Carbamylated-Oxidized LDL: Proatherosclerotic Effects on Endothelial Cells and Macrophages

Abstract

Aim Both oxidized LDL and carbamylated LDL are considered important for initiating atherosclerosis in patients with end-stage kidney disease through vascular endothelial cell dysfunction or injury. However their effects on each other and their relationship related to pro-atherosclerotic effects on endothelial cells and macrophages have not been investigated. In this study, we analyzed the competition between LDL carbamylation and oxidation, tested biological effects of carbamylated-oxidized LDL (coxLDL) toward the endothelial cells, assessed its ability to cause foam cell development, and determined the roles of scavenger receptors in this process. Methods Cross-competition between carbamylation and oxidation of LDL particles was tested using cell-free fluorescent ligand-receptor assay. Pro-atherogenic properties (cell proliferation, cytotoxicity, and foam cell formation) of all LDL isoforms were tested in vitro and ex vivo using endothelial cells and peritoneal macrophages. In addition, coxLDL was assessed in human sera and in vivo atherosclerotic plaques which were developed in mouse model of uremia-induced atherosclerosis. Results Our data suggest that there is potential competition between carbamylation and oxidation of LDL, and that oxidation is a much stronger inhibitor of carbamylation than vice versa. coxLDL is highly cytotoxic to endothelial cells and strongly induce their proliferation measured by DNA synthesis. All three tested LDL isoforms demonstrated strong ability for transformation of primary mouse peritoneal macrophages to foam cells using predominantly CD36 scavenger receptor. coxLDL was the most potent inducer of foam cell development and macrophages/foam cell injury assessed by cell count and TUNEL, respectively. Finally, LDL particles modified by oxidation and carbamylation were detected in blood and shown to co-localize in atherosclerotic plaques in mice. Conclusion Our study demonstrated that LDL particles can be simultaneously carbamylated and oxidized and modifications are likely coexisting in the same LDL particle. We also demonstrated pro-atherosclerotic properties of coxLDL and proposed its role in atherosclerosis.

Epidemiology and Mechanisms of Uremia-Related Cardiovascular Disease

“…Carbamylated low-density lipoprotein has also been shown to markedly accelerate atherosclerosis and to reduce myocardial function in mice. 80 Levels of carbamylated proteins provide a time-averaged indicator of urea concentration (analogous to the widespread use of hemoglobin A 1c to indicate glycemic control in individuals with diabetes mellitus). Berg et al 78 and Drechsler et al 81 reported that higher levels of carbamylated albumin (C-Alb) are associated with a higher risk of all-cause mortality and uremic heart failure in both incident and prevalent ESRD populations.…”

Modified Lipids and Lipoproteins in Chronic Kidney Disease: A New Class of Uremic Toxins

“…Thirdly, MPO catalyzed the addition of thiocyanate to the LDL (derived from the decomposition of urea) and leads to the formation of carbamylated-LDL (cLDL) [66,67]. These cLDL have potent pro-atherogenic effects such as the transformation of macrophages into foam cells [68] through their binding to the pro-atherogenic CD36 receptor [69] (upregulated in CKD [70]) and associated with endothelial toxicity [71,72] and platelet aggregation [73] through LOX-1 [74]. cLDL levels are raised by chronic uremia and were linked with atherosclerosis in CKD [75,76].…”

“…Protein carbamylation is associated with cardiovascular disease [186], mortality in CKD [187,188] and can also affect lipoproteins [75] and promote atherosclerotic complications [68,189] (Figure 3). …”

Are oxidised low-density lipoproteins the true inducers of inflammation in atherosclerosis?

“…In addition, the presence of AGES and glycosylated LDL in the lesion will be detected by RAGE expressed on macrophages and promote inflammation. Thus, the presence of other forms of modified LDL inside atheromas will be enough to justify a dangerous and accelerated evolution of atheromas in people with diabetes, smokers and patients with chronic kidney disease [99,127,128]. Several canonical ligands of TLRs can potentially be present in the lesions and mediate the polarisation of macrophages towards M1 functions.…”

“…In uremic patients and smokers these other forms of modified LDL seem to be much more dangerous than ox-LDL. It has been demonstrated that LDL can be simultaneously carbamylated and oxidised making them highly cytotoxic to endothelial cells [99].…”