Carbamylated LDL and endothelial dysfunction

In renal disease, elevated levels of urea lead to an increased concentration of cyanate (OCN ) that reacts irreversible with amino groups of proteins through carbamylation reactions. High concentrations of carbamylated low-density-lipoproteins (cLDL) have recently been reported to accumulate in plasma of patients with chronic renal failure, indicating that cLDL is by far the most abundant modified LDL found in human plasma. Of importance, carbamylated proteins may also be formed directly at sites of inflammation - even in the absence of renal disease - via the phagocyte derived inflammatory mediator myeloperoxidase (MPO). Active MPO is present in human atherosclerotic lesion material and has been shown to bind to low- and high-density lipoproteins. Thus, it can be assumed that MPO derived cyanate contributes to lipoprotein carbamylation at sites of inflammation. Recent clinical studies demonstrated that carbamylated proteins predict increased risk of coronary artery disease, future myocardial infarction, stroke and death. Both LDL isolated from uremic patients and LDL carbamylated in vitro have been reported to cause multiple pro- atherosclerotic effects, including diminished LDL receptor recognition, smooth muscle cell proliferation and endothelial cell activation and apoptosis. Therefore, a potential role in the evolution of vascular disease has been proposed for carbamylated LDL. One of the earliest changes in the development of atherosclerosis is an endothelial dysfunction, which is - at least in part - triggered by receptor dependent activities leading to initiation and/or perpetuation of the inflammatory response. However, no endothelial receptors or binding proteins that might trigger the potent pro-atherosclerotic effects of carbamylated LDL have yet been identified. Therefore, the aim of the proposed project is to identify endothelial receptors for carbamylated LDL and to systematically examine the impact of carbamylated LDL on the expression of inflammatory genes in human derived vascular endothelial cells.

The dramatic cardiovascular morbidity and mortality of chronic kidney disease patients is attributable in a significant proportion to endothelial dysfunction. While urea itself is innoxious, many molecules can be carbamylated through cyanate, a reactive decomposition product of urea that is formed in excess in chronic kidney disease. Carbamylation is a post- translational modification in which cyanate binds to molecules containing primary amine or thiol groups and forms carbamyl groups. Recent clinical studies provided evidence that protein carbamylation strongly predicts increased cardiovascular risk in patients with kidney disease. However, the underlying mechanisms remain unclear. The central hypothesis of the project was that cyanate and cyanate-modified lipoproteins are pro-atherogenic triggers promoting endothelial dysfunction and vascular inflammation. In this project, we observed that cyanate promotes intercellular cell adhesion molecule-1 (ICAM-1) expression in endothelial cells resulting in increased leukocyte binding to endothelial cells, a crucial step in atherogenesis. In mice, oral administration of cyanate induced marked ICAM-1 expression in the aortic arch. Most importantly, in patients with chronic kidney disease, the extent of plasma protein carbamylation (a marker for cyanate exposure) significantly correlated with plasma levels of soluble ICAM-1, suggesting that cyanate triggers vascular inflammation in these patients. Since ICAM-1 is known for its importance in mediating cellcell interactions and facilitating leukocyte endothelial transmigration, our results provide further insights to explain a part of the underlying mechanisms that contribute to the enhanced cardiovascular risk associated with chronic renal failure. A very interesting and important finding of the project was that carbamyllysine levels are markedly elevated in high-density lipoproteins (HDL - the so called good cholesterol) in the inflamed atherosclerotic vessel wall. We observed that cyanate induced carbamylation significantly impaired anti-oxidant, anti-inflammatory capabilities of HDL. Most importantly, one carbamyllysine residue per HDL-associated major apoprotein apoA-I was sufficient to induce cholesterol accumulation in cells through a pathway involving scavenger receptor class B, type 1 (SR-BI). Overall, our observations suggest that cyanate promotes human atherogenesis by activating endothelial cells and rendering good cholesterol dysfunctional and pro-atherogenic.