Myung Jin Son

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.

Determination of hypoxic region by hypoxia marker in developing mouse embryos in vivo: A possible signal for vessel development

Hypoxia is a well‐known signal for angiogenesis, but the recent proposal that hypoxia exists in developing embryonic tissues and that it induces vascular development remains to be proven. In the present study, we demonstrate the presence of hypoxia in normal developing embryos by means of a hypoxia marker, pimonidazole, and its associated antibody. Our data clearly show that hypoxia marker immunoreactivity was highly detected in developing neural tubes, heart, and intersomitic mesenchyme at an early stage of organogenesis, suggesting that hypoxia may exist in the early stages of embryo development. We also found that hypoxia inducible factor‐1α (HIF‐1α) and vascular endothelial growth factor (VEGF) were spatiotemporally co‐localized with possible hypoxic regions in embryos. Investigation of platelet endothelial cell adhesion molecule (PECAM) expression provides evidence that endothelial cells proliferate and form the vessels in the hypoxic region in developing organs. Furthermore, we found that hypoxia induced both HIF‐1α and VEGF in F9 embryonic stem and differentiated cells. Thus, we suggest that hypoxia may exist widely in developing embryonic tissues and that it may act as a signal for embryonic blood vessel formation in vivo.

KR-31831, a new synthetic anti-ischemic agent, inhibits in vivo and in vitro angiogenesis

Angiogenesis is considered to be an integral process to the growth and spread of solid tumors. Anti-angiogenesis therapy recently has been found to be one of the most promising anti-cancer therapeutic strategies. In this study, we provide several lines of evidences showing that KR-31831, a new benzopyran derivative, has anti-angiogenic activities. KR-31831 inhibited the proliferation, migration, invasion and tube formation of bovine aortic endothelial cells (BAECs), and suppressed the release of matrix metalloproteinase-2 (MMP-2) of BAECs. KR-31831 also inhibited in vivo angiogenesis in mouse Matrigel plug assay. Furthermore, the mRNA expressions of basic fibroblast growth factor (bFGF), fibroblast growth factor receptor-2 (FGFR-2), and vascular endothelial growth factor receptor-2 (VEGFR-2) were decreased by KR-31831. Taken together, these results suggest that KR-31831 acts as a novel angiogenesis inhibitor and might be useful for treating hypervascularized cancers.

Biochemical and molecular characterization of novel mutations in GLB1 and NEU1 in patient cells with lysosomal storage disorders.

Lysosomes are cytoplasmic compartments that contain many acid hydrolases and play critical roles in the metabolism of a wide range of macromolecules. Deficiencies in lysosomal enzyme activities cause genetic diseases, called lysosomal storage disorders (LSDs). Many mutations have been identified in the genes responsible for LSDs, and the identification of mutations is required for the accurate molecular diagnoses. Here, we analyzed cell lines that were derived from two different LSDs, GM1 gangliosidosis and sialidosis. GM1 gangliosidosis is caused by mutations in the GLB1 gene that encodes β-galactosidase. A lack of β-galactosidase activity leads to the massive accumulation of GM1 ganglioside, which results in neurodegenerative pathology. Mutations in the NEU1 gene that encodes lysosomal sialidase cause sialidosis. Insufficient activity of lysosomal sialidase progressively increases the accumulation of sialylated molecules, and various clinical symptoms, including mental retardation, appear. We sequenced the entire coding regions of GLB1 and NEU1 in GM1 gangliosidosis and sialidosis patient cells, respectively. We found the novel mutations p.E186A in GLB1 and p.R347Q in NEU1, as well as many other mutations that have been previously reported. We also demonstrated that patient cells containing the novel mutations showed the molecular phenotypes of the corresponding disease. Further structural analysis suggested that these novel mutation sites are highly conserved and important for enzyme activity.

cDNA representational difference analysis used in the identification of genes related to the aging process in rat kidney.

Aging is a complex physiological process by which the functions of many organ systems deteriorate. Growing evidence shows that age-related changes and damage are causally related to oxidative stress and inflammatory responses from reactive species. The aim of this study was to identify differentially expressed genes in old and young kidneys of Fisher 344 male rats during the aging process using complementary DNA representational difference analysis (cDNA RDA). cDNA RDA is a subtractive technique for identifying a focused set of differentially expressed genes. The distinctive advantage of this technique is its capability of detecting differences in gene expressions at less than one copy per cell and identifying genes not previously described in the database. Reverse transcription-polymerase chain reaction with specific primers was applied to confirm the differences found by RDA. Twenty-one putative differentially expressed genes were identified. Sixteen genes were up-regulated during aging and were associated with stress-response and inflammatory reactions, while five genes were down-regulated. These data suggested that the inflammatory process is a plausible cause of the aging process.

Upregulation of mitochondrial NAD|[plus]| levels impairs the clonogenicity of SSEA1|[plus]| glioblastoma tumor-initiating cells

Emerging evidence has emphasized the importance of cancer therapies targeting an abnormal metabolic state of tumor-initiating cells (TICs) in which they retain stem cell-like phenotypes and nicotinamide adenine dinucleotide (NAD+) metabolism. However, the functional role of NAD+ metabolism in regulating the characteristics of TICs is not known. In this study, we provide evidence that the mitochondrial NAD+ levels affect the characteristics of glioma-driven SSEA1+ TICs, including clonogenic growth potential. An increase in the mitochondrial NAD+ levels by the overexpression of the mitochondrial enzyme nicotinamide nucleotide transhydrogenase (NNT) significantly suppressed the sphere-forming ability and induced differentiation of TICs, suggesting a loss of the characteristics of TICs. In addition, increased SIRT3 activity and reduced lactate production, which are mainly observed in healthy and young cells, appeared following NNT-overexpressed TICs. Moreover, in vivo tumorigenic potential was substantially abolished by NNT overexpression. Conversely, the short interfering RNA-mediated knockdown of NNT facilitated the maintenance of TIC characteristics, as evidenced by the increased numbers of large tumor spheres and in vivo tumorigenic potential. Our results demonstrated that targeting the maintenance of healthy mitochondria with increased mitochondrial NAD+ levels and SIRT3 activity could be a promising strategy for abolishing the development of TICs as a new therapeutic approach to treating aging-associated tumors.