Post-translational modification and functional impairment of proteins through carbamylation is thought to promote vascular dysfunction during end-stage renal disease. Cyanate, a reactive species in equilibrium with urea, carbamylates protein lysine residues to form epsilon-carbamyllysine (homocitrulline), altering protein structure and function. We now report the discovery of an alternative and quantitatively dominant mechanism for cyanate formation and protein carbamylation at sites of inflammation and atherosclerotic plaque: myeloperoxidase-catalyzed oxidation of thiocyanate, an anion abundant in blood whose levels are elevated in smokers. We also show that myeloperoxidase-catalyzed lipoprotein carbamylation facilitates multiple pro-atherosclerotic activities, including conversion of low-density lipoprotein into a ligand for macrophage scavenger receptor A1 recognition, cholesterol accumulation and foam-cell formation. In two separate clinical studies (combined n = 1,000 subjects), plasma levels of protein-bound homocitrulline independently predicted increased risk of coronary artery disease, future myocardial infarction, stroke and death. We propose that protein carbamylation is a mechanism linking inflammation, smoking, uremia and coronary artery disease pathogenesis.

Malondialdehyde acetaldehyde (MAA) adducts are generated under oxidative stress and shown to be highly immunogenic. Our aim was to investigate the recognition of MAA adducts by human natural antibodies in newborns before or at the time of full-term pregnancy. Plasma samples of pre-term (n = 11) and full-term (n = 36) newborns were enriched in specific IgM binding to MAA adducts compared with the maternal plasma IgM levels. Umbilical cord blood lymphocyte phage display library was generated to clone Fabs that specifically recognized MAA adducts without cross-reactivity to malondialdehyde. Fab clones from the antibody libraries of the pre-term and full-term newborns showed high sequence homology to the germline genes encoding the variable regions of antibodies, confirming that these Fabs represented the natural antibody repertoire of human fetuses. The MAA-specific umbilical cord blood Fabs bound to apoptotic human endothelial cells and the binding was efficiently competed with MAA adducts. The MAA-specific Fabs also recognized epitopes on advanced atherosclerotic lesions, and the uptake of infrared (IR)-labeled MAA-low-density lipoprotein by mouse J774A.1 macrophages was significantly reduced in the presence of these Fabs. In conclusion, MAA adducts were identified as one of the major antigenic targets for human natural antibodies already before the time of birth. MAA-specific natural antibodies are suggested to regulate apoptotic cell clearance starting from fetal development and to participate in the immunomodulation of atherosclerosis development during adulthood.

Objective Increased risk for atherosclerosis is associated with infectious diseases including periodontitis. Natural IgM antibodies recognize pathogen-associated molecular patterns on bacteria, and oxidized lipid and protein epitopes on low-density lipoprotein (LDL) and apoptotic cells. We aimed to identify epitopes on periodontal pathogen Porphyromonas gingivalis recognized by natural IgM binding to malondialdehyde (MDA) modified LDL. Methods and Results Mouse monoclonal IgM (MDmAb) specific for MDA-LDL recognized epitopes on P. gingivalis on flow cytometry and chemiluminescence immunoassays. Immunization of C57BL/6 mice with P. gingivalis induced IgM, but not IgG, immune response to MDA-LDL and apoptotic cells. Immunization of LDLR −/− mice with P. gingivalis induced IgM, but not IgG, immune response to MDA-LDL and diminished aortic lipid deposition. On Western blot MDmAb bound to P. gingivalis fragments identified as arginine-specific gingipain (Rgp) by mass spectrometry. Recombinant domains of Rgp produced in E. coli were devoid of phosphocholine epitopes but contained epitopes recognized by MDmAb and human serum IgM. Serum IgM levels to P. gingivalis were associated with anti-MDA-LDL levels in humans. Conclusion Gingipain of P. gingivalis is recognized by natural IgM and shares molecular identity with epitopes on MDA-LDL. These findings suggest a role for natural antibodies in the pathogenesis of two related inflammatory diseases, atherosclerosis and periodontitis.

Low 25-hydroxyvitamin D levels correlate with the prevalence of diabetes; however, the mechanisms remain uncertain. Here, we show that nutritional deprivationresponsive mechanisms regulate vitamin D metabolism. Both fasting and diabetes suppressed hepatic cytochrome P450 (CYP) 2R1, the main vitamin D 25-hydroxylase responsible for the first bioactivation step. Overexpression of coactivator peroxisome proliferator-activated receptor g coactivator 1-a (PGC-1a), induced physiologically by fasting and pathologically in diabetes, resulted in dramatic downregulation of CYP2R1 in mouse hepatocytes in an estrogen-related receptor a (ERRa)-dependent manner. However, PGC-1a knockout did not prevent fasting-induced suppression of CYP2R1 in the liver, indicating that additional factors contribute to the CYP2R1 repression. Furthermore, glucocorticoid receptor (GR) activation repressed the liver CYP2R1, suggesting GR involvement in the regulation of CYP2R1. GR antagonist mifepristone partially prevented CYP2R1 repression during fasting, suggesting that glucocorticoids and GR contribute to the CYP2R1 repression during fasting. Moreover, fasting upregulated the vitamin D catabolizing CYP24A1 in the kidney through the PGC-1a-ERRa pathway. Our study uncovers a molecular mechanism for vitamin D deficiency in diabetes and reveals a novel negative feedback mechanism that controls crosstalk between energy homeostasis and the vitamin D pathway.

Lipid accumulation is a key characteristic of advancing atherosclerotic lesions. Herein, we analyzed the ultrastructure of the accumulated lipids in endarterectomized human carotid atherosclerotic plaques using three-dimensional (3D) electron microscopy, a method never used in this context before. 3D electron microscopy revealed intracellular lipid droplets and extracellular lipoprotein particles. Most of the particles were aggregated, and some connected to needle-shaped or sheet-like cholesterol crystals. Proteomic analysis of isolated extracellular lipoprotein particles revealed that apolipoprotein B is their main protein component, indicating their origin from low-density lipoprotein, intermediate-density lipoprotein, very-low-density lipoprotein, lipoprotein (a), or chylomicron remnants. The particles also contained small exchangeable apolipoproteins, complement components, and immunoglobulins. Lipidomic analysis revealed differences between plasma lipoproteins and the particles, thereby indicating involvement of lipolytic enzymes in their generation. Incubation of human monocyte-derived macrophages with the isolated extracellular lipoprotein particles or with plasma lipoproteins that had been lipolytically modified in vitro induced intracellular lipid accumulation and triggered inflammasome activation in them. Taken together, extracellular lipids accumulate in human carotid plaques as distinct 3D structures that include aggregated and fused lipoprotein particles and cholesterol crystals. The particles originate from plasma lipoproteins, show signs of lipolytic modifications, and associate with cholesterol crystals. By inducing intracellular cholesterol accumulation (ie, foam cell formation) and inflammasome activation, the extracellular lipoprotein particles may actively enhance atherogenesis.

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These data give insight into mechanisms of in vivo humoral recognition of post-translationally modified structures. Humoral IgG immune response to carbamylated proteins is suggested to play a role in conditions leading to enhanced carbamylation, such as uremia and smoking.

Background and Purpose: Many drugs and environmental contaminants induce hypercholesterolemia and promote the risk of atherosclerotic cardiovascular disease.We tested the hypothesis that pregnane X receptor (PXR), a xenobiotic-sensing nuclear receptor, regulates the level of circulating atherogenic lipids in humans and utilized mouse experiments to identify the mechanisms involved.Experimental Approach: We performed serum NMR metabolomics in healthy volunteers administered rifampicin, a prototypical human PXR ligand or placebo in a crossover setting. We used high-fat diet fed wild-type and PXR knockout mice to investigate the mechanisms mediating the PXR-induced alterations in cholesterol homeostasis.