Possible involvement of altered RGD sequence in reduced adhesive and spreading activities of advanced glycation end product-modified fibronectin to vascular smooth muscle cells

Although fibronectin (FN) modified by advanced glycation end products (AGEs ) has been shown to contribute to the development of diabetic vascular comp lications through its reduced adhesive activity to vascular cells, little i s known about changes in the cell binding domain of AGE-modified FN. Here w e examined the mechanism of reduced adhesive and spreading activities of AG E-modified FN to vascular smooth muscle cells (SMCs), particularly the cont ribution of modification of Ag-Gly-Asp (RGD) sequence. Incubation with gluc ose caused not only the formation of N-epsilon-carboxymethyllysine and pent osidine, but also polymerization of FN in a dose- and time-dependent manner . AGE-modified FN had significantly low adhesive and spreading activities t o cultured SMCs. On the other hand, multimeric FN formed by disulfide bonds did not show any effect on either cell adhesion or spreading. The adhesive activity of type I collagen, one of the RGD sequence-containing proteins, to SMCs also decreased by AGE-modification. The inhibitory effect of AGE-mo dification on cell adhesion was significantly greater in type I collagen th an in FN. Although the extent of AGE-modification of type I collagen was in distinguishable from that of FN, AGE-modification decreased the arginine co ntent of type I collagen by 69.5% and of FN by 30.6%, compared with their. non-glycated forms. The addition of RGD peptides caused a decrease in adhes ion of SMCs to non-glycated FN, but not to AGE-modified FN. Modification of RGD sequence with glyoxal eliminated its inhibitory effect on cell adhesio n. Our results suggest that a marked decrease in adhesive and spreading act ivities of AGE-modified FN to SMCs might largely be due to a modification o f its RGD sequence by AGE, thus suggesting a potential link between AGE mod ification of FN and the pathogenesis of diabetic angiopathy.