Effects of histamine and serotonin on TSP-1 expression. (A) Serum-starved HUVEC were stimulated with 10 μM of histamine, 1 μM of serotonin, or 10 ng/mL of VEGF-A¹⁶⁵ for indicated times. Cell extracts were immunoblotted with a polyclonal chicken antibody specific for mouse TSP-1 (left panel). Membranes were stripped and reprobed with an antibody against β-actin to confirm equal protein loading (right panel). Data are representative of 3 separate experiments. (B) Nu/Nu mice were implanted subcutaneously with pellets containing 10 μg of histamine or 1 μg of serotonin, or were injected intradermally with 1× 10⁷ pfu Ad-VEGF-A¹⁶⁴. At indicated times, tissue adjacent to pellets was collected and extracted, and immunoblots were prepared using the same anti-mouse TSP-1 antibody used in (A). Data are representative of 4 separate experiments. (C) Quantification of immunoblots from 3 independent HUVEC experiments and 4 separate in vivo experiments with Image J software. Data are presented as percent change (mean ± standard error) from time zero and were analyzed with the Kruskal-Wallis test. Regarding the HUVEC data, TSP-1 levels were significantly depressed from 4 to 8 hours, but not thereafter, in the case of both histamine and serotonin treatments, whereas TSP-1 was not significantly depressed in response to VEGF until 12 hours. Regarding the histamine and serotonin in vivo data (B), TSP-1 was significantly reduced from 4 to 8 days, corresponding to the period of angiogenesis; thereafter, TSP-1 levels rebounded, correlating with angiogenesis regression (Figure 4). Ad-VEGF-A¹⁶⁴ did not lead to reduced TSP-1 expression at any time point.