Carbamylated high-density lipoprotein and atherosclerosis

Under physiological conditions, high-density lipoprotein (HDL) mediates the reverse cholesterol transport, is vasculoprotective and anti-inflammatory. However, emerging evidence indicates that HDL can be modified under certain pathophysiological conditions and lose its protective activities or even become atherogenic. The underlying mechanisms responsible for generating dysfunctional HDL and the chemical and structural changes of HDL remain largely unknown. One potential mechanism involves covalent modification of apolipoprotein A-I through cyanate, a reactive carbonyl. Cyanate is formed at sites of inflammation by the myeloperoxidase (MPO)-H 2 O2 -thiocyanate system of activated phagocytes and reacts irreversibly with amino groups of amino acids transforming lysine to ?- amino-carbamyllysine (protein carbamylation). Since MPO is a component of HDL in the human atherosclerotic intima, MPO derived cyanate may selectively target HDL in the vessel wall. Most importantly, carbamylated proteins as well as MPO have been demonstrated to predict an increased cardiovascular risk. Therefore, the central hypothesis of this project is that carbamylation of HDL compromises its anti-atherogenic properties and that this mechanism is relevant under in vivo conditions. Therefore, the following aims will be addressed in detail: Aim I. Quantification of the carbamyllysine content of HDL recovered from human atherosclerotic lesions and serum Aim II Identification of specific carbamylation site(s) on HDL associated apolipoproteins Aim III. To determine the impact of cyanate on HDLs capability to mobilize cholesterol from macrophages Aim IV. To test whether cyanate affects antioxidant and anti-inflammatory properties of HDL Aim V. Impact of cyanate on reverse cholesterol transport of HDL in vivo

Carbamylation is a post-translational modification induced by cyanate. Cyanate targets primary amine or thiol groups and forms carbamyl groups. Recent clinical studies provided evidence that protein carbamylation strongly predicts cardiovascular mortality in patients with chronic kidney disease. However, the underlying mechanisms remain unclear. The central hypothesis of the project was that cyanate and cyanate-modified lipoproteins are pro-inflammatory mediators promoting endothelial dysfunction and vascular inflammation. An important finding of the project was that high-density lipoproteins (HDL - the so called good cholesterol) isolated from human atherosclerotic tissue were markedly carbamylated. We observed that cyanate significantly impaired anti-oxidant and anti-inflammatory capabilities of HDL. We observed that already minimal carbamylation of HDL generated a pro-atherogenic particle promoting cholesterol accumulation in macrophages through a pathway involving scavenger receptor class B, type 1. We were able to identify novel mechanisms inducing protein carbamylation. We observed that leukocyte derived reactive chlorinating species efficiently promote decomposition of urea and thiocyanate thereby generating cyanate that promotes carbamylation of HDL. Importantly, we observed that cyanate significantly impaired (i) HDL`s ability to activate lecithin-cholesterol acyltransferase; (ii) the activity of paraoxonase, a major HDL-associated anti-inflammatory enzyme and (iii) the antioxidative activity of HDL. In subsequent immunohistochemical analyses performed during the project, we observed that carbamylated epitopes were mainly associated with endothelial cells in early human atherosclerotic lesions. We therefore tested whether cyanate affects endothelial functionality. Of particular interest, we observed that cyanate treatment (i) reduced expression of endothelial nitric oxide synthase and (ii) increased tissue factor and (iii) plasminogen activator inhibitor-1 expression. In mice, administration of cyanate - promoting protein carbamylation at levels observed in uremic patients - attenuated arterial vasorelaxation of aortic rings in response to acetylcholine, without affecting sodium nitroprusside-induced relaxation. Total endothelial nitric oxide synthase and nitric oxide production were significantly reduced in aortic tissue of cyanate-treated mice. This coincided with a marked increase of tissue factor and plasminogen activator inhibitor-1 protein levels in aortas of cyanate-treated mice. Collectively, our data observed during the project provided clear evidence that cyanate and cyanate-modified lipoproteins promote endothelial dysfunction and vascular inflammation.