The C-terminal domain of thrombospondin-1 induces vascular smooth muscle cell chemotaxis

Objective: Thrombospondin-l (TSP-1), an acute-phase reactant implicated in vascular disease, is a 420-kd multifunctional glycoprotein chemotactic for vascular smooth muscle cells (VSMCs). TSP-I. has six domains of repeating h omologous amino acid sequences: N-terminal, procollagen homology, type 1 re peat, type 2 repeat, type 3 repeat/RGD (T3), and C-terminal (COOH). The pur pose of this experiment was to determine which domains of TSP-1 induce VSMC chemotaxis. Methods: A modified Boyden Chamber chemotaxis assay was used to assess VSMC migration. Serum-free medium, TSP-1, or each of the fusion proteins (10 an d 20 mug/mL) synthesized for the different domains were placed in the botto m wells. Quiescent bovine aortic VSMCs (50,000) were placed in the top well s. After 4 hours at 37 degreesC, migrated VSMCs were recorded as cells per five fields (400x) and analyzed with the paired t test. To verify the fusio n protein data, we performed chemotaxis assays with antibodies to each of t he domains (25 mug/mL) combined with TSP-1 (20 mug/mL) in the bottom wells and VSMCs in the top wells. Results: The COOH domain significantly stimulated VSMC chemotaxis (P = <.00 1). To a lesser extent, the N-terminal and T3 domains also induced chemotax is (P < .05). However, only the anti-COOH antibody (C6.7) and the anti-inte grin-associated protein portion of COOH antibody (D4.6) significantly inhib ited TSP-1-induced VSMC chemotaxis (by 85% and 92%, respectively). Conclusions: These results implicate the COOH domain as the portion of the TSP-1 molecule primarily responsible for VSMC chemotaxis. This experiment s uggests that future strategies in the prevention of VSMC migration, an init ial step in the development of vascular lesions, may involve selective inhi bition of the COOH domain of TSP-1.