In Vitro Interactions of Oxidatively Modified LDL With Type I, II, III, IV, and V Collagen, Laminin, Fibronectin, and Poly- d -Lysine

Abstract

Abstract The accumulation of LDL in the arterial intima is considered a key event in atherogenesis. We investigated the binding of oxidized LDL (ox-LDL) to microtiter plates coated with type I or II collagen, laminin, fibronectin, or poly- d -lysine. Oxidation of LDL, ¹²⁵ I-LDL, or Eu ³⁺ -LDL was performed with CuCl ₂ , varying the time of oxidation. Bound lipoprotein was assessed by counting radioactivity or fluorescence in the wells. Binding of highly ox-LDL in PBS followed the order: type I collagen>poly- d -lysine>type II collagen>laminin>fibronectin. Comparing various collagen types, the binding of ox-LDL followed the order: type I>type V and, type III>type IV>type II collagen. Binding of ox-LDL in PBS was dependent on an increase in negative charge of ox-LDL. Testing certain amino acids as competitors for binding of highly ox-LDL to type I collagen put lysine first, followed by arginine and histidine. On laminin, histidine competed most, followed by lysine and arginine. When studying the influence of Na ⁺ , K ⁺ , Ca ²⁺ , Mg ²⁺ (equivalent to their concentrations in the interstitial fluid), native LDL, moderately ox-LDL, and highly ox-LDL showed the same affinity to type I collagen. However, a fivefold dilution of the buffer increased the affinity of moderately and highly ox-LDL 3.9- and 10-fold compared with native LDL. Application of the F(ab′) ₂ from a monoclonal antibody to ox-LDL revealed a strong competition of the binding of highly ox-LDL to type II collagen (60%), laminin (35%), type I collagen (20%), and poly- d -lysine (15%), whereas the binding to fibronectin was not affected.