HUVEC were harvested by trypsinization, and 100,000 cells/well were seeded onto 24-well plate pre-coated with martrigel. Cells were incubated for 8 h at 37uC in respective HRMC conditioned media. 
The branching points were continuously monitored and the tube formation of cells was photographed in five random fields of view per well using a microscopy with CCD camera. Data Analysis The results are presented as mean 6 SEM. Statistical analyses were conducted using the Statistical Analysis statistical software package version 6.12. Significance was determined by one-way ANOVA, followed by Duncan’s multiplerange test for multiple comparisons. Differences were considered significant at P,0.05. Enzyme-linked Immunosorbent Assay Mouse plasma levels of VEGF and thrombospondin-1 was determined by using ELISA kits according to the manufacturer’s instructions. Results Suppressive Effects of PCE on Induction of VEGF and HIF1a This study examined whether HG-exposed mesangial cells facilitated expressions of proangiogenic factors in glomerular endothelial cells in a paracrine action, which was disrupted by PCE. Endothelial cells were experienced with respective mesangial cell conditioned media collected from HRMC cultured in DMEM/F-12 containing different glucose concentrations. The endothelial expression of VEGF and HIF-1a was enhanced by 6 h-culturing cells in HG -HRMC conditioned media. When 120 mg/ml PCE was supplemented to endothelial cells exposed to HG-HRMC-CM, such expression was dose-dependently attenuated. Furthermore, the in vivo study attempted to confirm that PCE blocked the induction of the proangiogenic factors of VEGF and HIF-1a in db/db mice. Oral administration of 10 mg/kg PCE reduced the plasma level of VEGF elevated in db/db mice. In addition, the tissue levels of VEGF and HIF-1a were diminished in PCE-treated mouse kidneys. Accordingly, hyperglycemia appeared 1685439 to result in a hypoxic milieu in mouse kidneys and foster neovascular angiogenesis in glomeruli, which was antagonized by treating PCE to diabetic mice. Immunofluorohistochemistry BAY41-2272 web Staining For the immunohistochemical analyses, paraffin-embedded integument sections were employed. The sections were placed on glass slides, deparaffinated, hydrated with xylene and graded alcohol, and pre-incubated in boiling sodium citrate buffer for the antigen retrieval. A specific primary antibody against VEcadherin, PECAM-1, Ki-67 or 14522929 VEGFR2 was incubated overnight with tissue sections. For the VE-cadherin visualization, the sections were incubated for 3 h with FITC-conjugated anti-goat IgG. For the double staining of PECAM-1 and Ki-67, the sections were incubated for 3 h with FITC-conjugated anti-goat IgG for PECAM-1 and Cy3-conjugated anti-goat IgG for Ki-67. VEGFR2 was visualized with 3,39-diaminobenzidine to produce a brown staining, being counterstained with hematoxylin. The stained tissue sections were examined using an optical Axiomager microscope system and five images were taken for each section. Protein levels of VE-cadherin, Ki-67 and VEGFR2 were quantified by image analysis program of the microscope system. 3 Purple Corn Extract and Glomerular Angiogenesis Inhibition of Glomerular Cell Proliferation by PCE Angiopathy characterized by abnormal angiogenesis is a major diabetic vascular complication in DN. Abnormal neovascularization is closely linked to glomerular hypertrophy and excess endothelial cell proliferation in DN. The expression of the endothelial adhesion molecules of PEC