Proteome Profiler Output.xlsx (25.52 kB)
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posted on 2022-05-31, 08:16 authored by Daniela Pedicino, Ramona VinciRamona Vinci

Human epicardial adipose tissue, a dynamic source of multiple bioactive factors, holds a close functional and anatomic relationship with the epicardial coronary arteries and communicates with the coronary artery wall through paracrine and vasocrine secretions. We explored the hypothesis that in patients with Non-ST segment Elevation Myocardial Infarction (NSTEMI) T-cell recruitment into epicardial adipose tissue (EAT) could be part of a specific antigen-driven response implicated in acute coronary syndrome onset and progression.

We enrolled 32 NSTEMI patients and 34 chronic coronary syndrome (CCS) patients undergoing coronary artery bypass grafting (CABG), and 12 mitral valve disease (MVD) patients undergoing surgery.


We performed EAT proteome profiling on pooled specimensfrom 3 NSTEMI and 3 CCS patients. In details, EAT biopsies from CCS (n = 3) and NSTEMI (n = 3) were collected before the surgery in all cases and immediately frozen at -80°C. In CCS EAT was collected near the proximal right coronary artery; in NSTEMI patients, EAT was collected near the culprit coronary artery, i.e. left coronary artery (LCA). 

EATs were homogenized in cold radioimmunoprecipitation (RIPA) buffer (1mg/100mL) supplemented with a protease and phosphates inhibitor cocktail (1:100) (HaltTM Protease and Phosphatase Inhibitor Cocktail, Thermo Fisher Scientific, USA) and by mechanical disruption (TissueLyserII, Qiagen, DE). Homogenized samples were centrifuged at 14000x g/10’/4°C and EAT protein concentration were quantified by Bradford method.

EAT samples were pooled for pro-inflammatory proteome profiling evaluation. A total of 200 micrograms per membrane of pooled EAT (CCS vs NSTEMI) were incubated. The proteome profile assay was performed according to manual user (ARY022B, Proteome Profiler Array, R&D, USA). After X-ray acquisition (ChemiDoc™, Biorad, USA), obtained spots were analyzed using Image Lab software (Biorad, USA).

We performed T-cell receptor (TCR) spectratyping and CDR3 sequencing in EAT and in peripheral blood mononuclear cells of 29 NSTEMI, 31 CCS and 12 MVD patients. We then used computational modeling studies to predict interactions of the TCR beta chain variable region (TRBV) and explore sequence alignments. The EAT proteome profiling displayed a higher content of pro-inflammatory molecules (CD31, CHI3L1, CRP, EMPRINN, ENG, IL-17, IL-33, MMP-9, MPO, NGAL, RBP-4, RETN, VDB) in NSTEMI as compared to CCS (p<0.0001). CDR3-beta spectratyping showed a TRBV21 enrichment in EAT of NSTEMI (12/29 patients;41%) as compared with CCS (1/31 patients; 3%) and MVD (none) (ANOVA for trend P<0.001). Of note, 11/12 (92%) NSTEMI patients with TRBV21 perturbation were at their first manifestation of ACS. Four patients with the first event shared a distinctive TRBV21-CDR3-sequence of 178b length and 2/4 were carriers of the human leukocyte antigen (HLA)-A*03:01 allele. A 3D analysis predicted the most likely epitope able to bind HLA-A3*01 and to interact with the TRBV21-CDR3-sequence of 178b length, while the alignment results were consistent with microbial DNA sequences.

Our study revealed a unique immune signature of the epicardial adipose tissue, which led to a 3D modeling of the TCRBV/peptide/HLA-A3 complex, in acute coronary syndrome patients at their first event, paving the way for epitope-driven therapeutic strategies. 


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