Secretome analysis for identification of proteins as potential markers for extracellular remodelling during pathogenesis in human atherosclerotic arteries

T Tejerina1, F De la Cuesta2, MG Barderas3, M Carnero4, J Navarro-Dorado1, M Ramajo1, G Alvarez-Llamas5, F Vivanco2,5. 1Universidad Complutense de Madrid, Facultad de Medicina, Pharmacology 28040, Spain, 2IIS-Fundacion Jimenez Diaz, Immunology 28040, Spain, 3Hospital Nacional de Paraplejicos, SESCAM, Toledo, Vascular Physiopathology 45071, Spain, 4Hospital Clínico San Carlos, Surgery 28040, Spain, 5Universidad Complutense, Biochemistry and Molecular Biology I 28040, Spain

Aims:Cardiovascular diseases are of increasing prevalence, and consequently, early detection and knowledge of the underlying mechanisms are essential to help limit and improve prognosis. Approaching directly the tissue will reveal key molecules implicated in the process. Secretome studies resemble more closely to the in vivo situation, showing a much narrower protein concentrations dynamic range than plasma. This study was aimed to the analysis of human arterial tissue (HAT) secretome as well as the quantitative comparison of healthy and atherosclerotic secretome to discover proteins with key roles in atherosclerosis development, which could be used as biomarkers of early diagnosis, disease progression or response to pharmacological intervention.

Methods and Results: Following a comprehensive optimization of experimental culture conditions, secretomes from three biological replicates of human atherosclerotic coronary arteries (APC), preatherosclerotic coronaries (PC) and mammaries (M) were analyzed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) in a Orbitrap instrument. A specific picture of proteins secreted by coronaries affected by atheromatous lesion, preatherosclerotic coronaries and mammaries is provided. Secretome collection from tissue in culture for 24h and was optimized and minimization of plasma proteins was checked by 2-DE. The identified proteins were submitted to Ingenuity Pathway Analysis (IPA) tool. Label-free MS/MS based quantification was performed and validated by immunohistochemistry. 64 proteins were identified in the 3 replicates of at least one of the 3 groups (M, PC, and AC), of which 14 secreted proteins have not been previously reported in plasma. By IPA, 15 molecules have been related to “cardiovascular system development and function” and the top network associated was “cellular movement, cell death, cellular growth and proliferation”. Four proteins were significantly released in higher amounts by mammary tissue: gelsolin, vinculin, lamin A/C and phosphoglucomutase 5. Results are coincident in both cases for vinculin and lamin A/C, in terms of groups between which they resulted to be significantly different and trends (increased or decreased). Gelsolin was found to be more abundant in the secretome from mammary than from preatherosclerotic coronary arteries according to both approaches and also than from coronaries with atherome plaque, although not significantly. Phosphoglucomutase 5 abundance was higher in mammary than in preatherosclerotic coronaries and in atherosclerotic coronaries, although with different signification level depending on the software tool.

Conclusion: We highlight the relevance of tissue secretome as reflected by the identification of proteins secreted by HAT which were not reported in plasma. The proteins described in this study, involved in the process of inflammation, cellular adhesion and arterial aging process had been identified as significantly regulated and could be potential therapeutic targets in clinical practice.

Grants:RECAVA RD06/0014/1007 (Spanish Ministry of Health).

RECAVA RD06/0014/1015 (Spanish Ministry of Health).

RD07/0064/0023 (Instituto de Salud Carlos III)