[Transcription of DNA-Methyltransferases in Endothelial Cells Exposed to Mitomycin C].

DNA-methyltransferases catalyze DNA methylation in the CpG sites, which play an important role in the maintenance of genome stability. The association between DNA methylation and genotoxic stress resulting in the action of various clastogens has been shown. Genotoxic stress is one of the triggers of endothelial dysfunction. In this study, the transcription of DNMT1, DNMT3A and DNMT3B genes in coronary (HCAEC) and internal thoracic (HITAEC) artery endothelial cells exposed to alkylating mutagen mitomycin C was studied using quantitative polymerase chain reaction. In HCAEC exposed to mitomycin C, DNMT1 transcription is 1.7-fold higher compared to the unexposed control. After elimination of the mutagen from the cultures followed by 24-hours of cultivation, a 2-fold increase of transcription of DNMT3B in HCAEC exposed to mitomycin C compared to the control was observed. At the same time, no changes in transcription of the studied DNA-methyltransferases were found in HITAEC exposed to the mutagen. Thus, increased transcription of DNA-methyltransferase may be a possible molecular mechanism underlying endothelial dysfunction in response to mutagenic load in an in vitro experiment.

The cytokine response of human coronary artery endothelial cells treated with doxorubicin: results of an in vitro experiment.

The cytokine profile of primary coronary artery endothelial cells cultivated in the presence of doxorubicin (2 µg/ml and 6 µg/ml) was evaluated using enzyme-linked immunosorbent assay and qPCR. Cultivation of cells in the presence of these concentrations of doxorubicin for 24 h, upregulated expression of the following genes: IL6 (by 2.30 and 2.66 times, respectively), IL1B (by 1.25 and 3.44 times), and CXCL8 (by 6.47 times and 6.42 times), MIF (2.34 and 2.28 times), CCL2 (4.22 and 3.98 times). Under these conditions the following genes were downregulated: IL10, IL1R2, TNF. Cultivation of cells in the presence of doxorubicin (2 µg/ml and 6 µg/ml) for 24 h also increased the secretion of IL-6.

[Genotoxic stress leads to the proinflammatory response of endothelial cells: an in vitro study]. / Genotoksicheskii stress vyzyvaet formirovanie éndotelial'nymi kletkami provospalitel'nogo fenotipa: issledovanie in vitro.

It was shown, that genotoxic stress can trigger endothelial disfunction and atherosclerosis, but the molecular genetic mechanisms of this process are poorly investigated. At the same time, inflammation also plays the important role in atherogenesis. This study aimed access of inflammatory marker expression in the endothelial cells exposed to alkylating mutagen mitomycin C (MMC). Primary human coronary (HCAEC) and internal thoracic artery endothelial cells (HITAEC) exposed to 500 ng/ml MMC (experimental group) and 0.9% NaCl (control) were used in this research. A gene expression profile was evaluated by quantitative reverse transcription PCR after 6 h exposure of endothelial cells to MMC (or 0.9% NaCl) followed by subsequent 24 h incubation in the mutagen-free cell growth media. The cytokine profile of endotheliocytes was studied by dot blotting. We found that MIF, IL-8, MCP-1, IP-10 and PDGFB were upregulated both in HCAEC and HITAEC, while MIP-1ß release remained unchanged. TIMP-2 was upregulated in HCAEC but not in HITAEC. sTNF RI was expressed only in HCAEC. According to gene expression analysis, HCAEC exposed to MMC are characterized by the increased mRNA level of IL-8, MCP-1 and IP-10; decreased expression of TIMP-2 and no differences in the expression of MIF, MIP-1ß and PDGFB compared to the control. In HITAEC, increased mRNA level of IL-8 and IP-10; decreased expression of MIF and TIMP-2, no differences in the expression of MCP-1, MIP-1ß and PDGFB was shown. TNF-RI expression was not detected in both cell lines. Thus, genotoxic stress in endothelial cells induced by MMC leads to differential inflammatory response that can trigger endothelial dysfunction.

Endothelial Dysfunction in the Context of Blood-Brain Barrier Modeling.

Here, we discuss pathophysiological approaches to the defining of endothelial dysfunction criteria (i.e., endothelial activation, impaired endothelial mechanotransduction, endothelial-to-mesenchymal transition, reduced nitric oxide release, compromised endothelial integrity, and loss of anti-thrombogenic properties) in different in vitro and in vivo models. The canonical definition of endothelial dysfunction includes insufficient production of vasodilators, pro-thrombotic and pro-inflammatory activation of endothelial cells, and pathologically increased endothelial permeability. Among the clinical consequences of endothelial dysfunction are arterial hypertension, macro- and microangiopathy, and microalbuminuria. We propose to extend the definition of endothelial dysfunction by adding altered endothelial mechanotransduction and endothelial-to-mesenchymal transition to its criteria. Albeit interleukin-6, interleukin-8, and MCP-1/CCL2 dictate the pathogenic paracrine effects of dysfunctional endothelial cells and are therefore reliable endothelial dysfunction biomarkers in vitro, they are non-specific for endothelial cells and cannot be used for the diagnostics of endothelial dysfunction in vivo. Conceptual improvements in the existing methods to model endothelial dysfunction, specifically, in relation to the blood-brain barrier, include endothelial cell culturing under pulsatile flow, collagen IV coating of flow chambers, and endothelial lysate collection from the blood vessels of laboratory animals in situ for the subsequent gene and protein expression profiling. Combined with the simulation of paracrine effects by using conditioned medium from dysfunctional endothelial cells, these flow-sensitive models have a high physiological relevance, bringing the experimental conditions to the physiological scenario.