BIOINFORMATIC, MULTI-OMICS ANALYSIS OF INTACT ARTERIAL ENDOTHELIAL CELLS

Highlights

• The most expressed endothelial phenotype markers are vWF, EPCR/CD201, MCAM/CD146, ICAM2/CD102, VE-cadherin/CDH5/CD144, and PECAM1/CD31; two-thirds of endothelial phenotype markers (30/45) are released into the extracellular space even without any stimulation.
• Intact endothelial cells exhibit low basal pro-inflammatory activity, yet having a high potential for pathological activation in response to appropriate stimuli due to the broad expression of inducible cytokine genes and because of rapid secretion of these molecules into the microenvironment.
• The high proportion of secreted proteins composing endothelial basement membrane and subendothelial extracellular matrix relative to the total amount synthesized confirms the pivotal role of endothelial cells in the formation of these histological structures.

Aim. To perform an unbiased bioinformatic analysis of the molecular phenotype of intact endothelial cells derived from the atherosusceptible coronary artery (HCAEC) and the atheroresistant internal mammary artery (HITAEC).

Methods. Lysates of HCAEC and HITAEC were analyzed by whole transcriptome sequencing (RNA-seq) and ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Conditioned medium from HCAEC and HITAEC was also analyzed by UHPLC-MS/MS. Mean transcripts per million was used as the metric of gene expression, whilst protein expression was quantified based on chromatographic peak areas. Bioinformatic analysis of transcriptomic and proteomic data was performed using the Gene Ontology and Reactome databases.

Results. The most highly expressed endothelial phenotype markers in secretome and intracellular proteome of intact arterial ECs were vWF, EPCR/CD201, MCAM/CD146, ICAM2/CD102, VE-cadherin/CDH5/CD144, and PECAM1/CD31. Notably, 30 out of 45 endothelial phenotype markers were released into the extracellular microenvironment even without pathological activation. Most endothelial cell adhesion molecules exhibited low basal expression and were inducible (ICAM1, NRCAM, ALCAM, SELE, VCAM1). Although arterial ECs expressed approximately 40 cytokine genes, only about 10 were synthesized and released at relatively significant levels (including MIF, PTX3, CSF1, CCL2, CCL14, IL-8/CXCL8, and CXCL1). ECs demonstrated high expression and secretion of almost all basement membrane components (approximately 20), as well as a substantial number of extracellular matrix components (approximately 65). Approximately 50 angiogenic and 65 hemostatic molecules were detected in the conditioned media of ECs. Higher number of transcribed genes and synthesized proteins involved in vasodilatory pathways compared to vasoconstrictive pathways indicated a more prominent role of intact ECs in vasodilation.

Conclusion. Intact ECs have high basal bioactivity in maintaining angiogenesis, hemostasis, and the synthesis of endothelial basement membrane and subendothelial extracellular matrix components. Albeit intact ECs exhibit low basal pro-inflammatory activity, they have a high potential for pathological activation in response to the respective stimuli.

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