells are more susceptible to vitamin C than any other tumor cells, but the precise mechanism remains to be elucidated. In the present study, the proliferation of B16F10 melanoma cells was suppressed by vitamin C, which induced growth arrest in a dose‐dependent manner without cytotoxic effects. Therefore, we investigated the changes in cell cycle distribution of B16F10 melanoma cells by staining DNAs with propidium iodide (PI). The growth inhibition of B16F10 melanoma by vitamin C was associated with an arrest of cell cycle distribution at G1 stage. In addition, the levels of p53‐p21Waf1/Cip1 increased during G1 arrest, which were essential for vitamin C‐induced cell cycle arrest. The increased p21Waf1/Cip1 inhibited CDK2. Moreover, the activity of p53‐p21Waf1/Cip1 pathway was closely related with the activation of checkpoint kinase 2 (Chk2). Inhibitor of the PI3K‐family, LY294002 and the ATM/ATR inhibitor, caffeine, blocked vitamin C‐induced growth arrest in B16F10 melanoma cells. These results suggest that vitamin C might be a potent agent to inhibit proliferative activity of melanoma cells via the regulation of Chk2‐p53‐p21Waf1/Cip1 pathway. J. Cell. Biochem. 102: 1002–1010, 2007.