Background-Previous work indicated that enzymatically remodeled LDL (E-LDL) might activate complement in atherosclerotic lesions via a C-reactive protein (CRP)-dependent and CRP-independent pathway. We sought to substantiate this contention and determine whether both pathways drive the sequence to completion. Methods and Results-E-LDL was prepared by sequential treatment of LDL with a protease and cholesteryl esterase.Trypsin, proteinase K, cathepsin H, or plasmin was used with similar results. Functional tests were used to assess total complement hemolytic activity, and immunoassays were used to demonstrate C3 cleavage and to quantify C3a, C4a, C5a, and C5b-9. E-LDL preparations activated complement to completion, independent of CRP, when present above a threshold concentration (100 to 200 g/mL in 5% serum). Below the threshold, all E-LDL preparations activated complement in dependence of CRP, but the pathway then halted before the terminal sequence. Native LDL and oxidized LDL did not activate complement under any circumstances tested. Immunohistological analyses corroborated the concept that CRP-dependent complement activation inefficiently generates C5b-9. Conclusions-Binding of CRP to E-LDL is the first trigger for complement activation in the atherosclerotic lesion, but the terminal sequence is thereby spared. This putatively protective function of CRP is overrun at higher E-LDL concentrations, so that potentially harmful C5b-9 complexes are generated.

Background-Treatment of low-density lipoprotein (LDL) with a protease and cholesterolesterase transforms the lipoprotein to an entity that resembles lipoprotein particles in atherosclerotic lesions, which have a high content of free cholesterol, reflecting extensive de-esterification in the intima. Because de-esterification would occur beneath the endothelium, we examined the effects of enzymatically modified LDL (E-LDL) on cultured endothelial cells. Methods and Results-Incubation of endothelial cells with E-LDL provoked selective accumulation of interleukin (IL)-8 mRNA and production of the cytokine. Chemical analyses and depletion experiments indicated that the effect was caused by the presence of free fatty acids in the altered lipoprotein. Reconstitution studies demonstrated that the oleic and linoleic acids associated with E-LDL are particularly effective IL-8 inducers. The effects of E-LDL on endothelial cells could be abrogated with albumin. Conclusion-IL-8 is required for rolling monocytes to adhere firmly to the endothelium; thus, the findings reveal a link between subendothelial entrapment of LDL, cleavage of cholesterol esters, and monocyte recruitment into the lesion.

Objective-Functionally interactive proteases of the plasminogen/plasmin and the matrix metalloproteinase (MMP) system degrade and reorganize the extracellular matrix of the vessel wall in atherosclerosis. Here we investigated whether such proteases are able to confer atherogenic properties onto low density lipoprotein by nonoxidative modification. Methods and Results-Similar to the recently described enzymatically-modified low-density lipoprotein (E-LDL), native LDL exposed to plasmin or matrix MMP-2 or MMP-9 and cholesterylester-hydrolase (CEH) showed extensive deesterification, with ratios of free cholesterol to total cholesterol rising to 0.8 compared with 0.2 in native LDL. When the ratio exceeded 0.6, both plasmin/CEH-LDL and MMP/CEH-LDL fused into larger particles. In parallel, they gained C-reactive protein-dependent complement-activating capacity. E-LDL produced with any protease/CEH combination was efficiently taken up by human macrophages, whereby marked induction of MMP-2 expression by E-LDL was observed. These in vitro findings had their in vivo correlates: urokinase-type plasminogen activator, MMP-2, and MMP-9 were detectable in both early and advanced human atherosclerotic lesions in colocalization with E-LDL. Conclusions-Plasmin and MMP-2/MMP-9 may not only be involved in remodeling of the extracellular matrix in progressing plaques, but they may also be involved in lipoprotein modification during genesis and progression of atherosclerotic lesions. Key Words: atherosclerosis Ⅲ lipoproteins Ⅲ macrophages Ⅲ metalloproteinases Ⅲ plasminogen activators I t is widely held that atherogenesis is triggered by enhanced entrapment of low-density lipoprotein (LDL) in the intima, which is followed by its uptake by macrophages. Both oxidative and nonoxidative processes can generate potentially atherogenic LDL derivatives. 1 We are pursuing the concept that enzymatic remodeling of the lipoprotein is a key modification, because proteolytic cleavage of apolipoprotein B (apoB) in conjunction with hydrolysis of cholesteryl esters generates lipoprotein particles that are similar to lesionderived LDL in structure, biological properties, and composition. 2,3 Enzymatically-remodeled LDL (E-LDL) binds C-reactive protein (CRP) and activates complement. 4 E-LDL induces foam cell formation in monocytes, 5 macrophages, 3 and smooth muscle cells, 6 stimulates MCP-1 production, 7 and directly promotes adhesion and transmigration of monocytes through endothelial cell monolayers. 8 These in vitro findings have their in vivo correlates: immunohistological analyses with specific monoclonal antibodies (mAbs) have revealed extensive extracellular deposits of E-LDL at the early stages of atherosclerotic lesion formation. 9 CRP and activated complement components are also present in colocalization with E-LDL. 4,9 Like in vitro-generated E-LDL, lesioned LDL has a high content of free cholesterol; 10 -12 therefore, it is apparent that extensive deesterification of cholesteryl esters must indeed occur in the lesions.We used trypsin in c...

Objective-Modification with proteases and cholesterylesterase transforms LDL to a moiety that resembles lipoproteins isolated from atherosclerotic lesions and possesses atherogenic properties.