Hammes H P, Weiss A, Hess S, Araki N, Horiuchi S, Brownlee M, Preissner K T
Third Department of Internal Medicine, Justus Liebig Universität, Giessen, Germany.
Lab Invest. 1996 Sep;75(3):325-38.
Early diabetic retinopathy is characterized by areas of acellular capillaries and the accumulation of advanced glycation end products (AGE). Endothelial cell maintenance, growth, and migration is regulated by a complex system involving the partitioning of growth factors between extracellular matrix-bound proteoglycans and cells, as well as by the activation and control of pericellular proteolysis. Using specific antibodies, we compared the immunolocalization of the vascular heparan sulfate proteoglycan (HSPG)-binding protein vitronectin (VN), HSPG, basic fibroblast growth factor, and collagen type 1 in retinae prepared from 11-month-old diabetic and nondiabetic Wistar rats. In normal rats, VN immunostaining was prominent in vascularized parts of the inner retina and colocalized with HSPG, which itself showed costaining with basic fibroblast growth factor in the extracellular matrix, vascular walls, and the inner limiting membrane. In contrast, the recognition of VN, HSPG, and basic fibroblast growth factor in diabetic retinae was greatly reduced in the inner limiting membrane and the extracellular matrix, where increased immunoreactive AGE colocalizing with VN were detectable. Capillary labeling of retinal vessel walls for plasminogen activator inhibitor-1 was moderate in both normal and diabetic retinae. AGE-VN was demonstrated in tissue extracts from retina by immunoprecipitation and Western blotting and presented a similar increase of AGE-related immunoreactivity compared with AGE-VN generated in vitro. AGE-VN in vitro had lost its native conformation, yielded high Mr SDS-resistant products, and was resistant to proteolysis because of modification of about 30% of lysines. Because binding of glycosaminoglycans, as well as interaction of type I collagen and the morphoregulatory proteins osteonectin and tenascin with AGE-VN, were reduced to at least 50% of control, alteration of basic residues in the heparin-binding domain of VN is plausible. In comparison to nonmodified VN, AGE-VN exhibited reduced cell attachment-promoting activity. Together, these in vitro results suggest that AGE-VN found in vivo is related to morphologic and functional changes in the diabetic retina and may contribute to the genesis of acellular capillaries in early diabetic retinopathy.
早期糖尿病视网膜病变的特征是无细胞毛细血管区域以及晚期糖基化终产物（AGE）的积累。内皮细胞的维持、生长和迁移受一个复杂系统调控，该系统涉及生长因子在细胞外基质结合蛋白聚糖与细胞之间的分配，以及细胞周蛋白水解的激活和控制。我们使用特异性抗体，比较了11月龄糖尿病和非糖尿病Wistar大鼠视网膜中血管硫酸乙酰肝素蛋白聚糖（HSPG）结合蛋白玻连蛋白（VN）、HSPG、碱性成纤维细胞生长因子和I型胶原的免疫定位。在正常大鼠中，VN免疫染色在内层视网膜的血管化部分很突出，并与HSPG共定位，而HSPG本身在细胞外基质、血管壁和内界膜中与碱性成纤维细胞生长因子呈共染色。相比之下，糖尿病视网膜中VN、HSPG和碱性成纤维细胞生长因子在内界膜和细胞外基质中的识别显著减少，在这些部位可检测到与VN共定位的免疫反应性AGE增加。视网膜血管壁纤溶酶原激活物抑制剂-1的毛细血管标记在正常和糖尿病视网膜中均为中等程度。通过免疫沉淀和蛋白质印迹在视网膜组织提取物中证实了AGE-VN，与体外产生的AGE-VN相比，其呈现出类似的AGE相关免疫反应性增加。体外AGE-VN失去了其天然构象，产生高分子量的耐SDS产物，并且由于约30%的赖氨酸被修饰而对蛋白水解具有抗性。由于糖胺聚糖的结合以及I型胶原与形态调节蛋白骨连接蛋白和腱生蛋白与AGE-VN的相互作用减少至对照的至少50%，VN肝素结合结构域中碱性残基的改变是合理的。与未修饰的VN相比，AGE-VN表现出降低的促进细胞黏附活性。总之，这些体外结果表明，体内发现的AGE-VN与糖尿病视网膜的形态和功能变化有关，可能有助于早期糖尿病视网膜病变中无细胞毛细血管的形成。
