Plaque Protein Carbamylation Is Associated with Atherosclerosis Progression and Uremia and Accumulates in Foam Cells

Valeria Saar-Kovrov¹Aleksandra Pawlowska¹Adrien Guillot²Marion Gijbels^1,3,4Judith Sluimer^1,5Barend Me Mees⁶Frank Tacke²Vera Jankowski⁷Joachim Jankowski⁷Marjo Donners^1*Erik A.L. Biessen^1*

• ¹Department of Pathology, School for Cardiovascular Diseases, Faculty of Health, Medicine and Life Sciences, Maastricht University, Maastricht, Netherlands
• ²Department of Hepatology and Gastroenterology, Charité University Medicine Berlin, Berlin, Baden-Württemberg, Germany
• ³GROW School for Oncology & Reproduction, Faculty of Health, Medicine and Life Sciences, Maastricht University, Maastricht, Netherlands, Netherlands
• ⁴Department of Medical Biochemistry, Academic Medical Center, Amsterdam, Netherlands
• ⁵Centre for Cardiovascular Science, University of Edinburgh, Edinburgh, Scotland, United Kingdom
• ⁶Department of Vascular Surgery, Maastricht University Medical Centre, Maastricht, Limburg, Netherlands
• ⁷Institute for Molecular Cardiovascular Research, University Hospital RWTH Aachen, Aachen, North Rhine-Westphalia, Germany

Carbamylation is a non-enzymatic post-translational protein modification common in patients with uremia that causes pro-atherogenic alterations in plasma proteins. It is abundantly present in late-stage atherosclerotic plaques; however, the pathogenic relevance and functional consequences of this accumulation are not known. Human atherosclerotic plaque tissue samples were stratified by plaques' stage and kidney function. Immunohistochemistry revealed a significantly higher carbamylated lysine (carb-lys) abundance in late-stage hemorrhaged plaques of chronic kidney disease patients compared to early-stage plaques, and a significant negative correlation to glomerular filtration rate for the advanced plaques. While we saw the difference in the total levels of carbamylation between early and advanced plaques, cellular carbamylation signal, studied in a parallel cohort of stable vs unstable plaques, did not differ between plaque stages but significantly correlated to CD68, PLIN2, and LGALS3 signals.Functional effects of carbamylated LDL (carbLDL) uptake on macrophages were studied in vitro on an in-house developed confocal-based microscale multi-assay platform to screen multiple cellular functions and demonstrated similar foam cell formation compared to the uptake of oxidized LDL (oxLDL). However, in contrast to oxLDL, carbLDL did not induce PPARγ reporter gene expression, suggesting differential capacity to induce lipogenic pathways. Moreover, unlike oxLDL, carbLDL did not induce apoptosis or ROS production.Taken together, our findings demonstrate an accumulation of carbamylated protein during plaque progression in patients with reduced kidney function. This can be, at least partially, explained by uptake of carbLDL particles by the macrophages. CarbLDL uptake, in turn, can induce foam cell formation but seems less cytotoxic than oxLDL.