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

Abstract

Objective: Thrombospondin-1 (TSP-1), an acute-phase reactant implicated in vascular disease, is a 420-kd multifunctional glycoprotein chemotactic for vascular smooth muscle cells (VSMCs). TSP-1 has six domains of repeating homologous amino acid sequences N-terminal, procollagen homology, type 1 repeat, type 2 repeat, type 3 repeat/RGD (T3), and C-terminal (COOH). The purpose 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 and 20 μg/mL) synthesized for the different domains were placed in the bottom wells. Quiescent bovine aortic VSMCs (50,000) were placed in the top wells. After 4 hours at 37°C, migrated VSMCs were recorded as cells per five fields (400×) and analyzed with the paired t test. To verify, the fusion protein data, we performed chemotaxis assays with antibodies to each of the domains (25 μg/mL) combined with TSP-1 (20 μg/mL) in the bottom wells and VSMCs in the top wells. Results: The COOH domain significantly stimulated VSMC chemotaxis (P = < .001). To a lesser extent, the N-terminal and T3 domains also induced chemotaxis (P < .05). However, only the anti-COOH antibody (C6.7) and the anti-integrin-associated protein portion of COOH antibody (D4.6) significantly inhibited 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 suggests that future strategies in the prevention of VSMC migration, an initial step in the development of vascular lesions, may involve selective inhibition of the COOH domain of TSP-1.