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Human mesenchymal stem cells (MSCs) are widely used in regenerative medicine. However, many questions on the role of different signaling pathways in the regulation of stem cell (SC) functional activity within the organism still remain unanswered. In lesion regions the level of cell death increases and DNA fragments from dead cells (cell-free DNA, cfDNA) are accumulated in blood. We have shown that in vitro contact of adipose-derived MSCs with cfDNA fragments the increase of transcription activity (evaluated by the increase of total cellular RNA and rRNA) is observed. GC-rich cfDNA fragments (GC-DNA) activated the TLR9-dependent signal pathway causing up-regulation of expression of TLR9 and MyD88, the TLR9-signaling pathway adapter. AT-rich DNA fragments did not increase the TLR9 expression, but did increase the level of MyD88 mRNA. So we suggest that AT-DNA acts via some other receptors, which, nevertheless, activate MyD88-dependent signalling in MSCs. We have also shown that cfDNA fragments decreased the activity of caspase, an apoptotic enzyme. Thus, cfDNA can significantly influence the functional activity of MSC by activating the TLR9- and MyD88-dependent signal pathways and by lowering the apoptosis level.

In this study we have investigated properties of blood plasma extracellular DNA (cell-free DNA, cfDNA) from patients with essential arterial hypertension (AH). Concentration of cell-free DNA was basically the same as in healthy donors, however, the content of the marker, CpG-rich cell-free DNA fragments (CpG-DNA) of the transcribed area of the ribosomal repeat (TArDNA, CpG-DNA) was higher in AH patients. For evaluation of the effect of CpG-DNA on the development of arterial hypertension 2-day-old SHR rat pups and corresponding controls of normotensive WKY rats received a single subcutaneous injection of human TArDNA (700 ng) to generate anti-CpG-DNA antibodies (and thus to alter the CpG-DNA content in total cfDNA). After 9 weeks blood pressure (BP) in SHR rats immunized with CpG-DNA was significantly lower than in control SHR rats and was basically the same as in WKY rats. However, subsequently, BP of the immunized SHR exhibited age-related increase, which reached the stably high values typical for mature SHR 8 weeks later compared with control SHR. Analysis of cfDNA has shown that in 17-week-old immunized SHR rats concentrations of cell-free DNA and its small DNA fragments are lower and the content of CpG-DNA (rat TArDNA) is higher than in corresponding controls. These changes were accompanied by a 3.5-fold increase in blood endonuclease activity and the decrease in content of free (unbound to cfDNA) anti-CpG-DNA antibodies. Total content of anti-CpG-DNA antibodies in the immunized rats was the same as in control animals. Thus, the delayed age-related increase in stable BP observed in immunized SHR rats is obviously not associated with increased generation of anti-CpG-DNA antibodies. Possible reasons of this effect are discussed.

We have developed a novel method for in vivo evaluation of cell death in patients with acute and/or chronic heart diseases, which are accompanied by apoptosis or cell necrosis. The method is based on the analysis of cell free DNA (cfDNA) in the blood serum (or plasma). The major parameters assessed in the method include total concentration of serum cfDNA, concentration of serum ribosomal repeat (rDNA), content of rDNA in total cfDNA, as well as factors of cfDNA elimination, such as nuclease activity and anti-DNA antibody. We demonstrated a fivefold increase in the serum cfDNA concentration and a 12-fold enhancement of serum rDNA concentration in patients with acute myocardial infarction compared with healthy individuals. In chronic coronary ischemia the serum cfDNA concentration was similar to that in the disease-free group. However, the content of rDNA in cfDNA was 4.8-fold higher, and the serum rDNA concentration was increased sevenfold. We hypothesize that one reason for accumulation of rDNA within cfDNA might be the previously reported resistance of rDNA to the ds-fragmentation by serum endonucleases. In both acute and chronic coronary disease the nuclease activity in the serum was substantially higher than that in the healthy cohort. Moreover, the titer of anti-DNA antibodies was elevated, with these antibodies being mostly bound to the cfDNA. Thus, the release of rDNA fragments into the blood not only reflects cellular death in the body but also determines the response of the organism to the disease-associated stress.