Biomarker Discovery in Serum from Patients with Carotid Atherosclerosis

Abstract

Background: Blood-based biomarkers of atherosclerosis have been used to identify patients at high risk for developing stroke. We hypothesized that patients with carotid artery disease would have a distinctive proteomic signature in blood as compared to a healthy control population without carotid artery disease. In order to discover protein biomarkers associated with increased atherosclerotic risk, we used two different strategies to identify biomarkers from patients with clinically defined atherosclerosis who were undergoing endarterectomy for atherosclerotic carotid artery disease. These patients were compared with healthy matched controls. Methods: Serum was obtained from patients undergoing endarterectomy (EA; n = 38) and compared to a group of age-matched healthy controls (n = 40). Serum was fractionated using anion exchange chromatography and three different surface-enhanced laser desorption/ionization (SELDI) chip surfaces and then evaluated with mass spectrometry (MS) and two-dimensional difference gel electrophoresis (2D-DIGE). Results: A random forest (RF) analysis of the SELDI-MS protein peak data distinguished these two groups with 69.2% sensitivity and 73.2% specificity. Four unique SELDI peaks (4.2, 4.4, 16.7 and 28 kDa, all p< 0.01) showed the greatest influence in the RF model. The EA patients with a history of prior clinical atherosclerotic plaque rupture manifested as either stroke or transient ischemic attack (symptomatic; n = 16) were compared to patients with carotid atherosclerosis but no clinical evidence of plaque rupture (asymptomatic; n = 22). Analysis of the SELDI spectra did not separate these two patient subgroups. A subgroup analysis using 2D-DIGE images obtained from albumin-depleted serum comparing symptomatic (n = 10) to asymptomatic EA patients (n = 10) found 4 proteins that were differentially expressed (p < 0.01) in the symptomatic patients. These proteins were identified as α₁-antitrypsin, haptoglobin and vitamin D binding protein that were downregulated and α₂-glycoprotein precursor that was upregulated in the symptomatic EA group. Conclusions: SELDI-MS data analysis of fractionated serum suggests that a distinct protein signature exists in patients with carotid atherosclerosis compared to age-matched healthy controls. Identification of 4 proteins in a subset of patients with symptomatic and asymptomatic carotid atherosclerosis suggests that these and other protein biomarkers may assist in identifying high-risk patients with carotid atherosclerosis.

Novel Multiomics Profiling of Human Carotid Atherosclerotic Plaques and Plasma Reveals Biliverdin Reductase B as a Marker of Intraplaque Hemorrhage

“…Moreover, it has been observed that IPH in carotid lesions can predict adverse events in other vascular territories, which underlined the notion that it should be investigated as a target for noninvasive imaging and diagnostic approaches (33) . We recently demonstrated that Hb-haptoglobin (Hp) binding as well as Hb metabolism were some of the most enriched functional categories in a global transcriptomic analysis of carotid plaques and peripheral blood mononuclear cells (11) , and the importance of Hb metabolism in unstable atherosclerosis has also been supported by epidemiological findings linking a common genetic variant of Hp to cardiovascular risk 34 , 35 , 36 . Here, the notion that IPH and Hb metabolism are central elements in end-stage unstable carotid atherosclerosis was confirmed as enrichment of many IPH-related molecules was found in plaques, whereas BLVRB was the only component of this pathway significantly reflected in plasma.…”

Gene expression signatures, pathways and networks in carotid atherosclerosis

“…Furthermore, the Hb-Hp complex was one of the most enriched GO categories in plaques and Hb metabolism was enriched in PBMCs. A common genetic variant of Hp has recently been associated with cardiovascular disease risk in multiple independent cohorts, both in plaques and in peripheral blood [56][57][58], and Hp genotyping has been recommended for risk identification and treatment optimization [56].…”

Serum cyclin-dependent kinase 9 is a potential biomarker of atherosclerotic inflammation

“…In the current view, accumulating evidences support a critical role for inflammation in the pathogenesis of atherosclerosis. Several studies have demonstrated that C-reactive protein [ 4 , 5 ], B-type natriuretic peptide (NT-pro-BNP) [ 6 ], and serum cardiac troponin I (cTN-I) [ 7 ] as robust serum biomarkers for CAD risk.…”