Thrombospondin-1 induces matrix metalloproteinase-2 activation in vascular smooth muscle cells.
BACKGROUND
Thrombospondin-1 (TSP-1), an extracellular matrix (ECM) glycoprotein, is associated with a variety of cellular processes relevant to atherosclerosis and intimal hyperplasia, including vascular smooth muscle cell (VSMC) migration. Matrix metalloproteinase-2 (MMP2) is associated with basement membrane and ECM degradation, important processes for cell migration. We hypothesized that TSP-1 modulates MMP2 activity in VSMCs and is critical for VSMC migration.


METHODS
Quiescent bovine aortic VSMCs (48 hours) were incubated in serum-free media (SFM) with or without TSP-1 (10 or 20 microg/mL). Gelatinase activity was measured with zymography to determine pro-MMP2 and MMP2 activity. MMP2 messenger RNA expression was determined with Northern blot analysis. Invasion assays were performed. A binding assay was used to determine the specificity of TSP-1 binding to MMP2. Blots were quantified with densitometry, and all comparisons were made with a paired t test.


RESULTS
TSP-1 induced production of activated forms of MMP2, as well as upregulation of pro-MMP2. MMP2 mRNA was upregulated 1.7-fold by TSP-1 at 10 and 20 microg/mL. GM6001, an MMP inhibitor, inhibited VSMC migration across the matrix barrier, whereas migration that occurred in the absence of the matrix barrier was unaffected. With a binding assay, TSP-1 interacted physically with MMP2, and TSP-1-bound MMP2 showed the strongest binding activity in comparison with collagen I, fibronectin, and elastin.


CONCLUSIONS
TSP-1 induced MMP2 activation through transcriptional and posttranslational mechanisms. These findings imply that MMP2 activation is relevant to the mechanism of TSP-1-induced VSMC migration.
Lee
Taeseung
T
Section of Vascular Surgery, Yale University School of Medicine and the VA Connecticut Healthcare System, New Haven, CT 06520, USA.
Esemuede
Nowokere
N
Sumpio
Bauer E
BE
Gahtan
Vivian
V
D000818:Animals; D002417:Cattle; D002465:Cell Movement / Q000502:physiology*; D002633:Chemotaxis; D066298:In Vitro Techniques; D020778:Matrix Metalloproteinase 2 / Q000096:biosynthesis / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D032389:Myocytes, Smooth Muscle / Q000378:metabolism / Q000502:physiology*; D019700:Thrombospondin 1 / Q000502:physiology*; D015854:Up-Regulation
D019700:Thrombospondin 1; D020778:Matrix Metalloproteinase 2