SECRETOME OF PRIMARY HUMAN CORONARY ARTERY AND INTRNAL THORACIC ARTERY ENDOTHELIAL CELLS AT PHYSIOLOGICAL STATE AND AT ENDOTHELIAL DYSFUNCTION

Highlights

• Dysfunctional endothelial cells are characterized by the pathological shedding of endothelial receptors markers and reduced release of endothelial basement membrane components and subendothelial extracellular matrix proteins into the cell culture medium.
• Primary human coronary artery endothelial cells demonstrate an elevated shedding of integrins, an increased production of platelet aggregation and activation components, and reduced release of basement membrane components into the cell culture medium as compared to human internal thoracic artery endothelial cells.
• Primary human internal thoracic artery endothelial cells have higher resistance to endothelial dysfunction triggers in comparison with human coronary artery endothelial cells.

Abstract

Aim. To compare the secretome from intact and dysfunctional primary human coronary artery endothelial cells (HCAEC) and human internal thoracic artery endothelial cells (HITAEC).

Methods. Here we investigated the secretome of HCAEC and HITAEC treated with either PBS (intact ECs, n = 6 per EC line) or calciprotein particles (dysfunctional ECs, n = 6 per EC line) in the serum-free medium for 24 hours. To achieve this task, we performed acetone precipitation of proteins from the cell culture supernatant, tryptic digestion of proteins to peptides (15 µg per sample), protein identification by high-performance liquid chromatography-tandem mass-spectrometry, and bioinformatics analysis of endothelial phenotype markers, endothelial basement membrane components and subendothelial extracellular matrix proteins, angiogenesis pathways, platelet activation and aggregation pathways, and response to oxidative or endoplasmic reticulum stress. Concentration of pro-inflammatory cytokines in the cell culture supernatant was measured by multi-analyte profiling (xMAP).

Results. Cell culture supernatant from dysfunctional ECs had increased levels of endothelial cell receptor soluble forms and reduced quantities of endothelial basement membrane components and subendothelial extracellular matrix proteins. Cell culture supernatant from HCAEC contained increased levels of integrins (similar to dysfunctional ECs) and platelet activation and aggregation proteins, as well as reduced amount of basement membrane components. Treatment with primary calciprotein particles provoked the release of 12 and 5 pro-inflammatory cytokines, respectively. Treatment with secondary calciprotein particles induced production of 30 and 10 pro-inflammatory cytokines in HCAEC and HITAEC, respectively.

Conclusion. Secretome of HCAEC is more similar to dysfunctional ECs whilst HITAEC have higher resistance to pro-inflammatory endothelial activation.

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