Effects of DMSA-Fe2O3 on HAEC tube formation The tube formation assay is one of the most widely used assays to model the reorganization FHPI stage of angiogenesis in vitro. The assay measures the ability of endothelial cells, plated at subconfluent densities with the appropriate extracellular matrix support, to form capillary-like structures. In this study, the Matrigel basement membrane matrix was used as extracellular matrix support to observe whether angiogenesis of HAEC can be intervened by DMSA-Fe2O3
or not. For HAEC tube formation, 50 μl/well of the Matrigel basement membrane matrix was added to a 96-well plate and allowed to gel for 60 min at 37°C. Then, HAECs were seeded at a density of 1.5 × 104 cells/well on the surface of the selleck gel in the presence or absence of conditioned DMSA-Fe2O3 and incubated for 14 h at 37°C in a CO2 incubator. Meanwhile, the high urea solution (6M urea) was used as a positive control for inhibition of tube formation. The cultures on the gel were fixed for 10 min
in 25% glutaraldehyde, washed, and stained with Mayer’s hematoxylin. Each well was inspected under a light microscope at ×100 magnification and captured more than three pictures from different fields. Image-Pro plus (IPP) 6.0 for Windows software (Media Cybernetics, Inc., Rockville, MD, USA) was used to measure the length of tube formation on each picture. The average data from the same well was calculated as its quantitative value. Statistical analysis Chorioepithelioma The data were represented as mean ± SD of no less than three independent experiments. Statistical analysis was performed using a student’s t test. A value of p < 0.05 was considered statistically significant. Results and discussion Endocytosis of DMSA-Fe2O3 by HAECs We
were able to recognize the DMSA-Fe2O3 inside the HAECs and distinguish them from the cellular structures by their high electron density on TEM. Figure 1 represents TEM micrographic images between HAECs incubation with 0.02 mg/ml of DMSA-Fe2O3 (Figure 1c,d) and HAECs without DMSA-Fe2O3 incubation (Figure 1a,b). The results indicate that the DMSA-Fe2O3 aggregates are readily absorbed by the cells without disrupting the integrity of the cellular membrane and dispersed in the cytoplasm. Figure 1 The TEM images of HAECs incubated with 0.02 mg/ml of DMSA-Fe 2 O 3 for 24 h. (a) HAEC without DMSA-Fe2O3 (×8,000). (b) HAEC without DMSA-Fe2O3 (×30,000). (c) HAEC incubated with DMSA-Fe2O3 (×5,000). (d) HAEC incubated with DMSA-Fe2O3 (×30,000). Abbreviations: n, nucleus; v, vesicle; Arrows denote the DMSA-Fe2O3 or particulate matter.