Sakata N, Sasatomi Y, Meng J, Ando S, Uesugi N, Takebayashi S, Nagai R, Horiuchi S
Department of Pathology, School of Medicine; Fukuoka University, 45-1, 7-chome Nanakuma, Jonan-ku, Fukuoka 814-0180, Japan.
Connect Tissue Res. 2000;41(3):213-28. doi: 10.3109/03008200009005291.
Although fibronectin (FN) modified by advanced glycation end products (AGEs) has been shown to contribute to the development of diabetic vascular complications through its reduced adhesive activity to vascular cells, little is known about changes in the cell binding domain of AGE-modified FN. Here we examined the mechanism of reduced adhesive and spreading activities of AGE-modified FN to vascular smooth muscle cells (SMCs), particularly the contribution of modification of Arg-Gly-Asp (RGD) sequence. Incubation with glucose caused not only the formation of N(epsilon) -carboxymethyllysine and pentosidine, but also polymerization of FN in a dose- and time-dependent manner. AGE-modified FN had significantly low adhesive and spreading activities to 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-modification on cell adhesion was significantly greater in type I collagen than in FN. Although the extent of AGE-modification of type I collagen was indistinguishable from that of FN, AGE-modification decreased the arginine content 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 adhesion of SMCs to non-glycated FN, but not to AGE-modified FN. Modification of RGD sequence with glyoxal eliminated its inhibitory effect on cell adhesion. Our results suggest that a marked decrease in adhesive and spreading activities of AGE-modified FN to SMCs might largely be due to a modification of its RGD sequence by AGE, thus suggesting a potential link between AGE modification of FN and the pathogenesis of diabetic angiopathy.
尽管晚期糖基化终末产物（AGEs）修饰的纤连蛋白（FN）已被证明因其对血管细胞的黏附活性降低而促进糖尿病血管并发症的发展，但关于AGE修饰的FN细胞结合域的变化却知之甚少。在此，我们研究了AGE修饰的FN对血管平滑肌细胞（SMC）黏附及铺展活性降低的机制，特别是对精氨酸 - 甘氨酸 - 天冬氨酸（RGD）序列修饰的作用。葡萄糖孵育不仅导致N（ε）-羧甲基赖氨酸和戊糖苷的形成，还使FN以剂量和时间依赖性方式发生聚合。AGE修饰的FN对培养的SMC具有显著降低的黏附及铺展活性。另一方面，由二硫键形成的多聚体FN对细胞黏附或铺展均无影响。含RGD序列的蛋白质之一I型胶原蛋白对SMC的黏附活性也因AGE修饰而降低。AGE修饰对I型胶原蛋白细胞黏附的抑制作用比对FN的作用显著更强。尽管I型胶原蛋白的AGE修饰程度与FN的难以区分，但与未糖基化形式相比，AGE修饰使I型胶原蛋白的精氨酸含量降低了69.5%，FN的精氨酸含量降低了30.6%。添加RGD肽导致SMC对未糖基化FN的黏附减少，但对AGE修饰的FN无此作用。用乙二醛修饰RGD序列消除了其对细胞黏附的抑制作用。我们的结果表明，AGE修饰的FN对SMC黏附及铺展活性的显著降低可能很大程度上是由于其RGD序列被AGE修饰，从而提示FN的AGE修饰与糖尿病血管病变发病机制之间存在潜在联系。
