The limitation of lipotransfection with plasmid vectors is its low efficiency and the short-term expression of introduced genes. This is particularly important when the synthesis of high amounts of therapeutic products is required. However, growth factors with paracrine action overcome this problem. The aim of our study was to check whether the amounts of vascular endothelial growth factor (VEGF) generated after plasmid lipotransfection into vascular smooth muscle cells (VSMC) can be sufficient to stimulate endothelial cell proliferation. Two plasmids, pSG5-VEGF 121 and pSG5-VEGF 165 , harboring human VEGF 121 and VEGF 165 iso-forms were constructed and lipotransfected into COS-7 cells or to rat VSMC. The transfection efficiency, estimated by the expression of control, b-galactosidase gene, was about 50% in COS- 7 but rarely exceeded 5% in VSMC. However, despite this, the smooth muscle cells generated high amounts of VEGF protein, up to 3 ng/ml medium. The biological activity of this VEGF was confirmed by enhanced proliferation of human umbilical vein and coronary artery endothelial cells, stimulated with conditioned media of pSG5-VEGF transfected cells. Thus, the low transfection efficiency does not preclude the generation of high amounts of VEGF by VSMC. After reaching the maximum at about 48 h after transfection, the generation of VEGF decreased in the following days. Such a situation may be sufficient for the gene therapy of restenosis when the long-term expression of therapeutic gene(s) is not necessary. Thus, we suggest that the pSG5-VEGF 121 and pSG5-VEGF 165 plasmids can be used for therapeutic application.
Stem cells exist and can do a lot. For several decades, bone marrow and umbilical cord blood transplants containing haematopoietic stem cells have been used in the treatment of blood diseases. Genetic modifications (gene therapy) of such cells help to cure complex immunodeficiencies and severe anaemias. The limbal stem cells taken from the eye and properly multiplied can regenerate the damaged cornea, and the epidermal stem cells help in the treatment of severe burns and some hereditary, severe skin diseases. Promising experimental research is under way on other uses of stem cells. However, these cells are appropriately selected, having real ability to differentiate into specialized cells whose malfunction is the cause of the disease. Therapeutic applications of stem cells are apparently limited to date. Meanwhile, the Internet is full of advertisements for supposedly miraculous treatments for almost any disease. Stem cells have become a modern synonym of the Holy Grail. A wonderful dish, transforming every drink into elixir of health, youth and long life. Stem cells from a single source, e.g., umbilical cord blood, or so-called cells, although without proven properties of stem cells, are offered in commercial private clinics as a panacea for autism, cerebral palsy, spina bifida, eye diseases, amyotrophic lateral sclerosis and dozens other disorders. Without justification for their action in these diseases, without convincing evidence of safety, but for a high fee. This article discusses stem cells and misunderstandings about including any cells among them. It draws attention to the real possibilities and confirmed uses of stem cells and presents the problems, doubts and dangers for patients associated with commercial offers of treatments using “stem” cells. The author cites the positions of scientific institutions and societies warning against premature commercialization of unjustified and potentially dangerous therapies
Summary: Background: Peroxisome proliferator-activated receptor-γ (PPARγ) ligands were shown to induce the vascular endothelial growth factor (VEGF) synthesis in several cell types. We tested the effect of ciglitazone, one of the most specific PPARγ agonists, on the angiogenic activity of VEGF. Methods: Experiments were performed on human umbilical vein endothelial cell (HUVEC), incubated for 12 to 48 h with human VEGF165 (30 ng/ml) in the presence or absence of ciglitazone (1 – 10 µM). Cell proliferation was tested by BrdU incorporation. Migration was measured in modified Boyden chambers. Morphogenesis and capillary outgrowth were assessed by tube formation assay and spheroid cell culture, respectively. Results: Treatment with ciglitazone inhibited by about 50 % VEGF-induced proliferation, migration, morphogenesis, and sprouting of capillaries. Conclusions: Thus we conclude that, despite augmentation of VEGF generation, ciglitazone may act as an antiangiogenic agent attenuating VEGF activity by directly influencing endothelial cells. Zusammenfassung: Grundlagen: Peroxisome proliferator-activated receptor-γ (PPARγ)-Liganden induzieren die Synthese von Vascular Endothelial Growth Factor (VEGF) in verschiedenen Zellen. Methodik: Es wurde die Wirkung von Ciglitazon, einer der potentesten PPARγ-Agonisten, auf die angiogenetische Wirkung von VEGF untersucht. HUVEC (human umblical endothelial cells) wurden für 12 bis 48 Stunden mit humanem VEGF165 (30 ng/ml) sowohl mit, als auch ohne Ciglitazon (1 – 10 µM) inkubiert. Ergebnisse: Ciglitazon hat die angiogenetischen Aktivitäten von Endothelzellen gehemmt. Die Behandlung mit Ciglitazon hat die VEGF-induzierte Proliferation und Migration von HUVEC unterdrückt. Weiteres konnte mit der Ciglitazon-Behandlung eine Reduktion der morphogenetischen Kapazität von Endothelzellen („tube formation assay”) sowie ein vermindertes Aussprossen der Kapillaren (Sphärenzellkultur) nachgewiesen werden. Schlußfolgerungen: Obwohl Ciglitazon die Synthese von VEGF erhöht, hat Ciglitazon eine anti-angiogenetische Wirkung, welche durch Hemmung der VEGF-Aktivität und direkten Einfluß auf Endothelzellen bedingt ist.
Summary: Background: Angiogenesis is a promising novel therapeutic strategy to provide new venues for blood flow in patients with severe ischaemic heart and peripheral vascular diseases. Among several stimulators, the vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2, basic FGF) have been most widely studied. Methods: Preclinical animal studies suggested the strong therapeutic potentials of VEGF, and such positive effects have also been confirmed in a limited number of phase I clinical trials. However, some animal studies suggest the existence of a considerable risk associated with angiogenic therapy. Results: VEGF is strongly expressed in human atherosclerotic plaques, it promotes atherosclerosis in animal models, and there is evidence indicating that the plaque growth is dependent on the formation of new blood vessels. Therefore, for a convincing demonstration of the safety and efficacy of VEGF gene therapy, the clinical effects need to be evaluated in larger randomized, double-blinded clinical trials. Conclusions: Further understanding of the mechanisms of activity of angiogenic growth factors and the formation of blood vessels is required, and improved gene transfer methods are necessary to introduce therapeutic angiogenesis in clinical practice. Zusammenfassung: Grundlagen: Die Angiogenese ist eine vielversprechende neue therapeutische Strategie bei Patienten mit Myokardischämie und peripheren Gefäßerkrankungen. Unter den verschiedensten Stimulatoren sind der Vascular Endothelial Growth Factor (VEGF) und der Fibroblast Growth Factor-2 (FGF-2, basic FGF) am besten erforscht. Methodik: Vorklinische Tierversuche lassen ein großes therapeutisches Potential erwarten, und diese positiven Effekte werden von einer begrenzten Zahl von Phase-I-Studien bestätigt. Einige der experimentellen Studien lassen erhebliche Risiken in Verbindung mit einer angiogenetischen Therapie vermuten. Ergebnisse: VEGF wird von humanen atherosklerotischen Plaques verstärkt gebildet, fördert Atherosklerose am Tiermodell, und es gibt Anzeichen, daß das Plaquewachstum von der Bildung neuer Blutgefäße abhängig ist. Daher sollten die klinischen Effekte, um die Sicherheit und Effizienz einer VEGF-Gentherapie zu zeigen, in großen, randomisierten, doppelblinden, klinischen Studien evaluiert werden. Schlußfolgerungen: Um eine therapeutische Angiogenese in der klinischen Praxis anwenden zu können, ist ein genaueres Verständnis der Mechanismen der Aktivität von angiogenetischen Wachstumsfaktoren und der Bildung von Blutgefäßen und die Verbesserung der Gentransfermethoden nötig.
