Extracellular DNA Affects NO Content in Human Endothelial Cells

Abstract

Fragments of extracellular DNA are permanently released into the blood flow due to cell apoptosis and possible de novo DNA synthesis. To find out whether extracellular DNA can affect the synthesis of nitric oxide (NO), one of key vascular tone regulators, we studied in vitro effects of three artificial DNA probes with different sequences and 10 samples of extracellular DNA (obtained from healthy people and patients with hypertension and atherosclerosis) on NO synthesis in endothelial cell culture (HUVEC). For detection of NO in live cells and culture medium, we used a NO-specific agent CuFL penetrating into the cells and forming a fluorescent product FL-NO upon interaction with NO. Human genome DNA fragments affected the content of NO in endothelial cells; this effect depended on both the base sequence and concentration of DNA fragments. Addition of artificial DNA and extracellular DNA from healthy people into the cell culture in a low concentration (5 ng/ml) increased the detected NO concentration by 4-fold at most. Cytosine-guanine (CG)-rich fragment of the transcribed sequence of ribosomal repeat was the most powerful NO-inductor. The effect of DNA fragments on NO synthesis was comparable with that of low doses of oxidizing agents, H(2)O(2) and 17β-estradiol. Extracellular DNA samples obtained from patients with hypertension and atherosclerosis decreased NO content in cells and medium by 1.3-28 times compared to the control; the effect correlated with the content of CG-rich sequences.

An Exposure to the Oxidized DNA Enhances Both Instability of Genome and Survival in Cancer Cells

“…B – the ratio of the levels of AIM1 [1] and TLR9 [2] – encoding RNAs to the levels TBP-encoding reference mRNA in cells exposed to gDNA or gDNA OX for 2 hrs (grey columns) and 48 hrs (black columns).…”

“…R: gated area. Panels C [2] and D [2]: median signal intensity of FL2 (R) in MCF-7 cells (mean value for three independent experiments). Panels C [3] and D [3]: relative proportions of AIM2- or TLR9-positive cells in R gates [1].…”

“…In confluent cultures, the proportion of cells with AIM2 increases 2-fold (Figure 2C[1,3]). These increases are paralleled by increases in AIM2 protein levels per cell (Figure 2C[2]), while the levels of AIM2 encoding mRNAs remain approximately the same (Figure 2B[1]). These observations may be explained by prevailing regulation of AIM2 activity at the level of the translation or its stability rather than at the level of transcription and await further investigation.…”

Oxidized Extracellular DNA as a Stress Signal in Human Cells

“…For example, when primary endotheliocytes (HUVECs) were exposed to cfDNA samples obtained from patients with hypertension and atherosclerosis, their NO contents substantially decreased, while the DNA samples obtained from healthy donors have no effect of NO release [28, 29]. In electrically paced cultures of ventricular neonatal rat myocytes, an exposure to the cfDNA of patients with acute myocardial infarction has produced a decrease in the frequency of contraction [108].…”

Endothelial nitric oxide synthase uncoupling: A novel pathway in OSA induced vascular endothelial dysfunction

“…No significant differences were observed from different levels within a biopsy. The CuFL technique has been widely applied for cellular and tissue measurements of NO(Efremova et al, 2010; Schreiber et al, 2011). We have previously applied and validated the CuFL fluorescence along with the electron paramagnetic resonance (EPR) measurement of NO (Yang et al, 2013).…”