Vol 6, No 3 (2011)

В связи с тем, что настоящий выпуск жур- нала посвящен перспективному направлению фундаментальной и клинической ангиологии - терапевтическому ангиогенезу, редакция обра- тилась к практикующим хирургам со следующи- ми вопросами: «Удовлетворены ли Вы как врач- клиницист имеющимся арсеналом хирургических и фармацевтических средств лечения поражений сосудов нижних конечностей? Какие пути до- стижения прогресса в этой области - продления жизни пациентов, уменьшения инвалидизации, повышения качества жизни Вы видите и како- во среди них место методов «терапевтического ангиогенеза»?

The number of peripheral artery disease remains in the world. Search for new methods of treatment is to improve the condition of the distal blood stream, reduce peripheral resistance and stimulating capillary network. One possible solution to this problem is to use cellular technologies stimulate angiogenesis. A new promising treatments is autologous cell therapy using BMMNC or PBMNC. This overview focuses on the exploration of autologous cell therapy in treating diseases of the peripheral arteries.

Currently a protein/peptide-mediated gene delivery has been considered a promising approach in non-viral gene transfer. The previous investigations have shown that histones and other nuclear proteins might be effective vectors for gene transfer into cells. Transfection of eukaryotic cells by nucleic acid and histone complexes (histonefection) effectively occurs with various histone proteins. The presence of DNA-binding domains and specific signal sequences of nuclear location allows to use histones (Н1/Н5, Н2А, Н2В, Н3, Н4) and other nuclear proteins (such as HMG family proteins and histonelike prokaryotic proteins) for recombinant genes transfer. The positive charge of histone protein molecules enables electrostatic interaction with negatively charged molecules of nucleic acids and charge neutralization that facilitates the complexes penetration through a negatively charged cell membrane. Thus, histonefection is a promising method for non-viral transfer of recombinant nucleic acids in gene therapy.

Мезенхимальные стволовые клетки жировой ткани (МСК-ЖТ) способны стимулировать ангиогенез посред- ством продукции факторов роста и стабилизации растущих сосудов. МСК-ЖТ являются перспективным материалом для аутологичной клеточной терапии. Однако с возрас- том пациентов активность их МСК-ЖТ, а, следовательно, и эффективность клеточной терапии могут снижаться. Целью нашей работы было оценить ангиогенные свойства МСК-ЖТ при старении. Материал и методы. МСК-ЖТ были выделены из под- кожной жировой ткани мышей линии BalB/с возраста 1-2, 12, 18 и 24 мес., а также 12 доноров трех возрастных групп (12 (5; 12) лет, 45 (41; 45) лет и 65 (59; 76) лет). МСК-ЖТ 2-го пассажа помещали на 48 ч в условия 1% или 20% содержания кислорода. Оценивали способность клеток стимулировать ангиогенез in vitro и in vivo. Результаты. Способность МСК-ЖТ мышей стимулиро- вать формирование капилляроподобных структур in vitro и васкуляризацию подкожных имплантатов Матригеля in vivo с возрастом снижалась в среднем на 60%. Содержание мРНК сосудистого эндотелиального фактора роста (VEGF) и плацентарного фактора роста (PlGF) в этих клетках сни- жалось, а фактора роста гепатоцитов (HGF) и компонентов системы внеклеточного протеолиза (рецептор к урокиназе, металлопротеиназы 2 и 9 типов, ингибитор активатора плазминогена-1) - возрастало с возрастом животных. Сти- муляция экспрессии VEGF, PlGF и HGF в условиях гипоксии была выражена слабее в случае МСК-ЖТ старых живот- ных. Экспрессия PlGF и ангиопоэтина-1, а также секреция VEGF и HGF МСК-ЖТ человека отрицательно коррелирова- ли с возрастом донора. Таким образом, при старении способность МСК-ЖТ сти- мулировать ангиогенез снижается в результате подавления экспрессии ключевых ангиогенных факторов.

The development of effective treatments for patients with peripheral nerve injury is an urgent task of biomedicine. «Gold» standard in restoring the integrity of nerve conduits is auto-nerve transplantation in which a peripheral nerve defect is corrected with autologous nerve graft. Here we propose a method for stimulating revascularization and regeneration of 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 that direct injection of plasmid pBud-VEGF-FGF2 in the proximal and distal segments of nerve, as well as in the auto-nerve graft, stimulates the regeneration of the rats sciatic nerve and restores motor activity.

The article deals with the results of a 6 month comparative follow-up of non-resectable patients with lower limb atherosclerosis having clinical symptoms as claudication (IIa-III stages according to the Fontaine- Pokrovsky classification) who received Neovasculgen as a part of conservative therapy. Neovasculgen, a gene therapy drug, is a plasmid construction with a vascular endothelial growth factor gene. 100 patients were enrolled into the study: 75 patients comprised the clinical group, while 25 ones being the controls. The patients underwent treatment in three clinical centers. The drug was injected intramuscular, maximally close to ischemia zones, twice in doses of 1.2 mg with the interval of 14 days. The following factors were controlled: pain free walking distance (PlWD), ankle-brachial index (ABI), transcutaneous partial pressure of oxygen (TcPO2), linear blood flow rate (LBFR) in the involved segment, angiography; also the integral treatment result on the scale «success/failure», quality of life SF-36 questionnaire were assessed. The study duration was 6 months. PlWD was determined to increase by 110% that shows statistically significant difference versus the control (P=0.000).

Therapeutic angiogenesis is a promising treatment modality in patients with chronic lower limb ischemia, for whom revascularization is not indicated. A number of studies demonstrated satisfactory therapeutic effects of gene and cell-based therapy targeted to stimulate angiogenesis. At the same time the factors affecting the efficacy of angiogenesis stimulation in clinical settings are under-investigated. In the given study 30 patients, aged 49-78 years with chronic lower limb ischemia of atherosclerotic origin (Fontaine IIb-IV) were randomized into groups of gene therapy (GT) or the control group (C). Plasmid DNA containing an encoding part of the vascular endothelial growth factor (VEGF-165) gene was inserted into ischemic limb muscles of the GT patients. All the patients had sessions of treadmill training walking. Besides clinical findings, angiography, scintigraphy, transcutaneous oxymetry, laser Doppler examination, duplex ultrasonography, treadmill test were carried out to evaluate the invention efficacy. Also circulating progenitor cell count was assessed by flow cytometry, ELISA was used to evaluate angiogenic growth factors. In 3 months limb perfusion rate, painless walking distance, collateral circulation indices increased. In 50% of patients the therapeutic effect was assessed as moderate (Rutherford +2), in 30% as minimal (Rutherford +1), 20% of patients showed no dynamics. Clinical laboratory tests had no significant dynamics in the group of standard therapy. No serious complications were noted. In a correlation test the efficacy of angiogenic therapy was associated with a patent ileac segment, shorter smoking history and higher ankle-brachial index.