In Vitro Study of Thrombin on Tubule Formation and Regulators of Angiogenesis

Abstract

Angiogenesis occurs within atherosclerotic plaques, and thrombin has been implicated in plaque progression by increasing smooth muscle cell proliferation and upregulating Vascular Endothelial Growth Factor (VEGF) receptor expression. This study investigated the effects of thrombin on key aspects of angiogenesis and expression of pro- and anti-angiogenic regulators: VEGF and Pigment Epithelial Derived Factor. Human umbilical smooth muscle cells (HUASMC) were exposed to vehicle or thrombin at 10 U/ml. To quantify cell proliferation, methyl tetrazolium salt (MTS) solution was added after exposure to thrombin for 24, 48 and 72 hours and the absorbance at 490nm recorded using a plate reader. For Real-time RT-PCR cells were exposed to thrombin for 24hr before analysis of VEGF and PEDF mRNA. A commercial Angiokit was used to construct an in vitro angiogenesis model to measure tubule formation and branching. After 12 days treatment with thrombin 10 U/ml, cells were fixed and the AngioSys 1.0 software was used to analyse tubule morphology. In comparison with controls, thrombin significantly increased HUASMC proliferation (p = 0.002, n = 11) and VEGF mRNA expression by 93% (n = 4, p = 0.024). However in the HUVEC/fibroblast co-cultures it decreased the number of junctions [132(9) vs 196(18), n = 6, p = 0.017] and tubules [537 (17) vs 589 (26), n = 6, p = 0.049], and tubule length [11393 (1601) vs 12195 (1014), n = 6, p = 0.044], indicating an anti-angiogenic effect. Thrombin stimulates vascular smooth muscle cell proliferation and VEGF expression, but attenuates endothelial cell-mediated growth of vascular tubules and branching of new vascular structures.