Proteomic signatures of serum albumin-bound proteins from stroke patients with and without endovascular closure of PFO are significantly different and suggest a novel mechanism for cholesterol efflux

Abstract

Background: The anatomy of PFO suggests that it can allow thrombi and potentially harmful circulatory factors to travel directly from the venous to the arterial circulation - altering circulatory phenotype. Our previous publication using high-resolution LC-MS/MS to profile protein and peptide expression patterns in plasma showed that albumin was relatively increased in donor samples from PFO-related than other types of ischemic strokes. Since albumin binds a host of molecules and acts as a carrier for lipoproteins, small molecules and drugs, we decided to investigate the albumin-bound proteins (in a similar sample cohort) in an effort to unravel biological changes and potentially discover biomarkers related to PFO-related stroke and PFO endovascular closure.

Methods: The method used in this study combined albumin immuno-enrichment with high resolution LC-MS in order to specifically capture and quantify the albumin-bound proteins. Subsequently, we measured cholesterol and HDL in a larger, separate cohort of PFO stroke patients, pre and post closure.

Results: The results demonstrated that a number of proteins were specifically associated with albumin in samples with and without endovascular closure of the PFO, and that the protein profiles were very different. Eight proteins, typically associated with HDL were common to both sample sets and quantitatively differently abundant. Pathway analysis of the MS results suggested that enhanced cholesterol efflux and reduced lipid oxidation were associated with PFO closure. Measurement of total cholesterol and HDL in a larger cohort of PFO closure samples using a colorimetric assay was consistent with the proteomic predictions.

Conclusions: The collective data presented in this study demonstrate that analysis of albumin-bound proteins could provide a valuable tool for biomarker discovery on the effects of PFO endovascular closure. In addition, the results suggest that PFO endovascular closure can potentially have effects on HDL, cholesterol and albumin-bound ApoA-I abundance, therefore possibly providing benefits in cardioprotective functions.

Keywords: Albumin; Biomarker; Cerebrovascular disease; Discovery; Ischemic stroke; Mass spectrometry; PFO; Patent foramen ovale; Proteomics; Stroke.

Figure 1

Specificity of anti-albumin MSIA enrichment: Binding characteristics of peptide VQH[Oxid]LGA[A_42]PVT[Phosphoryl]LRAS[Phosphoryl]YLEIYNEQVR from Kinesin-like protein to MSIA micro columns activated with either anti-albumin or anti-beta lactoglobulin, antibodies.

Figure 2

Distribution of albumin-bound proteins in PFO closure vs no-closure sample sets. Specificity for albumin-binding was assumed when binding to the negative control (anti-BL Ab) was less than binding to the anti-albumin Ab (ratio <0.5). Eight proteins were present in both sample sets and their abundance ratios and ROC AUC scores are given in Table 3. All 8 proteins present in both sample sets have been previously identified in HDL (26).

Figure 3

IPA downstream analysis of differently abundant albumin-bound proteins in PFO closure vs no-closure sample sets. The IPA biological function downstream analysis is based on z-score and p-value. In our analyses, the z-score values indicated that a function was expected to be increased (positive z-score) or decreased (negative z-score) in PFO closure relative to no-closure samples.

Figure 4

HDL and Cholesterol levels measured by colorimetric assay in samples before and after stroke–related PFO closure. HDL and cholesterol were measured in a separate validation cohort (n = 104, Table 1). All patients had CLIO certified cholesterol and HDL measured pre and post PFO closure at the same time interval as the exploratory cohort. Conditional logistic regression analysis (Table 4) demonstrated that cholesterol levels were statistically decreased (p-value 0.0007) and HDL levels were statistically increased (p-value 0.0031) post PFO closure when compared to baseline levels.