Identification of Key Genes and Pathways in Genotoxic Stress Induced Endothelial Dysfunction: Results of Whole Transcriptome Sequencing

Abstract

Atherosclerosis is a leading cause of cardiovascular morbidity and mortality worldwide. Endothelial disfunction underlying the atherogenesis can be triggered by genotoxic stress in endothelial cells. In the presented research whole transcriptome sequencing (RNA-seq) of human coronary artery (HCAEC) and internal thoracic artery (HITAEC) endothelial cells in vitro exposed to 500 ng/mL mitomycin C (treatment group) or 0.9% NaCl (control group) was performed. Resulting to bioinformatic analysis, 56 upregulated differentially expressed genes (DEGs) and 6 downregulated DEGs with absolute fold change ≥ 2 and FDR p-value < 0.05 were selected in HCAEC exposed to mitomycin C compared to the control group; in HITAEC only one upregulated DEG was found. According to Gene Ontology enrichment analysis, DEGs in HCAEC were classified into 25 functional groups of biological processes, while in HITAEC we found no statistically significant (FDR p-value < 0.05) groups. The four largest groups containing more than 50% DEGs (“signal transduction”, “response to stimulus”, “biological regulation”, and “regulation of biological process”) were identified. Finally, candidate DEGs and pathways underlying the genotoxic stress induced endothelial disfunction have been discovered that could improve our understanding of fundamental basis of atherogenesis and help to justification of genotoxic stress as a novel risk factor for atherosclerosis.

Multi-Omics Profiling of Human Endothelial Cells from the Coronary Artery and Internal Thoracic Artery Reveals Molecular but Not Functional Heterogeneity

“…This task requires large amounts of ATP to rearrange the intercellular junctions and maintain intracellular Ca 2+ metabolism, which is also indispensable for the enzymatic digestion and biosynthesis of nitric oxide and prostacyclin, the potent vasodilators [121][122][123][124]. With regards to nucleotide metabolism, DNA repair defects have been associated with age-related and premature endothelial dysfunction which develops as reduced nitric oxide release and chronic low-grade inflammation [125][126][127][128][129][130][131]. The same has been reported for decreased ubiquitination and impaired autophagy, as removal of dysfunctional organelles, supramolecular complexes, and molecules also becomes compromised with age [132][133][134], and ubiquitination participates in regulating rearrangements of intercellular junctions and integrity of the endothelial barrier [135,136].…”

Proteomic Profiling of Endothelial Cells Exposed to Mitomycin C: Key Proteins and Pathways Underlying Genotoxic Stress-Induced Endothelial Dysfunction

“…308K-05a, Cell Applications Inc., San Diego, CA, USA) cryopreserved at the 2nd passage were used in the present research. All manipulations with cells were performed as described previously [27]. Briefly, the cells were seeded into fibronectin-coated T-75 flasks (Greiner Bio-One GmbH., Kremsmünster, Austria) containing 15 mL of a Human MesoEndo Cell Growth Medium (Cell Applications Inc., San Diego, CA, USA) and incubated in a humidified atmosphere with 5% CO 2 at 37 • C. After 3 passages, the cells were reseeded into new T-75 flasks (Greiner Bio-One GmbH., Kremsmünster, Austria) and refed (after reaching 80% confluency) with another 15 mL of a Human MesoEndo Cell Growth Medium (Cell Applications Inc., San Diego, CA, USA) containing 500 ng/mL of MMC (AppliChem, Barcelona, Spain, CAS No.…”

“…Whole-transcriptome sequencing (RNA-seq) and ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS/MS) are the modern high-throughput methods allowing for the identification of any signaling pathways determining the acquisition of certain phenotypes by cells in response to various stimuli. Resulting from RNA-seq, MMC-induced genotoxic stress in primary human coronary artery endothelial cells (HCAEC) and internal thoracic artery endothelial cells (HITAEC) leads to the upregulation of genes involved in the p53, GAS6/AXL, JNK/SAPK, PI3K/AKT, DNA damage, oxidative stress and inflammatory response signaling pathways, the inflammatory activation of endothelial cells, endothelial migration and differentiation, the adhesion of mononuclear blood fractions to the plasma membrane of endothelial cells and apoptosis, and to the downregulation of genes involved in angiogenesis [27]. At the same time, the proteomic profiling of the endothelial cells incubated under a genotoxic load has still not been performed.…”

Atorvastatin Can Modulate DNA Damage Repair in Endothelial Cells Exposed to Mitomycin C

“…Recently, it was reported that genotoxic stress induced by 6 h of exposure of endothelial cells to 500 ng/mL MMC is associated with proinflammatory activation of endothelium and endothelial dysfunction [ 17 , 18 , 19 ] underlying atherosclerosis [ 20 , 21 ], a leading cause of cardiovascular morbidity and mortality worldwide [ 22 , 23 ]. HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) reductase inhibitors, also known as statins, are small molecules that are the rate-controlling enzyme of the mevalonate pathway.…”