Yung-Jin Kim

SSeCKS regulates angiogenesis and tight junction formation in blood-brain barrier.

The blood-brain barrier (BBB) is essential for maintaining brain homeostasis and low permeability. BBB maintenance is important in the central nervous system (CNS) because disruption of the BBB may contribute to many brain disorders, including Alzheimer disease and ischemic stroke. The molecular mechanisms of BBB development remain ill-defined, however. Here we report that src-suppressed C-kinase substrate (SSeCKS) decreases the expression of vascular endothelial growth factor (VEGF) through AP-1 reduction and stimulates expression of angiopoietin-1 (Ang-1), an antipermeability factor in astrocytes. Conditioned media from SSeCKS-overexpressing astrocytes (SSeCKS-CM) blocked angiogenesis in vivo and in vitro. Moreover, SSeCKS-CM increased tight junction proteins in endothelial cells, consequently decreasing [3H]sucrose permeability. Furthermore, immunoreactivity to SSeCKS gradually increased during the BBB maturation period, and SSeCKS-expressing astrocytes closely interacted with zonula occludens (ZO)-1-expressing blood vessels in vivo. Collectively, our results suggest that SSeCKS regulates BBB differentiation by modulating both brain angiogenesis and tight junction formation.

Melatonin suppresses tumor angiogenesis by inhibiting HIF-1α stabilization under hypoxia

Abstract:  Angiogenesis is an important mediator of tumor progression. As tumors expand, diffusion distances from the existing vascular supply increases, resulting in hypoxia in the cancer cells. Sustained expansion of a tumor mass requires new blood vessel formation to provide rapidly proliferating tumor cells with an adequate supply of oxygen and nutrients. The key regulator of hypoxia‐induced angiogenesis is the transcription factor known as hypoxia‐inducible factor (HIF)‐1. HIF‐1α is stabilized by hypoxia‐induced reactive oxygen species (ROS) and enhances the expression of several types of hypoxic genes, including that of the angiogenic activator known as vascular endothelial cell growth factor (VEGF). In this study, we found that melatonin, a small lipophilic molecule secreted primarily by the pineal gland, destabilizes hypoxia‐induced HIF‐1α protein levels in the HCT116 human colon cancer cell line. This destabilization of HIF‐1α resulted from the antioxidant activity of melatonin against ROS induced by hypoxia. Moreover, under hypoxia, melatonin suppressed HIF‐1 transcriptional activity, leading to a decrease in VEGF expression. Melatonin also blocked in vitro tube formation and invasion and migration of human umbilical vein endothelial cells induced by hypoxia‐stimulated conditioned media of HCT116 cells. These findings suggest that melatonin could play a pivotal role in tumor suppression via inhibition of HIF‐1‐mediated angiogenesis.

Quercetin Activates an Angiogenic Pathway, Hypoxia Inducible Factor (HIF)-1-Vascular Endothelial Growth Factor, by Inhibiting HIF-Prolyl Hydroxylase: a Structural Analysis of Quercetin for Inhibiting HIF-Prolyl Hydroxylase

We investigated a molecular mechanism underlying quercetin-mediated amelioration of colonic mucosal injury and analyzed chemical structure contributing to the quercetins effect. Quercetin up-regulated vascular endothelial growth factor (VEGF), an ulcer healing factor, not only in colon epithelial cell lines but also in the inflamed colonic tissue. VEGF derived from quercetin-treated colon epithelial cells promoted tube formation. The VEGF induction was dependent on quercetin-mediated hypoxia-inducible factor-1 (HIF-1) activation. Quercetin delayed HIF-1α protein disappearance, which occurred by inhibiting HIF-prolyl hydroxylase (HPH), the key enzyme for HIF-1α hydroxylation and subsequent von Hippel Lindau-dependent HIF-1α degradation. HPH inhibition by quercetin was neutralized significantly by an elevated dose of iron. Consistent with this, cellular induction of HIF-1α by quercetin was abolished by pretreatment with iron. Two iron-chelating moieties in quercetin, -OH at position 3 of the C ring and/or -OH at positions 3′ and 4′ of the B ring, enabled the flavonoid to inhibit HPH and subsequently induce HIF-1α. Our data suggest that the clinical effect of quercetin may be partly attributed to the activation of an angiogenic pathway HIF-1-VEGF via inhibiting HPH and the chelating moieties of quercetin were required for inhibiting HPH.

Anti-angiogenic and anti-tumor invasive activities of tissue inhibitor of metalloproteinase-3 from shark, Scyliorhinus torazame.

In order to investigate the anti-angiogenic activity of shark TIMP-3 (sTIMP-3) in endothelial cells, angiogenic assays including in vitro invasion assay, migration assay, zymogram assay and tube formation assay were performed. We observed that the overexpression of sTIMP-3 decreased the invasive capacity by about 70%, the migratory activity by about 50% and the production of gelatinase A in bovine aortic endothelial cells (BAECs). In addition, the overexpression of sTIMP-3 interfered with the formation of capillary-like network in endothelial cells. We also examined whether sTIMP-3 shows the anti-invasive activity in cancer cells. We found that the overexpression of sTIMP-3 diminished the invasive ability of the human fibrosarcoma HT1080 cells by about 40%. Also, the production of specific gelatinases was suppressed in the cancer cells. Therefore, we propose that sTIMP-3 acts as the inhibitor of angiogenesis in endothelial cells and the suppressor of tumor invasion in human fibrosarcoma HT1080 cells.

Identification of process operating state with operational map in municipal wastewater treatment plant.

This work was performed to develop an operational map for the objective diagnosis of the process operating states of a municipal wastewater treatment plant, for which multivariate statistical analysis techniques were applied. PCA (principal component analysis) was used to reduce the dimension of the data sets obtained from the field municipal wastewater treatment plant. A K-means clustering analysis was used to classify the group according to the property of the process operating state. A Fishers linear discriminant analysis was used to derive the discriminant function of each classified group. An operational map was developed by scatter-plotting the derived principal components (PCs) on a two-dimensional coordinate according to the classified groups. Using the new data sets not used for developing the operational map, the practical usefulness of the operational map and discriminant function in diagnosing the process operating state were evaluated. Hence, the process operating state could be easily and quickly diagnosed and the dynamic trend of the process operating state was also able to be estimated using the operational map.

Thromboxane A2 increases endothelial permeability through upregulation of interleukin-8

Thromboxane A(2) (TXA(2)), a major prostanoid formed from prostaglandin H(2) by thromboxane synthase, is involved in the pathogenesis of a variety of vascular diseases. In this study, we report that TXA(2) mimetic U46619 significantly increases the endothelial permeability both in vitro and in vivo. U46619 enhanced the expression and secretion of interleukin-8 (IL-8), a major inducer of vascular permeability, in endothelial cells. Promoter analysis showed that the U46619-induced expression of IL-8 was mainly regulated by nuclear factor-kappaB (NF-kappaB). U46619 induced the activation of NF-kappaB through IkappaB kinase (IKK) activation, IkappaB phosphorylation and NF-kappaB nuclear translocation. Furthermore, the inhibition of IL-8 or blockade of the IL-8 receptor attenuated the U46619-induced endothelial cell permeability by modulating the cell-cell junctions. Overall, these results suggest that U46619 promotes vascular permeability through the production of IL-8 via NF-kappaB activation in endothelial cells.

C-terminal region of HBx is crucial for mitochondrial DNA damage

HBx is strongly associated with hepatocellular carcinoma development through transcription factor activation and reactive oxygen species (ROSs) production. However, the exact role of HBx during hepatocellular carcinogenesis is not fully understood. Recently, it was reported that C-terminal truncated HBx is associated with tumor metastasis. In the present study, we confirmed that the C-terminal region of HBx is required for ROS production and 8-oxoguanine (8-oxoG) formation, which is considered as a reliable biomarker of oxidative stress. These results suggest ROS production induced by the C-terminal region of HBx leads to mitochondrial DNA damage, which may play a role in HCC development.

Identification of novel anti-angiogenic factors by in silico functional gene screening method

Angiogenesis, the formation of new blood vessels out of pre-existing capillaries, occurs in a variety of pathophysiological conditions, and is regulated by a balance of angiogenic activators and inhibitors. To identify novel angiogenic factors, we developed a gene screening method by combining the prediction analysis of transcription factor (TF) binding site and the chromosomal localization analysis. First, we analyzed the promoter sequences from known angiogenesis-related factors using the MATINSPECTOR program in TRANSFAC database. Interestingly, we found that the binding site of LMO2 complex is highly conserved in the promoter regions of these factors. Second, we analyzed chromosome loci based on the hypothesis that angiogenesis-related factors might be co-localized in a specific chromosomal band. We found that angiogenesis-related factors are localized in specific 14 chromosomal bands including 5q31 and 19q13 using AngioDB and LocusLink database mining. From these two approaches, we identified 32 novel candidates that have the LMO2 complex binding site in their promoter and are located on one of 14 chromosomal bands. Among them, human recombinant troponin T and spectrin markedly inhibited the neovascularization in vivo and in vitro. Collectively, we suggest that the combination of the prediction analysis of TF binding site and the chromosomal localization analysis might be a useful strategy for gene screening of angiogenesis.

Reduced expression of FASN through SREBP‐1 down‐regulation is responsible for hypoxic cell death in HepG2 cells

Cells under hypoxic stress either activate an adaptive response or undergo cell death. Although some mechanisms have been reported, the exact mechanism behind hypoxic cell death remains unclear. Recently, increased expression of fatty acid synthase (FASN) has been observed in various human cancers. In highly proliferating cells, tumor‐associated FASN is considered necessary for both membrane lipids production and post‐translational protein modification, but the exact mechanisms are not fully understood. Further, FASN overexpression is associated with aggressive and malignant cancer diseases and FASN inhibition induces apoptosis in cancer cells. For this reason, FASN is emerging as a key target for the potential diagnosis and treatment of various cancers. Here, we observed decreased FASN expression under hypoxic cell death conditions in HepG2 cells. Thus, we examined the effect of decreased FASN expression on hypoxia‐induced cell death in HepG2 cells and also investigated the mechanism responsible for reduction of FASN expression under hypoxic cell death conditions. As a result, reduction of FASN expression resulted in hypoxic cell death via malonyl‐CoA accumulation. In addition, SREBP‐1 restored FASN reduction and hypoxia‐induced apoptosis. Taken together, we suggest that hypoxic cell death is promoted by the reduced expression of FASN through SREBP‐1 down‐regulation. J. Cell. Biochem. 113: 3730–3739, 2012.

Pyruvate promotes tumor angiogenesis through HIF-1-dependent PAI-1 expression

Cancer cells usually obtain energy from a high rate of glycolysis rather than oxidative phosphorylation under normoxia as well as hypoxia. Under these circumstances, pyruvate, the end-product of glycolysis, accumulates in cancer cells. We have previously reported that pyruvate activates endothelial cells and induces angiogenesis. Here, we examined the angiogenic activity of pyruvate in tumor cells. Plasminogen activator inhibitor-1 (PAI-1), the gene most upregulated by pyruvate, showed a pro-angiogenic activity, which was abolished by a PAI-1 neutralizing antibody. Moreover, stabilization of hypoxia-inducible factor-1α (HIF-1α) by pyruvate was required for induction of PAI-1 transcription through direct binding to hypoxia response element-2 (HRE-2) on the promoter. These results suggest that pyruvate can activate the angiogenic activity of cancer cells under normoxia and that PAI-1 may act as a pro-angiogenic factor in pyruvate-induced angiogenesis.