Inhibition of Endothelial Cell Migration by Gene Transfer of Tissue Inhibitor of Metalloproteinases-1

Background.Angiogenesis requires degradation of the vessel's basal lamina and endothelial cell migration into the tissue stroma. Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) play important roles in this process. MMP activity is tightly regulated during vessel growth. This work was designed to characterize the effect of TIMP-1 upregulation on endothelial cell invasion of the extracellular matrix. Methods.We constructed replication-deficient recombinant adenoviruses that encode either TIMP-1 (Ad.TIMP-1) orEscherichia colilac Z (Ad.βgal) cDNA. Bovine aortic endothelial (BAE) cells were infected with 100 infectious particles/cell. Gene expression was assessed by Northern and Western blotting. TIMP-1 activity in cell-conditioned media was measured by a resorufin-labeled casein protease assay. BAE cell migration was measured by Boyden chamber assays with 0.2% gelatin-coated, 8.0-μm polycarbonate membranes. Results.TIMP-1 was overexpressed by Ad.TIMP-1-infected BAE cells relative to control, Ad.βgal-infected or uninfected cells. TIMP-1 activity in Ad.TIMP-1 cell-conditioned medium was 2.8-fold higher than in control cells. By Boyden chamber assays with gelatin-coated membranes, Ad.TIMP-1-infected BAE cells showed 89.97 ±1.64% (mean ± SEM) reduction in migration relative to Ad.βgal-infected cells (P< 0.02) and 90.53 ± 1.12% relative to uninfected cells (P< 0.02). Without gelatin coating, migration was equivalent in all groups. Conclusion.The replication-deficient recombinant adenovirus we constructed affords rapid and efficient upregulation of functional TIMP-1 in endothelial cells. Infection results in a dramatic decrease in cell migration and invasion of extracellular matrix. Thus, such a recombinant vector may provide a useful tool for the gene therapy of vascular remodeling and inhibition of angiogenesis.