Biomedical subjects

Dong-Ku Kang

Publications and source records attributed to Dong-Ku Kang.

7 recordsLinked to original sources

PubMed2006

Angiogenin-induced protein kinase B/Akt activation is necessary for angiogenesis but is independent of nuclear translocation of angiogenin in HUVE cells.

Angiogenin, a potent angiogenic factor, binds to endothelial cells and is endocytosed and rapidly translocated to and concentrated in the nucleolus where it binds to DNA. In this study, we report that angiogenin induces transient phosphorylation of protein kinase B/Akt in cultured human umbilical vein endothelial (HUVE) cells. LY294002 inhibits the angiogenin-induced protein kinase B/Akt activation and also angiogenin-induced cell migration in vitro as well as angiogenesis in chick embryo chorioallantoic membrane in vivo without affecting nuclear translocation of angiogenin in HUVE cells. These results suggest that cross-talk between angiogenin and protein kinase B/Akt signaling pathways is essential for angiogenin-induced angiogenesis in vitro and in vivo, and that angiogenin-induced PKB/Akt activation is independent of nuclear translocation of angiogenin in HUVE cells.

Active Transport, Cell Nucleus↗

PubMed2006

Protein nanoarray on Prolinker surface constructed by atomic force microscopy dip-pen nanolithography for analysis of protein interaction.

Protein nanoarrays are addressable ensembles of nano-scale protein domain on solid surfaces. This method can serve as a useful platform for ultraminiaturized bioanalysis. In this study, we investigated single molecular nanopatterning and molecular interaction of proteins that were immobilized on Prolinker surface of gold-coated silicon wafer by using dip-pen nanolithography (DPN) method. Contact force and humidity were optimized at 0.01 nN and 80%, respectively. The domain features of protein nanoarrays were developed at the contact time of 5 s. The optimized conditions for the nanoarray process were applied to create protein nanoarray using integrin alpha(v)beta3 and angiogenin. Constructed protein nanoarrays using integrin alpha(v)beta3 have single molecular monolayer with regular domain shape (height 15 +/- 5 nm). The changed height value due to the single molecular interaction between integrin alpha(v)beta3 and vitronectin was approximately 30 +/- 5 nm on Prolinker surface as measured with atomic force microscopy tip. Taken together, these results suggest that protein nanoarray on Prolinker surface fabricated by well-controlled DPN process can be used to analyze single molecular interaction of protein.

Gold↗

PubMed2006

Profiling of differential protein expression in angiogenin-induced HUVECs using antibody-arrayed ProteoChip.

ProteoChip has been developed as a novel protein microarray technology. So far it has been applied in new lead screening and molecular diagnostics and we expect its role to grow in the field of biology. Here, we investigated the application of ProteoChip for the study of differential protein expression profiles in angiogenin-induced human umbilical vein endothelial cells (HUVECs). Antibody microarrays constructed by immobilizing 60 distinct antibodies against signal-transducing proteins on ProteoChip base plates were used to analyze the expression pattern of cell-signaling proteins in HUVECs treated with angiogenin. The antibody microarray approach showed that angiogenin induced the up- and down-regulation of several cellular regulators related with cell proliferation. Changes in the expression of signaling proteins determined by antibody microarray were validated by Western blot analysis. In this experiment, ten up-regulated proteins and six down-regulated proteins were identified and confirmed by immunoblot analysis. Taken together, these data suggest that antibody microarrays using ProteoChip technology can be a powerful tool for high-throughput analysis of proteomes in biological samples.

Angiogenesis Inducing Agents↗

PubMed2006

Myoseverin is a potential angiogenesis inhibitor by inhibiting endothelial cell function and endothelial progenitor cell differentiation.

Myoseverin, a new microtubule-binding molecule, acts reversibly on myoblast proliferation without the cytotoxic effects displayed by nonpurine-based microtubule-disrupting molecules, like taxol, vinblastine, nocodazole, and the colchicines. In this study, we examined the effects of myoseverin on in vitro function of endothelial cells and endothelial progenitor cell differentiation in order to explore the possibility for the application of myoseverin as a reversible antiangiogenic agent. Myoseverin potently inhibited proliferation of human umbilical vein endothelial cells (HUVECs) in a dose-dependent manner with an IC50 of approximately 8 microM. When myoseverin was removed after treatment for 3 days, all the cells pretreated at a concentration range of 2.5-80 microM resumed the cell growth. It also inhibited VEGF-induced HUVEC migration dose dependently. When mononuclear cells (MNCs) isolated from human cord blood were cultured on fibronectin-coated plates for 7 days, myoseverin decreased the number of adherent cells in a dose-dependent manner with IC50 of approximately 9 microM. It also suppressed the development of ac-LDL uptake ability as well as the expression of endothelial lineage markers, KDR, CD31, and vWF. Finally, it inhibited formation of HUVECs or ex vivo cultivated EPCs into capillary-like structure on Matri-gel and in vivo angiogenesis on the chick chorioallantoic membrane. Therefore, these results suggest that myoseverin can be effectively used for the inhibition of new vessel growth by inhibiting endothelial cell function and differentiation of progenitor cells.

Angiogenesis Inhibitors↗

PubMed2004

High level production of bovine angiogenin in E. coli by an efficient refolding procedure.

Recombinant bovine angiogenin (rbAng) was expressed in E. coli at up to 30% of total cell proteins but was produced as inclusion bodies. By investigating the effect of various factors on the refolding yield, we obtained about 60% refolding. After chromatographic purification, about 60 mg purified angiogenin was obtained from 1 l culture. The purified recombinant bovine angiogenin was identical to native bovine angiogenin (nbAng) obtained from cow's milk. Our approach is highly efficient and can be generally used for the production of various types of angiogenin for functional and structural studies as well as therapeutic purposes.

Animals↗

PubMed2004

High-throughput screening of novel peptide inhibitors of an integrin receptor from the hexapeptide library by using a protein microarray chip.

Protein microarray is an emerging technology that makes high-throughput analysis possible for protein-protein interactions and analysis of proteome and biomarkers in parallel. The authors investigated the application of a novel protein microarray chip, ProteoChip, in new drug discovery. Integrin alpha(v)beta(3) microarray immobilized on the ProteoChip was employed to screen new active peptides against the integrin from multiple hexapeptide sublibraries of a positional scanning synthetic peptide combinatorial library (PS-SPCL). The integrin alpha(v)beta(3)-vitronectin interaction was successfully demonstrated on the integrin microarray in a dose-dependent manner and was inhibited not only by the synthetic RGD peptide but also by various integrin antagonists on the integrin microarray chip. Novel peptide ligands with high affinity to the integrin were also identified from the peptide libraries with this chip-based screening system by a competitive inhibition assay in a simultaneous and high-throughput fashion. The authors have confirmed antiangiogenic functions of the novel peptides thus screened through an in vitro and in vivo angiogenesis assay. These results provide evidence that the ProteoChip is a promising tool for high-throughput screening of lead molecules in new drug development.

Cell Movement↗

PubMed2003

Anti-angiogenic activity of the recombinant kringle domain of urokinase and its specific entry into endothelial cells.

Urokinase plasminogen activator (uPA) belongs to a family of proteins that contains kringle domain and plays an important role in inflammation, tissue remodeling, angiogenesis, and tumor metastasis by pericellular plasminogen activation. Kringle domains of plasminogen have been shown to demonstrate anti-angiogenic and anti-tumor activities. Here, we report our investigation of the kringle domain of uPA for anti-angiogenic activity and a possible cellular mechanism of action. The recombinant kringle domain of uPA (Asp(45)-Lys(135)) (UK1) inhibited endothelial cell proliferation stimulated by basic fibroblast growth factor, vascular endothelial growth factor (VEGF), or epidermal growth factor. It also inhibited migration of endothelial cells induced by VEGF or uPA, and in vivo angiogenesis on the chick chorioallantoic membrane. It did not block plasminogen activation by activated uPA in clot lysis and chromogenic substrate assays. Neither binding of UK1 to immobilized uPA receptor nor competitive inhibition of uPA binding were confirmed by real-time interaction analysis. However, internalization of UK1 followed by translocation from cytosol to nucleus was determined to be specific to endothelial cells. It also elicited a transient increase of Ca(2+) flux of more than 2-fold within 2 min of exposure in an endothelial cell-specific manner. These results suggest that the kringle domain of uPA exhibits anti-angiogenic activity and that its anti-angiogenic activity may occur through a different mechanism from inhibition of uPA-uPA receptor interaction or uPA proteolytic activity and may be associated with endothelial-cell specific internalization not mediated by the uPA receptor.

Amino Acid Sequence↗