The number of peripheral artery disease remains in theworld. Search for new methods of treatment is to improvethe condition of the distal blood stream, reduce peripheralresistance and stimulating capillary network. One possiblesolution to this problem is to use cellular technologies stimulateangiogenesis. A new promising treatments is autologous celltherapy using BMMNC or PBMNC. This overview focuses onthe exploration of autologous cell therapy in treating diseasesof the peripheral arteries.

Gene therapeutic approaches to the restoration of theischemic tissue perfusion are considered very promising,but to this time the molecular mechanisms which allow thetherapeutic gene encoding plasmid to transfect the targetcell and underlie the positive clinical effects remain unknown.In this review the possible molecular mechanisms of theangiogenic factor VEGF encoding plasmid penetration into thecytoplasm and the nucleus of the target cell are discussed,and also the methods for better transfection and the gene ofinterest expression are proposed.

Currently a protein/peptide-mediated gene delivery hasbeen considered a promising approach in non-viral genetransfer. The previous investigations have shown that histonesand other nuclear proteins might be effective vectors for genetransfer into cells. Transfection of eukaryotic cells by nucleicacid and histone complexes (histonefection) effectively occurswith various histone proteins. The presence of DNA-bindingdomains and specific signal sequences of nuclear locationallows to use histones (Н1/Н5, Н2А, Н2В, Н3, Н4) and othernuclear proteins (such as HMG family proteins and histonelikeprokaryotic proteins) for recombinant genes transfer.The positive charge of histone protein molecules enableselectrostatic interaction with negatively charged moleculesof nucleic acids and charge neutralization that facilitatesthe complexes penetration through a negatively chargedcell membrane. Thus, histonefection is a promising methodfor non-viral transfer of recombinant nucleic acids in genetherapy.

The search of new way of cell injection that can providemaximal angiogenesis in ischemic limbs is urgent aim ofinvestigation for the right way of cell delivery other thingsbeing equal is the main request for cell therapy efficiency.On experimental model of critical ischemia (by angiographicmethod and evaluation of capillary density on histologicalsections) it was shown that cell therapy independently fromthe way of cell injection (intramuscularly, intra-arterialy andcombined) induces angiogenesis, the rate and intensity ofwhich depend on the way of cell delivery. The optimum variantof cell therapy of critical ischemia is combined (intramusculararterial)method of cell injection, uniting the advantages ofintramuscular and intra-arterial ways.

The paper presents the results of a pilot clinical study ofthe use of injection intramiokardial mononuclear fraction ofcells autologous bone marrow as an additional procedure tothe standard surgical treatment in 10 patients of heart valvedefects in order to assess opportunities to improve blood flowin areas of severe hypoperfusion and myocardial scarring.In terms of monitoring of patients to 72 months after celltransplantation demonstrated the safety of this treatment,the improvement of regional perfusion and myocardialcontractility in the areas of injection autologous mononuclearfraction of bone marrow cells.

The development of effective treatments for patientswith peripheral nerve injury is an urgent task of biomedicine.«Gold» standard in restoring the integrity of nerve conduits isauto-nerve transplantation in which a peripheral nerve defectis corrected with autologous nerve graft. Here we proposea method for stimulating revascularization and regenerationof auto-nerve graft by a local injection of plasmid pBud-VEGFFGF2,expressing vascular endothelial growth factor (VEGF)and basic fibroblast growth factor (FGF2). It is shown thatdirect injection of plasmid pBud-VEGF-FGF2 in the proximaland distal segments of nerve, as well as in the auto-nervegraft, stimulates the regeneration of the rats sciatic nerveand restores motor activity.

The aim is to study the phenotypic characteristics andcytokine-producing properties mobilized drug administrationG-CSF from bone marrow mononuclear cells of peripheralblood of patients with heart failure, which developed afteracute myocardial infarction, in connection with the efficiencyof intramyocardial stem cell therapy. The study included 67patients with CHD and III-IV functional class congestiveheart failure (NYHA), receiving current standard therapy.Shown that the introduction of the drug G-CSF results inmobilization of endothelial progenitor cells (EPC) from bonemarrow into peripheral blood (CD34+/CD133+and CD34+/KDR+populations). Intramyocardial cell injection resulted inimproved perfusion at the injection site in 76% of patients.Patients who responded to improved perfusion, the number ofCD34+CD133+PC in 3,2 times higher than in patients withouteffect or impairment. Mononuclear cells after administrationof G-CSF in a 48 hour culture secrete cytokines, Epo,GM-CSF, TNF-ƒ, contribute to the improvement of myocardialperfusion. Peripheral blood is a readily available source of EPС,and mononuclear cells after mobilization are able to exertreparative effects on the ischemic myocardium.

The article deals with the results of a 6 monthcomparative follow-up of non-resectable patients withlower limb atherosclerosis having clinical symptoms asclaudication (IIa-III stages according to the Fontaine-Pokrovsky classification) who received Neovasculgenas a part of conservative therapy. Neovasculgen, a genetherapy drug, is a plasmid construction with a vascularendothelial growth factor gene. 100 patients were enrolledinto the study: 75 patients comprised the clinical group,while 25 ones being the controls. The patients underwenttreatment in three clinical centers. The drug was injectedintramuscular, maximally close to ischemia zones, twice indoses of 1.2 mg with the interval of 14 days. The followingfactors were controlled: pain free walking distance (PlWD),ankle-brachial index (ABI), transcutaneous partial pressureof oxygen (TcPO2), linear blood flow rate (LBFR) in theinvolved segment, angiography; also the integral treatmentresult on the scale «success/failure», quality of life SF-36questionnaire were assessed. The study duration was 6months. PlWD was determined to increase by 110% thatshows statistically significant difference versus the control(P=0.000).

The controlled study results of using therapeuticangiogenesis in multimodality therapy of patients with chroniclower limb ischemia (CLLI) are presented. 134 patients (74ones comprised the target group and 60 patients were thecontrols) with CLLI IIb-III stages according to the Fontaine-Pokrovsky classification were included into the study.Angiogenesis stimulation was used both as a separatetreatment modality in combination with routine conservativetherapy and as an adjuvant to reconstructive vascularsurgery. It is proved that genetically engineered angiogenesisstimulators can be used in a multimodality therapy of patientswith CLLI, demonstrating satisfactory tolerance and safety.Therapeutic angiogenesis technologies have been shown tobe applied as an independent therapy and as in combinationwith reconstructive vascular surgery to improve a long-termoutcome of the latter.

Therapeutic angiogenesis is a promising treatmentmodality in patients with chronic lower limb ischemia, forwhom revascularization is not indicated. A number of studiesdemonstrated satisfactory therapeutic effects of gene andcell-based therapy targeted to stimulate angiogenesis. At thesame time the factors affecting the efficacy of angiogenesisstimulation in clinical settings are under-investigated.In the given study 30 patients, aged 49-78 years withchronic lower limb ischemia of atherosclerotic origin (FontaineIIb-IV) were randomized into groups of gene therapy (GT) orthe control group (C). Plasmid DNA containing an encodingpart of the vascular endothelial growth factor (VEGF-165)gene was inserted into ischemic limb muscles of the GTpatients. All the patients had sessions of treadmill trainingwalking. Besides clinical findings, angiography, scintigraphy,transcutaneous oxymetry, laser Doppler examination, duplexultrasonography, treadmill test were carried out to evaluatethe invention efficacy. Also circulating progenitor cell countwas assessed by flow cytometry, ELISA was used to evaluateangiogenic growth factors. In 3 months limb perfusionrate, painless walking distance, collateral circulation indicesincreased. In 50% of patients the therapeutic effect wasassessed as moderate (Rutherford +2), in 30% as minimal(Rutherford +1), 20% of patients showed no dynamics.Clinical laboratory tests had no significant dynamics in thegroup of standard therapy. No serious complications werenoted. In a correlation test the efficacy of angiogenic therapywas associated with a patent ileac segment, shorter smokinghistory and higher ankle-brachial index.