Dis Markers. 2015;2015:465242.
Site-specific secretome map evidences VSMC-related markers of coronary atherosclerosis grade and extent in the hypercholesterolemic swine.
Silvia Rocchiccioli § 1, Antonella Cecchettini§ 1, 2, Nadia Ucciferri1, Marianna Terreni1, Federica Viglione1, Maria Giovanna Trivella1, Lorenzo Citti1, Oberdan Parodi1*, Gualtiero Pelosi1
1 National Research Council, Institute of Clinical Physiology, Pisa, Italy
2 University of Pisa, Department of Clinical and Experimental Medicine, Pisa, Italy
§these two authors contribute equally to this work
*corresponding author: Oberdan Parodi, MD, Institute of Clinical Physiology, Via Moruzzi 1, 56124, Pisa, Italy, tel: +39 050 3152721, fax: +39 050 3153327 email: email@example.com
A drawback in coronary atherosclerosis (ATS) research is the difficulty of investigating the early phase of plaque growth in the clinical context and associate it to specific features and factors. In this study secreted proteins from atherosclerotic coronary arteries in a hypercholesterolemic swine model were characterized by a proteomics approach and their expression was related to site-specific ATS stage and extent. A wide coronary artery map of secreted proteins has been obtained in high fat diet (HF) induced ATS and significantly different expression of many vascular smooth muscle cell (VSMC) activation/migration related proteins evidenced. A significant association with ATS stage of HF coronary lesions and secreted protein expression was found for some VSMC-derived proteins and this association was validated for Chitinase 3 like protein 1 (CHI3L1) by tissue immunoexpression and a direct correlation (R2 = 0.85) evidenced with intima to media thickness ratio values. ELISA assay of CHI3L1 confirmed higher blood concentrations in HF cases.
These findings confirm the pivotal role of VSMC in coronary plaque development and demonstrate a strong site-specific relation between VSMC-secreted CHI3L1 and lesion grade, suggesting this biomarker as a useful tool for diagnosing and staging of atherosclerotic lesions in coronary artery disease.
Keywords: atherogenesis, secretome profiling, high fat diet swine model, VSMC related proteins
Experimental design and results
Coronary atherosclerosis (ATS) is the underlying pathology of major acute events associated to thrombotic obstruction of vessel lumen and a leading cause of morbidity and mortality in western countries [1,2].
The identification of molecular factors related to disease and associated to ATS grade, extent and lesion progression could have relevant implications for diagnosis, prevention and treatment of overt coronary artery disease (CAD). With our high performance, gel- and label-free LC-MS/MS workflow  we can produce a complete secretome profile, which is constituted of all proteins actively secreted from cells and/or passively released from a tissue.
In the present study, the overall workflow, as depicted in Figure 1, has been exploited for the study of the secretome of swine coronary arterial segments in order to characterize the early phases of plaque formation and growth, to identify molecular markers associated with ATS severity and to evaluate their translation into the clinical context.
Aims of the study were:
- Identifying a molecular map of proteins secreted by intact and atherosclerotic coronary arteries of pigs fed with standard (CTRL) or hypercholesterolemic (HF) diet, based on a hypothesis-free approach able to reconstruct a complete protein profile.
- Comparing protein expression of CTRL and HF samples to evidence differentially secreted proteins correlated with ATS onset and progression.
- Associating the most relevant differentially expressed proteins with histomorphometrically characterized lesions and assessing their cellular localization by immunohistochemistry.
The protocol has been performed in 12 male domestic pigs of 8 to 12 weeks of age. Femoral artery (FA) and the proximal tract of right coronary artery (RCA) were isolated and 25-30 mm long segments excised and quickly placed in serum free medium for secretome. HPLC-MS/MS was performed on collected proteins and subsequently tissues were processed for histology (plaque morphometry) and immunohistochemistry of anti-αSM-actin (alpha smooth muscle actin, clone 1A4 ADB, Serotec) as a VSMC phenotype marker, anti-S100 A4 (rabbit polyclonal antibody diluted 1:200, Novus Biologicals) as a marker of synthetic VSMC phenotype , and anti-CHI3L1 (goat polyclonal antibody diluted 1:40, R&D Systems) as a marker associated with secretome data.
Figure 1. Study workflow: coronary sampling, secretome processing, LC-MSMS identification of secreted proteins and (immuno)histology of right coronary artery (RCA) segments from pigs on standard diet (CTRL) and on high fat hypercholesterolemic diet (HF)
In RCA specimens used for secretome and in adjacent RCA samples of HF group, atherosclerotic changes of the intima were evidenced, ranging from lesions classifiable as Stary type I-III in 3 cases (pre-atheroma changes, pre-ATH) to those resembling type IV-V human atheromas in the other 3 cases (atheroma stage, ATH) . Minimal or no intimal changes were observed in HF FA samples.
LC-MSMS analysis of the whole secretome allowed to identify 224 proteins.
T-test evidenced 20 proteins differentially secreted when all HF RCA samples were compared to CTRL ones. HF FA samples were used to distinguish between diet-related tissue-secreted and ATS-related tissue-secreted proteins: T-test comparison between HF FA and CTRL groups showed significant diet-dpendent differential expression in glyceraldehyde 3-phosphate dehydrogenase, desmin, prelamin A/C, glutathione peroxidase 1 and apolipoproteins A I and A IV.
The comparison between RCA segments of CTRL and HF groups evidences that the most represented differentially expressed proteins are VSMC intracellular proteins and ECM factors related to VSMC activation and synthesis: when comparing pre-ATH (N=3) and ATH (N=3) segments of HF RCA samples, a statistical significant (p value < 0.05) difference was observed for CHI3L1 and for Vinculin.
Figure 2. Top panel: secretome expression levels of CH3L1, S100A6 and moesin, showing significant higher values of these proteins in pre-ATH (mild) and ATH (severe) RCA segments of HF cases as compared to CTRL RCA and HF FA segments. Mid and bottom panels: correlations between mass spectrometric expression of CH3L1, S100A6 and moesin and average IT of RCA samples from HF cases.
The most represented pathways in secretome were cell proliferation, migration and VSMC activation and many proteins resulted involved in atherogenesis and vascular diseases. Among these proteins CHI3L1, S100A6 and Moesin increase their secretome expression according to ATS severity and for all three a significant individual correlation between protein expression level and mean coronary IT value of RCA atherosclerotic lesions in HF cases is observed (Figure2). Moreover, tissue immunohistochemistry evidences that CH3L1 positive areas and cells coincide with aSMA-actin and S100A4 positivity demonstrating tissue and cell co-distribution respectively, a finding that supports the VSMC origin of this protein (Figure 3).
Figure 3: Tissue co-distribution of anti-CH3L1, anti-aSMA-actin and anti-S100A4 antibodies at low power (2x magnification, top panels) and cell co-distribution at higher magnification (insets, 20x) in two microscopic fields (mid and bottom panels) of three adjacent sections from the RCA of a ATH HF case.
The most relevant up-regulated protein in atherosclerotic RCA samples of high fat diet treated animals resulted CHI3L1, which was differentially expressed in CTRL, pre-ATH and ATH samples, was strongly associated with plaque size/extent and Stary’s stage, and was immunohistologically related to a prevalent VSMC plaque composition. The immunohistochemical characterization enabled to establish tissue and cell colocalization of CHI3L1 immunoexpression with αSM-actin /S100A4 positive migratory VSMC phenotype.
ELISA assay of CHI3L1 was performed on plasma samples of CTRL and HF animals and its higher circulatory expression in HF cases consolidates the association between this marker and the presence of coronary ATS.
Altogether, these data confirm that VSMC activation towards a migratory/synthetic phenotype may play a pivotal role in atheroma formation and suggest that VSMC derived/secreted molecules can be usefully exploited in CAD as biomarkers of plaque stage and size.
- VSMC-related proteins involved in cell activation, migration and proliferation are identified in a swine model of CAD and are differentially expressed as compared to controls.
- CHI3L1 protein is the most strongly associated with coronary ATS grade and extent.
- Immunohistochemistry suggests a prevalent CHI3L1 localization in S100 A4 positive VSMCs and supports the stage-specific distribution of this marker in the full spectrum of coronary lesions, from initial fatty streaks to raised fibroatheromas.
- ELISA assay of CHI3L1 confirms a statistical significant association between its circulatory level and the presence of CAD.
- A general reorganization of VSMC-based pathways related to CAD severity is suggested, supporting the role of activated VSMCs in the evolution of coronary lesions: this would help identifying circulating markers of early plaque development and/or of fast growth, and contribute to risk assessment in CAD patients.
This work was supported by ARTreat FP7 project [Grant Agreement FP7 224297].
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