Secreted proteins from carotid endarterectomy: an untargeted approach to disclose molecular clues of plaque progression

Abstract

Background: Atherosclerosis is the main cause of morbidity and mortality in Western countries and carotid plaque rupture is associated to acute events and responsible of 15-20% of all ischemic strokes. Several proteomics approaches have been up to now used to elucidate the molecular mechanisms involved in plaque formation as well as to identify markers of pathology severity for early diagnosis or target of therapy. The aim of this study was to characterize the plaque secretome. The advantage of this approach is that secretome mimics the in vivo condition and implies a reduced complexity compared to the whole tissue proteomics allowing the detection of under-represented potential biomarkers.

Methods: Secretomes from carotid endarterectomy specimens of 14 patients were analyzed by a liquid chromatography approach coupled with label free mass spectrometry. Differential expression of proteins released from plaques and from their downstream distal side segments were evaluated in each specimen. Results were validated by Western blot analysis and ELISA assays. Histology and immunohistochemistry were performed to characterize plaques and to localise the molecular factors highlighted by proteomics.

Results: A total of 463 proteins were identified and 31 proteins resulted differentially secreted from plaques and corresponding downstream segments. A clear-cut distinction in the distribution of cellular- and extracellular-derived proteins, evidently related to the higher cellularity of distal side segments, was observed along the longitudinal axis of carotid endarterectomy samples. The expressions of thrombospondin-1, vitamin D binding protein, and vinculin, as examples of extracellular and intracellular proteins, were immunohistologically compared between adjacent segments and validated by antibody assays. ELISA assays of plasma samples from 34 patients and 10 healthy volunteers confirmed a significantly higher concentration of thrombospondin-1 and vitamin D binding protein in atherosclerotic subjects.

Conclusions: Taking advantage of the optimized workflow, a detailed protein profile related to carotid plaque secretome has been produced which may assist and improve biomarker discovery of molecular factors in blood. Distinctive signatures of proteins secreted by adjacent segments of carotid plaques were evidenced and they may help discriminating markers of plaque complication from those of plaque growth.

Figure 1

Longitudinal section of an undivided CEA specimen at artery center line level showing the cutting line for P and DS segments separation. Prevalent complicated plaque features of P (lipid-necrotic core, calcium deposits, fibrosis and haemorrage) and milder changes of its downstream side DS (prevalent VSMCs and collagen component, small lipid and calcium deposits) are evidenced by Masson’s trichrome and α-SMA immunostaining (from top to bottom). Original magnification 2×, insets 10×.

Figure 2

Left: P and DS sections stained with (a) Masson’s trichrome stain, (b) α-SMA and (c) CD68 immunostain (original magnification 2×, insets 10×). P is a type VI plaque with a necrotic core, calcifications, intraplaque thrombus and a thin fibrous cap composed of collagen and αSM-actin positive cells; minor intraplaque CD68 positive staining is present and α-SMA -actin positive contractile VSMCs are also visible on the outer border of the specimen (opposite to the lumen) due to surgical cleavage of plaque from the intact tunica media. In DS, a much thinner lesion (equivalent to Stary type III) with extensive α -SMA and CD68 cellular positivity is present. Right: From top to bottom. Average values and SD of lesional area (mm²), intralesional α-SMA and CD68 stain positivity (% of lesional area) of P and DS segments of all specimens’ sections: a smaller lesional area and a markedly higher positivity of both cellular markers in the distal side of the plaque is evident (*p<0.05, **p<0.02 paired t-test).

Figure 3

Pie charts of the total identified proteins from P and DS. A) Identified proteins are divided on the basis of their localization in intracellular or extracellular space based on Gene Ontology. B) Identified proteins are evaluated with SecretomeP software to compute their secretion potential. They were divided in 1) classically secreted 2) not-classically secreted and 3) not-predicted.

Figure 4

Differential protein expression obtained by mass spectrometric analysis of P and DS. Four of the 31 proteins reported in table 3 are chosen and comparative expression analysis is presented as Box plots. Box plots show median value, standard deviation (SD), 50%, minimum and maximum intensity values and were exported from Marker View software.

Figure 5

Left: Western blot analysis of P and DS secretomes. Densitometric quantification of vinculin and thrombospondin-1 using Quantity One 1D analysis software. Representative images: 10 μg of proteins were separated on SDS-PAGE and blotted on nitrocellulose membrane. Immunoblots were probed with antibodies against vinculin and thrombospondin-1. Right: ELISA assay for vitamin D binding protein expression in secretome samples (P and DS).

Figure 6

Top: Double immunostaining of thrombospondin-1 (red) and vinculin (brown) in P (A) and DS (B) sections of a fibrocalcific type Vb plaque. Low magnification (2×, left) and high magnification (10× to 40×) microscopic fields (insets) are shown. Neither cellular nor extracellular co-distribution of the two antibodies is present. A selective cellular binding of vinculin both on P an DS sections is evident, while thrombospondin-1 is almost exclusively located in the fibrocalcific core of P. Bottom: Average values and SD of thrombospondin-1 and vinculin positivity (single immunostaining) in lesional area of all CEA specimens showing a markedly greater stain in P as compared toDS for thrombospondin-1 and an opposite pattern for vinculin. *p<0.02 paired t-test.

Figure 7

ELISA assay for thrombospondin-1 and vitamin D binding protein expression in plasma samples of 34 CEA patients classified as symptomatic and asymptomatic and 10 healthy controls.

Figure 8

Ingenuity Pathway Analysis (IPA) of a subset of 56 proteins identified in the secretome of CEA specimens. Pathways were generated based on the information stored in IPA Knowledge base. Nodes correspond to the 56 proteins and are reported with their Gene Codes. Colors correspond to differentially released proteins between P and DS reported in Table 3. Red: up-released by P. Green: down-released by P. Extracellular space, cytoplasm and plasma membrane proteins are shown based on IPA classification.