The proliferation of vascular smooth muscle cells (VSMCs) is controlled by growth factors and by the interaction of cells with the extracellular matrix (ECM). Our goal is to understand the role of the fibronectin matrix in controlling VSMC proliferation. We have found that a recombinant fragment of fibronectin (protein III1-C) inhibits VSMC fibronectin matrix assembly and also inhibits VSMC proliferation. Moreover, III1-C blocks both growth factor-induced and adhesion-induced MAP kinase activation in VSMCs. To determine whether inhibition of fibronectin matrix assembly blocks MAP kinase activation, we will test whether other methods of inhibiting fibronectin matrix assembly also inhibit VSMC proliferation and MAP kinase signaling. It is possible that III1-C inhibits VSMC proliferation by some activity other than inhibition of fibronectin matrix assembly. We will therefore test whether III1-C interacts with proteins other than fibronectin on the surface of VSMCs. This will be accomplished by several methods, including crosslinking of labeled III1-C to cell surface proteins and III1-C affinity chromatography to isolate and identify cell surface proteins that interact with III1-C. The molecular mechanism by which III1-C blocks MAP kinase signaling will be analyzed by systematically testing the effects of III1-C on the activities of the known members of the Ras/MAP kinase pathway, thereby allowing us to determine where III1- C disrupts this signaling pathway. We will also test the effect of III1-C on VSMC proliferation in vivo in a restenosis model. III1-C will be inserted into an adenovirus expression construct that includes a recently discovered SMC-specific promoter. This will allow high levels of expression and secretion of III1-C specifically in VSMCs in vivo. The III1-C-adenovirus vector will be tested in the rat carotid artery balloon injury model. If III1-C has the same effect on VSMC proliferation in vivo as it does in cell culture, then the III1-C- adenovirus vector should inhibit neointima formation in this rat carotid artery restenosis model. The work proposed in this grant will help establish the importance of the ECM, and in particular of the fibronectin matrix in regulating VSMC growth both in culture and in vivo. By understanding the role of the ECM in pathological processes such as restenosis and atherosclerosis we may begin to design more effective treatments for these conditions.

R01HL059962

1999-01-01

2003-12-31