Thrombin stimulates mitogenesis in pig cerebrovascular smooth muscle cells involving activation of pro-matrix metalloproteinase-2

Generation of thrombin is associated with vascular remodeling that involves proliferation of vascular smooth muscle cells (SMCs) and activation of pro-matrix metalloproteinases (pro-MMPs). The present study was to investigate whether thrombin would induce mitogenesis and activation of pro-MMPs in cerebrovascular SMCs (CSMCs), and if so, whether MMP activity would contribute to the CSMC mitogenesis. CSMCs were cultured from pig middle cerebral arteries and stimulated with thrombin. Thrombin (0.1-5U/ml), in a dose-dependent fashion, stimulated mitogenesis in CSMCs as detected by bromo-2'-deoxy-uridine (BrdU) incorporation. Additionally, zymographic analyses showed that thrombin stimulated the appearance of the active form of MMP-2 (MMP-2) in a concentration-dependent manner, but not the release of pro-MMP-2. Thrombin did not affect expression of cell-associated pro-MMP-2 protein as evaluated by Western blot analysis. Treatment with the synthetic MMP inhibitor GM6001 or antibodies to MMP-2 significantly reduced thrombin-induced BrdU incorporation in CSMCs. In conclusion, thrombin activates pro-MMP-2 in the absence of elevated pro-MMP-2 expression and secretion in CSMCs, and thrombin induces CSMC mitogenesis involving its action on MMP-2. These findings suggest that thrombin may have relevance in cerebrovascular remodeling associated with brain atherosclerosis and atherothrombotic ischemic stroke through a mechanism involving MMP-dependent CSMC mitogenesis.