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Volume 2012 (2012), Article ID 941465, 8 pages
VEGF Is Involved in the Increase of Dermal Microvascular Permeability Induced by Tryptase
1Department of Physiology and Pathophysiology, Fudan University Shanghai Medical College, Shanghai 200032, China
2Department of Cardiology, Shanghai Jiaotong University Affiliated Sixth People's Hospital, Shanghai 200032, China
Received 5 February 2012; Accepted 13 March 2012
Academic Editors: E. Pasmatzi and A. Zalewska
Copyright © 2012 Qianming Bai et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
All HDMECs gave typical confluent cobblestone appearance (Supplemental Figure 1(a)), and had positive reactions to the antibodies against vWF (Supplemental Figure 1(b)) and CD34 (Supplemental Figure 1(c)). Negative control without first antibody exhibited no staining (Supplemental Figure 1(d)). The expressions of vWF and CD34 were also quantified with flow cytometry. Exceed 90% cells were positive for vWF and CD34, which suggested the purity of the primary cells exceeded 90%.
Measurement of the tryptase activity of HMC-1 supernatant: To affirm the existence of tryptase in the conditioned medium, we incubated the HMC-1 supernatant made with HMC-1 suspension in the presence and absence of pro-degranulating agent a23187 (1 µg/mL) with substrate (t6140, N-Tosylglycyl-L-prolyl-L-lysine 4-nitroanilide acetate salt, 8 mmol/L) for 10 minutes in the reaction buffer (40 mM HEPES，0.12 M NaCl，pH 7.4). OD value of the reactions were detected by spectrophotometer at 405 nm each 30 seconds. The control is set with only the reaction buffer. As shown in Supplementary Figure 2(a), the change of OD405 (formation of t6140-derived product digested by tryptase) was linear for at least 10 minutes. Then 5 minutes was chosen to be the reaction time. Tryptase was released in the HMC-1 supernatant, which is increased dramatically by pro-degranulating agent a23187 (Supplementary Figure 2(b)). A23187 stimulated HMC-1 cells to release tryptase dose-dependently (Supplementary Figure 2(c)). On the other way, tryptase was released from HMC-1 cells by 1 µg/mL a23187 in density-dependent manner (Supplementary Figure 2(d)). In the following experiments, HMC-1 supernatant was prepared using 1 × 107 HMC-1 cells/mL treated with 1 µg/mL a23187.