Advances in Clinical and Experimental Medicine

Title abbreviation: Adv Clin Exp Med
JCR Impact Factor (IF) – 1.736
5-Year Impact Factor – 2.135
Index Copernicus  – 166.39
MEiN – 70 pts

ISSN 1899–5276 (print)
ISSN 2451-2680 (online)
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Advances in Clinical and Experimental Medicine

2010, vol. 19, nr 3, May-June, p. 347–359

Publication type: original article

Language: English

Angiogenic Therapy for Critical Lower Limb Ischemia

Angiogenna terapia w leczeniu krytycznego niedokrwienia kończyn dolnych

Anna Sadakierska-Chudy1,, Jan Skóra2,, Piotr Barć2,, Dagmara Baczyńska3,, Urszula Kasprzykowska4,, Artur Pupka2,, Marek Ussowicz5,, Piotr Szyber2,, Tadeusz Dobosz1,

1 Molecular Techniques Unit, Department of Forensic Medicine, Wroclaw Medical University, Poland

2 Department and Clinic of General and Vascular Surgery and Transplantology, Wroclaw Medical University, Poland

3 Institute of Biochemistry and Molecular Biology, Department of Cell Pathology, Wroclaw Medical University, Poland

4 Department of Microbiology, Wroclaw Medical University, Poland

5 Department of Pediatric Bone Marrow Transplantation, Oncology, and Hematology, Wroclaw Medical University, Poland

Abstract

Background. In several preclinical and clinical studies, VEGF gene therapy has proven successful in the treatment of critical limb ischemia (CLI). CLI is estimated to develop in 500–1000 individuals/million/year. It is a severe disease associated with a high risk of amputation and mortality. Therapeutic angiogenesis is a novel concept consisting of the use of proangiogenic growth factors to promote collateral artery development in ischemic tissues. To test a potential clinical application of this method, the authors prepared a nonviral expression vector encoding VEGF165 protein.
Objectives. The purpose of this study was to construct a functional expression vector encoding isoform VEGF165 which can be used for therapeutic angiogenesis in no-option patients with critical limb ischemia.
Material and Methods. Total RNA was extracted from vein and RT-PCR was used to prepare a cDNA sequence for cloning into the plasmid vector. The naked pcDNA3/VEGF165 plasmid was administered alone (group I) or combined with mononuclear cells (group II) directly into the skeletal muscle of the ischemic lower limb. The vector’s ability of expression in mammalian cells, clinical outcome, serum level of VEGF protein, and endothelial cell proliferation in muscle tissue were evaluated.
Results. pcDNA3/VEGF165 was expressed in transfected CHOPro5 cells. Due to the gene transfer, 9 of 24 patients did not require amputation. A higher serum VEGF concentration was observed than in healthy controls and the level of cytokine increased on day 14 and decreased on day 90 after plasmid administration. Histological and immunohistochemical analysis of the muscles derived from amputated limbs revealed the presence of VEGF protein and signs of new blood vessel formation.
Conclusion. This therapy is safe, but a single intramuscular gene transfer of VEGF165 is insufficient to promote angiogenesis efficiently. Better results were observed when combined therapy was used.

Streszczenie

Wprowadzenie. Wiele badań przedklinicznych i klinicznych udowodniło, że terapia genowa z wykorzystaniem genu VEGF może być z powodzeniem stosowana w leczeniu krytycznego niedokrwienia kończyn. Szacuje się, że niedokrwienie kończyn rozwija się u 500–1000 osób na 1 milion w ciągu roku. Choroba niesie ze sobą duże ryzyko amputacji i śmiertelności. Nowym sposobem leczenia może być terapeutyczna angiogeneza z wykorzystaniem proangiogennych czynników wzrostu, które pobudzają rozwój naczyń obocznych w niedokrwionej tkance. Aby zbadać możliwość klinicznego zastosowania tej metody, przygotowano niewirusowy wektor ekspresyjny kodujący białko VEGF165.
Cel pracy. Konstrukcja funkcjonalnego wektora ekspresyjnego kodującego izoformę VEGF165, który mógłby być wykorzystany w terapii angiogennej u pacjentów z krytycznym niedokrwieniem niemających innej możliwości leczenia.
Materiał i metody. RNA całkowite izolowano z żyły, sekwencję cDNA, którą wklonowano w wektor, przygotowano, wykorzystując reakcję RT-PCR. Bezpośrednio do mięśni szkieletowych niedokrwionej kończyny podawano albo nagi plazmid pcDNA3/VEGF165 (grupa I), albo komórki jednojądrzaste szpiku kostnego inkubowane z plazmidem (grupa II). W tym badaniu autorzy oceniali zdolność wektora do ekspresji w komórkach ssaczych, wynik kliniczny, stężenie białka VEGF w surowicy oraz proliferację komórek śródbłonka w tkance mięśniowej.
Wyniki. Wykazano zdolność plazmidu pcDNA3/VEGF165 do ekspresji w transfekowanych komórkach jajnika chomika chińskiego (CHOPro5). Po podaniu genu lub genu i komórek szpiku 9 z 24 pacjentów nie wymagało amputacji. Zaobserwowano większe stężenie cytokiny VEGF w surowicy u pacjentów z niedokrwieniem w porównaniu z osobami zdrowymi z grupy kontrolnej, stężenie zwiększyło się w 14. dniu, a zmniejszyło się w 90. dniu po zastosowaniu terapii. Badanie histologiczne i immnohistochemiczne mięśni z amputowanych kończyn wykazało obecność białka VEGF i ślady tworzenia nowych naczyń krwionośnych.
Wnioski. Przeprowadzone badanie wykazało, że terapia jest bezpieczna, pojedynczy domięśniowy transfer genu VEGF165 okazał się jednak niewystarczający do wydajnego pobudzenia angiogenezy. Lepsze wyniki uzyskano, gdy zastosowano terapię łączoną.

Key words

angiogenic cytokine, isoform VEGF165, expression vector, gene transfer, critical lower limb ischemia

Słowa kluczowe

cytokina angiogenna, izoforma VEGF165, wektor ekspresyjny, transfer genu, krytyczne niedokrwienie kończyn dolnych

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