LIGAND OCCUPANCY OF THE ALPHA-V-BETA-3 INTEGRIN IS NECESSARY FOR SMOOTH-MUSCLE CELLS TO MIGRATE IN RESPONSE TO INSULIN-LIKE GROWTH-FACTOR-I

Smooth muscle cells (SMCs) have been shown to migrate in response to i nsulin-like growth factor I (IGF-I), However, the mechanism mediating this response has not been determined, The migration rates of porcine and human vascular SMCs were assessed in a monolayer wounding assay. I GF-I and IGF-II induced increases of 141% and 97%, respectively, in th e number of cells that migrated in 4 days, The presence of 0.2% fetal bovine serum in the culture medium was necessary for the IGFs to stimu late migration over uncoated plastic surfaces, However, if vitronectin was used as the substratum, IGF-I stimulated migration by 162% even i n the absence of serum, To determine the role of integrins in mediatin g this migration, SMC surface proteins were labeled with I-125 and imm unoprecipitated with specific anti-integrin antibodies, Integrins cont aining alpha V (vitronectin receptor), alpha 5 (fibronectin receptor), and alpha 3 (collagen/laminin receptor) subunits were the most abunda nt, IGF-I treatment caused a 73% reduction in alpha 5-integrin subunit protein and a 25% increase in alpha V subunit. More importantly, liga nd binding of alpha V beta 3 was increased by 2.4-fold, We therefore e xamined whether the function of the alpha V beta 3 integrin was import ant for IGF-I-mediated migration, The disintegrin kistrin was shown by affinity crosslinking to specifically bind with high affinity to alph a V beta 3 and not to alpha 5 beta 1 or other abundant integrins, The related disintegrin echistatin specifically inhibited I-125-labeled ki strin binding to alpha V beta 3, while a structurally distinct disinte grin, decorsin, had 1000-fold lower affinity, The addition of increasi ng concentrations of either kistrin or echistatin inhibited IGF-I-indu ced migration, whereas decorsin had a minimal effect. The potency of t hese disintegrins in inhibiting IGF-I-induced migration paralleled the ir apparent affinity for the alpha V integrin, Furthermore, an alpha V beta 3 blocking antibody inhibited SMC migration by 80%, In summary, vitronectin receptor activation is a necessary component of IGF I-medi ated stimulation of smooth muscle migration, and alpha V beta 3 integr in antagonists appear to be important reagents for modulating this pro cess.