UvA-DARE ( Digital Academic Repository ) High-density lipoprotein proteome dynamics in human endotoxemia Levels

Background: A large variety of proteins involved in inflammation, coagulation, lipid-oxidation and lipid metabolism have been associated with high-density lipoprotein (HDL) and it is anticipated that changes in the HDL proteome have implications for the multiple functions of HDL. Here, SELDI-TOF mass spectrometry (MS) was used to study the dynamic changes of HDL protein composition in a human experimental low-dose endotoxemia model. Ten healthy men with low HDL cholesterol (0.7+/-0.1 mmol/L) and 10 men with high HDL cholesterol levels (1.9+/-0.4 mmol/L) were challenged with endotoxin (LPS) intravenously (1 ng/kg bodyweight). We previously showed that subjects with low HDL cholesterol are more susceptible to an inflammatory challenge. The current study tested the hypothesis that this discrepancy may be related to differences in the HDL proteome. Results: Plasma drawn at 7 time-points over a 24 hour time period after LPS challenge was used for direct capture of HDL using antibodies against apolipoprotein A-I followed by subsequent SELDI-TOF MS profiling. Upon LPS administration, profound changes in 21 markers (adjusted p-value < 0.05) were observed in the proteome in both study groups. These changes were observed 1 hour after LPS infusion and sustained up to 24 hours, but unexpectedly were not different between the 2 study groups. Hierarchical clustering of the protein spectra at all time points of all individuals revealed 3 distinct clusters, which were largely independent of baseline HDL cholesterol levels but correlated with paraoxonase 1 activity. The acute phase protein serum amyloid A-1/2 (SAA-1/2) was clearly upregulated after LPS infusion in both groups and comprised both native and N-terminal truncated variants that were identified by twodimensional gel electrophoresis and mass spectrometry. Individuals of one of the clusters were distinguished by a lower SAA-1/2 response after LPS challenge and a delayed time-response of the truncated variants. Conclusions: This study shows that the semi-quantitative differences in the HDL proteome as assessed by SELDITOF MS cannot explain why subjects with low HDL cholesterol are more susceptible to a challenge with LPS than those with high HDL cholesterol. Instead the results indicate that hierarchical clustering could be useful to predict HDL functionality in acute phase responses towards LPS. Introduction High-density Lipoprotein (HDL) cholesterol levels are inversely associated with a risk for cardiovascular disease [1]. LDL cholesterol lowering drugs have been proven to be successful, however up to 65% of cardiovascular death cannot be prevented which explains the strong interest in HDL increasing strategies [2]. Intensive research over the past decade has elucidated the involvement of HDL in multiple pathways in atherogenesis. Besides hyperand hypolipidemia, oxidation of lipoproteins and coagulation and fibrinolysis [3], Iinflammation is suggested to be one of the fields in which HDL and HDL-associated proteins [4] affect the course of acute infection. As a result, the consequential systemic inflammatory response may have a causal impact on atherosclerosis [5]. Proteomic biomarker search has been recently applied to HDL to achieve more insight into the functions and protein composition of this lipoprotein class [6-8]. Assessment of 1-dimensional electrophoresis (DE) and 2-DEmatrix-assisted laser desorption ionisation-time of flight, Isotope-coded Affinity Tag and Western-blot analysis has * Correspondence: h.levels@amc.uva. nl Department of Experimental Vascular Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands Full list of author information is available at the end of the article Levels et al. Proteome Science 2011, 9:34 http://www.proteomesci. com/content/9/1/34 © 2011 Levels et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. led to the identification of 56 HDL-associated proteins [9,10]. Importantly, Vaisar et. al. identified 42 HDL-associated proteins that differed substantially in amount between HDL from CAD patients and controls [11]. Serial sample analysis with the above mentioned approaches are labor intensive which prompted us establish a highthroughput HDL profiling technique [9]. Moreover, serial profiling of the HDL proteome might be a welcome addition to reveal information about the dynamic processes that occur compared to single “snapshot” approaches. The current study focuses on the HDL protein dynamics by means of SELDI-TOF MS after an endotoxin intervention in healthy volunteers.