Immunohistochemical Demonstration of Enzymatically Modified Human LDL and Its Colocalization With the Terminal Complement Complex in the Early Atherosclerotic Lesion

Abstract

Abstract-Treatment of low density lipoprotein (LDL) with degrading enzymes transforms the molecule to a moiety that is micromorphologically indistinguishable from lipoproteinaceous particles that are present in atherosclerotic plaques, and enzymatically modified LDL (E-LDL), but not oxidized LDL (ox-LDL), spontaneously activates the alternative complement pathway, as do lesion lipoprotein derivatives. Furthermore, because E-LDL is a potent inducer of macrophage foam cell formation, we propose that enzymatic degradation may be the key process that renders LDL atherogenic. In this article, we report the production of two murine monoclonal antibodies recognizing cryptic epitopes in human apolipoprotein B that become exposed after enzymatic attack on LDL. One antibody reacted with LDL after single treatment with trypsin, whereas recognition by the second antibody required combined treatment of LDL with trypsin and cholesterol esterase. In ELISAs, both antibodies reacted with E-LDL produced in vitro and with lesion complement activator derived from human atherosclerotic plaques, but they were unreactive with native LDL or ox-LDL. The antibodies stained E-LDL, but not native LDL or ox-LDL, that had been artificially injected into arterial vessel walls. With the use of these antibodies, we have demonstrated that early human atherosclerotic coronary lesions obtained at autopsy as well as lesions examined in freshly explanted hearts always contain extensive extracellular deposits of E-LDL. Terminal complement complexes, detected with a monoclonal antibody specific for a C5b-9 neoepitope, colocalized with E-LDL within the intima, which is compatible with the proposal that subendothelially deposited LDL is enzymatically transformed to a complement activator at the earliest stages in lesion development. (Arterioscler Thromb Vasc Biol. 1998;18:369-378.)

Enzymatically Modified Low‐Density Lipoprotein Is Present in All Stages of Aortic Valve Sclerosis: Implications for Pathogenesis of the Disease

“…In parallel to findings in early atherosclerotic lesions, we could demonstrate immunohistochemical staining for C3d and C5b‐9,11, 13, 33 the terminal membrane attack complex, in all stages of aortic valve sclerosis. Complement activation in aortic stenosis has already been detected before,34 but in the present study we were able to show close scrutiny of the staining patterns of complement components with eLDL for the first time, and our semiquantitative immunohistochemical analysis of eLDL and C5b‐9 suggests that C5b‐9 deposits reflect the presence of extensively modified LDL, whereby other possible complement activating mechanisms are not excluded.…”

“…Certainly, modified lipoproteins are intimately involved 3. eLDL is thought to be one of the initiators of atherosclerosis 7, 8, 10, 11. The novel findings of the present study are the evidence for the presence of this modified lipoprotein in all stages of aortic valve sclerosis, its role as a possible complement activator, and its cellular uptake by VICs/myofibroblasts.…”

“…The concept that we have been pursuing proposes extracellular enzymatic degradation of LDL to represent a key step leading to generation of an atherogenic lipoprotein 7, 10, 11, 30. eLDL generated by all tested proteases is endowed with the same atherogenic properties as the originally described trypsin‐generated eLDL 23, 31.…”

“…After blocking, slides were incubated with primary antibodies listed in Table 2 for 30 minutes. In particular, the characterization of the murine monoclonal antibody AIL‐3 recognizing cryptic epitopes in human apolipoprotein B that become exposed after enzymatic degradation of LDL has been reported 11. Antigen retrieval of CD68 was achieved by heating the sections in target retrieval solution (Dako) in a steamer for 20 minutes.…”

Complement C6 Deficiency Protects Against Diet-Induced Atherosclerosis in Rabbits

“…These antibodies reacted with E-LDL and LCA, but not with native or oxidized LDL, and it became possible to demonstrate extensive extracellular deposition of E-LDL in all early human atherosclerotic lesions. 15 Significantly, C5b-9 almost invariably colocalized with E-LDL, corroborating the hypothesis that LDL is enzymatically transformed to a complement-activating moiety at an early stage in atherogenesis. In contrast to E-LDL, oxidized LDL is not endowed with complement-activating properties.…”