SYNTHESIS OF PRO-INFLAMMATORY CYTOKINES BY HUMAN AORTIC VALVE, INTERNAL THORACIC ARTERY, SAPHENOUS VEIN, AND ADIPOSE TISSUE-DERIVED MICROVASCULAR ENDOTHELIAL CELLS

Highlights

• The list of cytokines produced by the primary human valvular, arterial, venous, and microvascular endothelial cells (ECs) is restricted to MIF, IL-6, IL-8/CXCL8, MCP-1/CCL2, RANTES/CCL5, MIP-3α/CCL20, GM-CSF, G-CSF, GRO-α/CXCL1, ENA-78/CXCL5, IP-10/CXCL10, and PTX3.
• These cytokines can be classified into those with a high (MCP-1/CCL2, IL-8/CXCL8, GROα/CXCL1, MIF, and pentraxin-3), moderate (IL-6), and low expression (GM-CSF, G-CSF, RANTES/CCL5, MIP-3α/CCL20, ENA-78/CXCL5, and IP-10/CXCL10).
• Human internal thoracic artery endothelial cells show the highest inflammatory activity in comparison with saphenous vein endothelial cells, adipose tissue-derived microvascular endothelial cells, and aortic valve endothelial cells.

Abstract

Aim. To define pro-inflammatory cytokines secreted by distinct primary endothelial cells (ECs) into the cell culture medium.

Methods. Primary human aortic valve endothelial cells (HAVEC) were obtained from the patients with aortic stenosis (n = 3). Primary human saphenous vein endothelial cells (HSaVEC, n = 3), internal thoracic artery endothelial cells (HITAEC, n = 3), and subcutaneous adipose tissue-derived microvascular endothelial cells (HMVEC, n = 3) were isolated from the patients with coronary artery disease. Expression of MIF, IL6, CXCL8, CCL2, CCL5, CCL20, CSF2, CSF3, CXCL1, CXCL5, CXCL10, PTX3, SERPINE1, VCAM1, ICAM1, SELE, and SELP genes was performed by reverse transcription-quantitative polymerase chain reaction. The levels of 109 pro-inflammatory cytokines in the serum-free cell culture medium were measured by the semi-quantitative dot blot profiling with the chemiluminescent detection and densitometry. Statistical analysis was carried out using Kruskal-Wallis test with the further Dunn’s multiple comparisons test.

Results. MIF, IL-6, IL-8/CXCL8, MCP-1/CCL2, RANTES/CCL5, GM-CSF, GROα/CXCL1, ENA-78/CXCL5, and PTX3 were detected in the cell culture supernatant from all EC lines, whilst G-CSF, MIP-3α/CCL20, and IP-10/CXCL10 were exclusively observed in the cell culture supernatant from HITAEC. As compared with other cell lines, HITAEC had significantly elevated expression of CCL2, CCL20, CSF2, CSF3, and CXCL10 genes and showed a trend to the increased expression of CXCL5 and SERPINE1 genes. In contrast, HSaVEC and HMVEC did not demonstrate significant increase in the expression of any of the genes encoding pro-inflammatory cytokines. CCL2, CXCL8, CXCL1, MIF, PTX3, and IL6 genes had higher expression in comparison to other cytokine-encoding genes regardless of the EC line. Count of the expression ranks per each of these pro-inflammatory cytokines showed a significant relative distance between HITAEC and other EC lines.

Conclusion. We defined a list of endothelial pro-inflammatory cytokines (MIF, IL-6, IL-8/CXCL8, MCP-1/CCL2, RANTES/CCL5, MIP-3α/CCL20, GM-CSF, G-CSF, GRO-α/CXCL1, ENA-78/CXCL5, IP-10/CXCL10, and PTX3) and suggested a higher pro-inflammatory status of HITAEC as compared with HAVEC, HSaVEC, and HMVEC.

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